Masters of asymmetry – lessons and perspectives from 50 years of septins

Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.

Fifty years ago, Nobel laureate Lee Hartwell published the first three genes from his pioneering screen for mutants that alter the cell division cycle (cdc) of the budding yeast Saccharomyces cerevisiae (Hartwell et al., 1970). Among them, cdc3 and subsequently cdc10, cdc11, and cdc12 were all reported to develop multiple elongated buds, failing to undergo cytokinesis (Hartwell et al., 1970; Hartwell, 1971). Electron microscopy observations indicated that the products of these genes formed a filamentous network at the mother–bud cortex (Byers and Goetsch, 1976). Further characterization and cloning of these genes by John Pringle, who named them septins, marked the birth of a new class of GTP-binding proteins with important functions in the spatial organization of eukaryotic cells (Pringle, 2008).Septins comprise a family of paralogous genes, which arose early in eukaryotic evolution and expanded in fungi and animals, while largely absent from plants (Pan et al., 2007). In mice and humans, 13 septin genes express a diversity of paralogues and isoforms, which are classified under four groups (SEPT2, SEPT6, SEPT7, and SEPT3) and consist of a conserved core GTP-binding domain with variable N- and C-terminal extensions (Figure 1A; Kinoshita, 2003; Mostowy and Cossart, 2012). Opening a new era for the septin field, the x-ray crystal structure of the mammalian SEPT2/6/7 complex revealed that septins dimerize in tandem via their GTP-binding domains by utilizing two different binding interfaces, which alternate every other monomer (Sirajuddin et al., 2007). Through a serial head-to-head and tail-to-tail binding mode, septins assemble linearly into nonpolar oligomers and polymers (Figure 1B). The SEPT2/6/7 structure suggested that the minimal septin unit is a palindromic hexamer, in which SEPT2/6/7 trimers are arranged symmetrically from a central homodimeric SEPT2-SEPT2 interface (Sirajuddin et al., 2007). New evidence, however, shows that SEPT2/6/7 assembles into a hexamer with SEPT7, or SEPT9 in the case of the hetero-octameric SEPT2/6/7/9 complex, forming the central homodimeric contact (McMurray and Thorner, 2019; Mendonca et al., 2019; Soroor et al., 2019). This order is consistent with the arrangement of the budding yeast hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) with Cdc10, the yeast septin most homologous to SEPT9, being the central homodimer (Bertin et al., 2008; McMurray and Thorner, 2019). In contrast to the symmetric end-to-end arrangement of their subunits, septin multimers are characterized by a top–bottom asymmetry with their C- and N-terminal extensions extending orthogonally toward opposite sides of the linear axis of multimerization (Figure 1C; John et al., 2007; Sirajuddin et al., 2007; Bertin et al., 2008). Although it is not functionally understood, this asymmetry may underlie an anisotropic mode of interaction with cellular components and protein complexes, which could be further modulated by the identity and combination of septin subunits.Open in a separate windowFIGURE 1:Structure and assembly of septin GTPases. (A) Schematic shows the main domains of septin GTPases, which consist of a highly conserved GTP-binding domain with G1 (GxxxxGK[ST]), G3 (DxxG), and G4 (AKAD) motifs, and a septin unique element. Septins contain a catalytic threonine residue (T*), which corresponds to the Thr of the G2 motif of Ras GTPases and is absent from septin paralogues that lack GTPase activity. The N- and C-terminal extensions of the septin GTP-binding domain vary in sequence and length, and contain proline-rich and coiled-coil domains, respectively. Domains of cytoskeleton- and membrane-binding (amphipathic helices and polybasic sequences) are denoted and differ among septin paralogues. (B, C) The x-ray crystal structure of SEPT2/6/7 (PDB: 2qag; B) shows the NC-NC and G-G interfaces of dimerization, which alternate between the GTP-binding domains of consecutive septin subunits. The C- and N-terminal ends of the G-domains are located at the top and bottom of the dotted vertical lines, respectively, and the guanine nucleotide is depicted in orange. A surface representation of the SEPT2/6/7 heterohexamer (C) shows the symmetric (nonpolar) end-to-end arrangement of septin paralogues from the central homodimeric SEPT7 interface and the asymmetric positioning of the C- and N-terminal ends across the horizontal plane of multimerization. (D) Schematic of the assembly of yeast septin complexes, which is driven by successive events of homo- and heterodimerization that are determined by GTP binding and hydrolysis. (E) Schematic shows the subunit order and identity of mammalian septin hetero-octamers, and the interchangeability of paralogue subunits of the same septin group (Kinoshita rule).Septins are structurally and phylogenetically related to the small GTPases of the Ras superfamily (e.g., Ras, Rho, Rab; Leipe et al., 2002), but most septins hydrolyze and turn over GTP slowly and there are septin paralogues (e.g., SEPT6 group) that lack GTPase activity (Vrabioiu et al., 2004; Sirajuddin et al., 2009; Zent and Wittinghofer, 2014; Abbey et al., 2019). Growing evidence indicates that GTP-binding and hydrolysis stabilize the dimeric interface, determine the order of assembly by selecting for specific dimerization partners, and induce allosteric conformational changes, which could affect the assembly and localization of septin complexes (Figure 1D; Sirajuddin et al., 2009; Zent and Wittinghofer, 2014; Weems and McMurray, 2017; Castro et al., 2020). Based on the activity and structure of their GTPase domains, paralogues of the same group occupy and exchange within the same position of the septin complex—a posit known as the Kinoshita rule (Figure 1E; Kinoshita, 2003). However, high-order septin complexes of homomeric and alternative compositions have also been reported (Mendoza et al., 2002; Mizutani et al., 2013; Sellin et al., 2014; Karasmanis et al., 2018). Higher-order septin structures exhibit slow subunit turnover and thereby persist in place longer than dynamic cytoskeletal polymers (Hu et al., 2008; Hagiwara et al., 2011; Bridges et al., 2014). Hitherto, in the absence of any known septin guanine nucleotide activating and exchange factors (GTPase-activating proteins [GAPs]/ guanine nucleotide exchange factor [GEFs]), septin dynamics and turnover are modulated by posttranslational modifications (Hernandez-Rodriguez and Momany, 2012; Ribet et al., 2017).Septins function broadly as scaffolds and barriers that recruit and exclude proteins, respectively, controlling the localization and interactions of membrane and cytoskeletal proteins (Caudron and Barral, 2009; Mostowy and Cossart, 2012; Spiliotis, 2018). This fundamental property of septins has been adopted by a diversity of molecular mechanisms and cellular processes. In the face of an ever-growing number of biological roles, it is often overlooked that septins function primarily as a module of spatial organization. After 50 years of research, we recount here how septins promote cellular asymmetries in fungi and animals, highlighting the evolutionary adaptation of septins as effectors of asymmetry from fungal cell membranes to the mammalian cytoskeleton.Septin roles in fungal cell asymmetrySeptins perform essential functions in nearly all aspects of the asymmetric growth and division of budding yeast (Figure 2). Prior to budding, a regulatory interplay between septins and the Rho family small GTPase Cdc42 enables yeast cells to polarize growth at a single site. GTP-bound Cdc42 initially recruits septins to a cloudy patch (Gladfelter et al., 2002), where a transient direct interaction between the septin Cdc11 and Cdc24, a GEF for Cdc42, creates a short-term positive feedback loop to reinforce septin recruitment and Cdc42 activation (Chollet et al., 2020). Targeted delivery of Cdc42-containing but septin-free vesicles to the center of the septin patch transforms it into a ring (Gladfelter et al., 2002; Okada et al., 2013). Septin polymerization into circular filaments is thought to render Cdc11 inaccessible to Cdc24 (Chollet et al., 2020). Concomitantly, septins begin to recruit Cdc42 GAPs, which inhibit Cdc42, and they promote the activation of formins, which drive actin cable polymerization (Buttery et al., 2012; Okada et al., 2013). As actin cables direct more vesicles to the center of the septin ring, the nascent bud grows and concomitantly Cdc42 pushes away from septins and localizes to the bud tip, where it promotes further growth (Okada et al., 2013). Hence, septins reinforce the formation of a Cdc42 polar cap during bud emergence. During bud growth, septins pattern the localization of the actin- and formin-binding protein Hof1 along the bud cortex in a manner that aligns actin cables along the mother–bud axis, which is critical for the asymmetric growth of the bud (Garabedian et al., 2020). Septin-mutant cells lose the mother- or bud-specific asymmetric localization of a number of cortical proteins as well as mother–bud asymmetry of actin patch stability and overall cell growth (Barral et al., 2000), though the mechanistic details of how septins control these asymmetries in wild-type cells are unclear.Open in a separate windowFIGURE 2:Septin localization and function in the asymmetric growth and division of the budding yeast S. cerevisiae. The schematic shows in a clockwise order the different stages of the lifecycle of vegetative haploid and diploid, and sporulating S. cerevisiae cells. The localization and organization of septin complexes in vegetative (Cdc11/Shs1-Cdc12-Cdc3-Cdc10) and sporulating (Spr28-Spr3-Cdc3-Cdc10) cells are depicted as single or double rings, an hourglass-shaped collar, and dots. Shaded boxes highlight septin functions in polarized membrane growth and the asymmetric partitioning and inheritance of membrane and cytoplasmic components in different stages of the budding yeast lifecycle.At the mother–bud neck, the septin ring expands into an hourglass collar and splits into two rings before cytokinesis (Figure 2). The hourglass structure provides a scaffold for the assembly of the actomyosin ring (Bi et al., 1998; Schneider et al., 2013) and the asymmetric distribution of many septin interactors, which localize to the mother or bud side of the neck and in between (Gladfelter et al., 2001; McMurray and Thorner, 2009). On the mother side, septins recruit chitin synthase for the generation of the bud scar, whose underlying membrane provides a landmark and spatial memory for the budding site of the next cell cycle (DeMarini et al., 1997; Kozubowski et al., 2005). On the bud side, septins scaffold kinases of the morphogenesis checkpoint, which monitors proper bud growth for entry into mitosis (Barral et al., 1999; Shulewitz et al., 1999; Theesfeld et al., 2003). At the interface of the mother–bud membranes, the double septin ring acts as a barrier to prevent the exocyst complex and membrane remodeling factors from diffusing out of the gap between them, delimiting the site of membrane growth that occurs in late cytokinesis (Dobbelaere and Barral, 2004).Septin barriers appear to be dispensable for cytokinesis (Wloka et al., 2011), but they are essential for mother–daughter asymmetry in organelle content and age. Septins compartmentalize the endoplasmic reticulum (ER) associated with the plasma membrane of the bud neck (Luedeke et al., 2005). The septin ring excludes ribosomes, creating smooth ER at the bud neck (Luedeke et al., 2005). Moreover, as the cortical ER moves into the growing bud, the septin ring protects the bud from inheriting ER membranes with aggregates of misfolded proteins (Clay et al., 2014). Septin-ring–dependent enrichment of sphingolipids in bud neck membranes is required for the diffusion barrier, but it is unknown how septins may control sphingolipid localization (Clay et al., 2014). Septins also act as a sphingolipid-dependent barrier for the diffusion of nuclear pore complexes (NPCs) from the mother to bud ER membranes, which are continuous with the nuclear envelope (NE; Shcheprova et al., 2008). Lack of NPC diffusion correlated with the asymmetric retention of nonchromosomal DNA circles within the mother compartment, which reduces replicative span (Shcheprova et al., 2008). However, the shape of the dividing nucleus and length of mitosis were also proposed to restrict the diffusion of DNA circles, which entered the bud upon artificial tethering to nuclear pores (Gehlen et al., 2011; Khmelinskii et al., 2011). Reconciling these findings, subsequent work showed that in aged cells DNA circles are coupled to only a subset of NPCs by the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, hindering diffusion into the bud beyond the constraints imposed by the nuclear shape (Denoth-Lippuner et al., 2014). Thus, the septin-dependent ER/NE diffusion barrier plays a fundamental role in yeast aging.Polarized budding yeast growth occurs not only intrinsically, taking place adjacent to or opposite the site of the preceding cell division, but also in response to mating pheromones, which override intrinsic signals. Septins encircle polarity factors at the polar cap of the shmoo-shaped tips that grow toward the pheromone gradient, and septin mutants are defective in pheromone tracking and polarized morphogenesis (Kelley et al., 2015). Upon mating, a distinct septin structure at the site of cell fusion limits diffusion of organelles and large cytoplasmic complexes between the mating partners, which promotes the asymmetrical inheritance of mitochondrial DNA by diploid buds (Tartakoff et al., 2013).Septins are similarly required for the polarized membrane growth that occurs during sporulation (Onishi et al., 2010; Heasley and McMurray, 2016). Spore membrane biogenesis is like inside-out budding as new membranes and cell walls form around the four haploid nuclei, which result from the meiosis of a diploid cell. Each membrane emerges from a single point, the pole of a postmeiosis II spindle, and grows outward as a cup before closing at another single point to form a sphere (Figure 2). Septins localize to bar- and horseshoe-shaped structures along the growing membranes. In septin mutants the membranes grow in the wrong directions and often fail to close (Onishi et al., 2010; Heasley and McMurray, 2016). After their formation, spores bud in a prepolarized manner (Joseph-Strauss et al., 2007). Strikingly, a septin spot is the only known polarity marker that demarcates the site of new membrane and wall synthesis. A septin ring of unknown function encircles the site of membrane outgrowth before a new septin ring forms where the spore first buds, completing the S. cerevisiae life cycle (Joseph-Strauss et al., 2007).While budding yeasts have taught us much about septins and asymmetry, we have learned many fundamental lessons from other fungi. In the fission yeast Schizosaccharomyces pombe, septins act as scaffolds and/or diffusion barriers to guide the orderly recruitment of the cytokinetic machinery to the division site, which occurs at the equatorial plane of the cell. Septin mutants undergo cytokinesis, but fail in cell separation due to defects in recruiting the cell wall degrading enzymes (Zheng et al., 2018). In filamentous fungi, multiple distinct septin-based structures are found in a single cell, and function in limiting the polarity sites and branching of hyphae (Khan et al., 2015; Momany and Talbot, 2017). In Ashbya gossypii, septins form rings and lateral bundles, and localize to the membrane curvature of hyphal branch points, which depends on a C-terminal amphipathic helix (Cannon et al., 2019). Notably, septin recognition of micron-scale membrane curvature is a conserved property of septins from fungi to animals (Bridges et al., 2016). The pathogenic rice blast fungus Magnaporthe oryzae undergoes polarized growth to develop a specialized structure called the appressorium, which contacts the plant surface and uses extreme pressure to penetrate the host (Momany and Talbot, 2017). Here, a septin ring acts as a diffusion barrier to retain actin-polymerizing proteins, and scaffolds actin assembly and the localization of the exocyst complex (Dagdas et al., 2012; Gupta et al., 2015). Overall, fungi have provided compelling systems to explore septin assembly and functions in cell asymmetry.Septins promote asymmetry in the plasma membrane of animal cellsStudies of septins in animal cells have revealed a remarkable conservation with their fungal counterparts in promoting cell asymmetry. Septins are required for plasma membrane protrusions and compartments (e.g., uropodia, filopodia, cilia) that underlie the morphogenesis of distinct cell types and tissues and arise from local membrane asymmetries, microdomains of distinct protein enrichment (Caudron and Barral, 2009; Tooley et al., 2009; Hu et al., 2010, 2012; Dolat et al., 2014a). Homozygous deletions of several septin genes in mice cause early embryonic lethality and male sterility, demonstrating how essential septins are for development and physiology (Dolat et al., 2014a).On the plasma membrane of animal cells, septin assembly and localization involves signaling cues and selective binding to microdomains with distinct phospholipid content and curvature (Figure 3). The signaling pathways that instruct the membrane sites of septin assembly in multicellular organisms are not as well studied as in fungi. The WD repeat-containing planar polarity effector and the PAR complex have been implicated in the localization of membrane septins (Cui et al., 2013; Park et al., 2015; Jordan et al., 2016). Septins have intrinsic preferences for select phospholipids including phosphoinositides (e.g, phosphatidylinositol 4,5-bisphosphate) and cardiolipin, a membrane curvature–specific lipid (Zhang et al., 1999; Krokowski et al., 2018). Taken together with a strong affinity for membrane micron-scale curvatures that is mediated by amphipathic helices (Bridges et al., 2016; Cannon et al., 2019), septins become enriched on the inner saddle-like areas of the plasma membrane, outlining the base-neck border of protrusions such as filopodia, lamellipodia, and dendritic spines (Figure 3).Open in a separate windowFIGURE 3:Septins associate with cell membranes and function in plasma membrane asymmetry. Septins associate preferentially with membrane domains of micron-scale curvature with a radius (R) of ∼0.5–1.5 μm or curvature parameter (k) of ∼0.67–2 μm⁻¹. In addition, septins bind preferentially phosphoinositides and cardiolipin, a cone-shaped lipid that localizes in curved membrane domains. Septins promote asymmetry by acting as barriers of lateral diffusion, membrane rigidifiers, and regulators of the organization and dynamics of cortical actin. Septins are essential components of diffusion barriers at the base of primary cilia, dendritic spines, and the midpiece of spermatozoa, where the annulus structure is located. By rigidifying specific subdomains of the plasma membrane, septins delimit areas of membrane activity, which is critical for the position of membrane protrusions in the rear (uropodia) and front (lamellipodia, filopodia) of migrating cells. In gastrulating frog embryos and C. elegans zygotes, septins locally regulate the organization and dynamics of cortical actin, which is critical for the asymmetric contraction of actomyosin that underlies convergent extension and cytokinesis.Septins promote asymmetry by restricting lateral diffusion, enhancing membrane rigidity and spatially regulating membrane–cytoskeleton interactions. Septins are required for the maintenance of diffusion barriers at the base of primary cilia, dendritic spines, and the midpiece of spermatozoa (Hu et al., 2010; Kwitny et al., 2010; Ewers et al., 2014). Diffusion of transmembrane and inner leaflet-anchored proteins across the midbody of dividing cells may also be impeded by septins (Schmidt and Nichols, 2004). It is unclear whether septin filaments directly impede lateral diffusion or whether they are an essential component of a larger actin–spectrin skeleton with barrier properties. Immuno-EM studies indicate that septins are indeed part of the membrane actin skeleton (Hagiwara et al., 2011). Moreover, in the axon initial segment, which functions as a diffusion barrier, septins interact with ankyrin G (Hamdan et al., 2020). Septins have also been proposed to rigidify the plasma membrane, suppressing cortical protrusions and blebs, which are asymmetrically confined to septin-free membrane areas (Gilden and Krummel, 2010). For example, T-cell uropods are delimited by a septin corset-like arrangement, which braces the perinuclear cortex and enables the front–back polarity of migrating T-cells (Gilden et al., 2012).Membrane septins bend actin filaments into circular arrays and scaffold the localization and activation of myosin-II, and thus they can promote asymmetry in membranes by locally regulating the cortical actomyosin (Joo et al., 2007; Mavrakis et al., 2014). In gastrulating frog embryos, septins localize to the vertices of mediolateral cell–cell contacts, where they stabilize actin and maintain the planar polarity of phosphorylated myosin along the anteroposterior contacts (Shindo and Wallingford, 2014). Thereby, actomyosin contraction is spatially restricted along the anteroposterior junctions, which promotes convergent extension by pulling mediolateral contacts closer to one another (Figure 3). In Caenorhabditis elegans embryos, septins polarize to the anterior pole away from the contractile actomyosin ring, inhibiting actin assembly outside the plane of cytokinesis (Jordan et al., 2016). Moreover, C. elegans septins are required for the asymmetric ingression of the cleavage furrow, which progresses directionally with a dorsal-to-ventral orientation (Maddox et al., 2007). Independently of their roles in cytokinesis, cortical septins provide spatial cues for the orientation of the axis of division in Drosophila larval neuroblasts, which is determined by the position of the last-born daughter cell (Loyer and Januschke, 2018). Notably, in neuronal crest cells, which generate dorsal root ganglia neurons after cell division, cortical septins provide a spatial memory for the outgrowth of axons from premitotic sites of membrane protrusions (Boubakar et al., 2017).In sum, septins associate preferentially with micrometer-scale membrane domains of negative Gaussian curvature and distinct phospholipid content. While it is unclear how they impede the lateral diffusion of proteins and lipids, septins promote macroscale asymmetries by enabling molecular partitioning across adjacent membrane compartments. At the nexus, septins also facilitate asymmetries of submicrometer scale by clustering membrane proteins or lipids. For example, septins are required for the organization of PI(4,5)P2 into clusters that surround the calcium release-activated calcium channel ORAI1 at plasma membrane–ER junctions (Sharma et al., 2013). How septins interact with and organize on membrane bilayers, and how they modulate the mobility and distribution of plasma membrane proteins and lipids are key questions. As septins associate with the membranes of various intracellular organelles, it is also unclear how septins function in the generation and/or maintenance of organelle membrane domains and membrane–membrane contacts (Akil et al., 2016; Dolat and Spiliotis, 2016; Pagliuso et al., 2016; Sirianni et al., 2016; Song et al., 2019).Septin roles in cytoskeleton-based mechanisms of cell asymmetryCell asymmetry requires the assembly of local microtubule and actin networks with unique organization and dynamics. Due to their structural polarity and dynamic growth, microtubules and actin filaments can spatially bias membrane traffic and determine the location, shape, and/or directionality of membrane protrusions and adhesions. Hence, microtubules and actin filaments are keys for morphing cells into asymmetric shapes.Septins localize to subsets of microtubules and actin filaments, and promote cell asymmetry by regulating the spatial organization of the cytoskeleton and membrane traffic (Figure 4). In a diversity of mammalian cell types, septins colocalize with microtubules and actin filaments of the perinuclear and peripheral cytoplasm (Spiliotis, 2018). It is not well understood how this region-specific association is established, but it may involve effectors of Rho signaling and depend on the composition (subunit isotypes) and posttranslational modifications of microtubules and actin filaments. Alternatively, cytoskeletal sites of binding might be indirectly determined by septin binding and turnover on proximal membranes. Thus, septin roles in cytoskeleton-based mechanisms of cell asymmetry are not mutually exclusive of their functions in membrane asymmetry, which feeds back locally to the cytoskeleton.Open in a separate windowFIGURE 4:Septins roles in cytoskeleton-based mechanisms of apicobasal, axonodendritic, and front–rear polarity. Microtubule-associated septins guide microtubule organization and membrane traffic along the apicobasal axis of polarizing epithelia. In neuronal dendrites, septin 9 reinforces the polarity of neuronal membrane traffic by impeding the transport of axonal cargo of kinesin-1/KIF5 and enhancing the anterograde movement of dendritic cargo of kinesin-3/KIF1A. In cells undergoing epithelial-to-mesenchymal transition, septins associate with the actin stress fibers of the leading lamellae and promote the asymmetric organization of the actin network and focal adhesion turnover, which is critical for the front–rear polarity of cell migration.In epithelia and neurons, microtubule-associated septins are functionally important for apicobasal and axon–dendrite polarity (Spiliotis et al., 2008; Bowen et al., 2011; Karasmanis et al., 2018). In polarizing epithelia, septins associate with subsets of microtubules, steering microtubule plus end growth and microtubule–­microtubule interactions (Bowen et al., 2011). Hence, septins provide a navigation mechanism for microtubule organization along the developing apicobasal axis of polarity. In addition, microtubule-associated septins are required for Golgi-to-plasma membrane transport of vesicles with apical and basolateral membrane proteins (Spiliotis et al., 2008). Septin depletion abrogates exocytic membrane traffic, disrupting the growth and differentiation of the epithelial cell membrane into apical and basolateral domains (Spiliotis et al., 2008). In fully polarized epithelia, it is unknown whether specific septin paralogues and complexes function exclusively in the apical or basolateral routes of membrane traffic. In hippocampal neurons, however, septin 9 localizes to dendritic microtubules, reinforcing the polarity of membrane traffic by hindering the transport of axonal cargo of kinesin-1/KIF5 and promoting the anterograde movement of dendritic cargo of kinesin-3/KIF1A (Karasmanis et al., 2018). This differential regulation of kinesin-driven transport is critical for axon–dendrite polarity, and in principle may constitute a general mechanism by which septins polarize membrane traffic.Growing evidence indicates that septins function in the mechanisms of mechanotransduction that induce and sustain front–rear polarity in migrating cells (Lam and Calvo, 2019). Cells migrate toward stiffer extracellular matrices and chemoattractants by forming a leading protrusive front and retracting rear. Front–rear polarity is largely supported by an asymmetry in the organization of the actomyosin stress fibers and the turnover of focal adhesions. Septins colocalize with actin stress fibers in a diversity of cell types, and alterations in septin expression impair directional migration. In cells undergoing epithelial-to-mesenchymal transition, septins are enriched in the leading lamella localizing at the interphase of a contractile network of curved actin stress fibers (transverse arc stress fibers) and the radial stress fibers, which are anchored to focal adhesions (Dolat et al., 2014b). Septin depletion collapses the transverse arc actin network with concomitant loss of stability and front-to-rear maturation of focal adhesions. Taken together with loss of directional cell migration, this phenotype underscores a critical role of septins in the front–rear asymmetry of cell migration. In cancer-associated fibroblasts, septins function together with the Cdc42 effector Cdc42EP3 in actin organization in a mechanosensitive manner, responding to stiffer matrices via association with perinuclear actin stress fibers (Calvo et al., 2015). Of note, septins may function in nuclear mechanotransduction, nuclear movement, and/or the regulation of subnuclear focal adhesions (Verdier-Pinard et al., 2017; Lam and Calvo, 2019). In parallel with their functions in the actin cytoskeleton, septins also likely contribute to the front–rear asymmetry of migrating cells by spatially regulating membrane–microtubule interactions (Shindo et al., 2018).Septin association with subsets of actin filaments and microtubules is a salient characteristic that enables local regulation of cytoskeleton-based processes, a key functionality in the generation of cellular asymmetries. Progress in understanding how septins interact with actin and microtubules, and how they mechanistically impact cytoskeletal dynamics has been slow. High-resolution cryoelectron microscopy studies of septins in complex with actin and microtubules are necessary to provide structural insights into how septins modulate cytoskeletal dynamics and interactions with actin-binding and microtubule-associated proteins. Septins have yet to be studied in the context of the basic mechanisms of actin nucleation and polymerization, which can have important implications for actin assembly and symmetry breaking at membrane sites of septin enrichment. Advances in these areas and better understanding of the signaling pathways that determine the cytoskeletal locales of septin function will provide a fuller picture of septins as cytoskeletal agents of cell asymmetry.Principles and unknowns of septin function as a symmetry-breaking moduleThe spatial organization of eukaryotic cells requires microscale asymmetries, which are established and maintained in a region-specific manner, and at the macroscale level promote the polarization of cellular shapes and processes. It is little understood how these asymmetries arise amidst membrane and cytoplasmic fluidity. Part small GTPase-like regulators and part cytoskeleton-like polymers, septins are inherently tailored to break symmetry by facilitating protein localization and interactions that persist in place and time. In summary of past and recent findings, there are four major principles of septin function as a symmetry-breaking module:

1. Septins are spatio-specific and modular. Septins assemble on select intracellular regions (e.g., micron-scale membrane curvatures, perinuclear microtubule bundles, peripheral transverse arc stress fibers). Septin assembly is modular, consisting of alternative combinations of paralogue and isoform subunits, which in turn determine intracellular localization and interactions.
2. Septins form higher-order structures, which turn over slowly and thereby persist spatially and temporally. Thus, septins can serve as imprints or landmarks of previous structures and molecular events, providing a type of spatial memory.
3. Septins scaffold protein localization and interactions. Septin scaffolds induce molecular asymmetries by recruiting and clustering protein interactors, and facilitating the assembly of macromolecular complexes.
4. Septins partition proteins in distinct membrane or cytoskeletal domains. Septins exclude proteins locally by impeding lateral diffusion or binding.

While most evident in membrane septins, these principles also apply to septins that associate with actin and microtubules and the membrane–cytoskeleton interface. On microtubule lattices, where many microtubule-associated proteins (MAPs) undergo diffusion through transient electrostatic interactions, septins selectively inhibit or promote microtubule binding of specific MAPs and kinesin motors (Spiliotis et al., 2008; Karasmanis et al., 2018). Similar modulation is likely to take place on actin filaments, where septins could specify domains of unique actin-binding protein composition promoting the formation of actin networks of distinct architecture and dynamics. A region-specific control of cytoskeletal organization may also occur by membrane-associated septins sequestering actin-­nucleating promoting factors or directly interacting with the dynamic ends of actin filament or microtubules (Dagdas et al., 2012; Hu et al., 2012; Nolke et al., 2016; Nakos et al., 2019). In the cytoplasm, a templated assembly of septins along subpopulations of microtubules and actin filaments may provide a form of cytoskeletal memory as previously proposed for vimentin intermediate filaments (Gan et al., 2016). By outlasting their shorter-lived templates, septins are likely to provide cytoplasmic landmarks for orienting microtubule and actin growth along previously held patterns of spatial organization. In polarized cell types, this region-specific patterning would be critical for the continuous organization of cytoskeletal networks along the axis of cell polarity.Despite much progress over the last two decades, we have merely begun discovering septins as a core mechanism for the spatial organization of cell biology. Many unknowns remain. The signaling inputs that interface with the assembly of mammalian septins are virtually unknown. The spatio-specificity of septin assembly on subnetworks and regions of the cytoskeleton is poorly understood. Septin modularity is little explored, and more work is needed to determine how different combinations of septin paralogues and isoforms bestow alternative localizations and properties. Deciphering this septin code is critical for determining which septin complexes do what, and whether certain septin paralogues can function as homomers independently of the canonical model of heteromeric assembly. In light of GTP hydrolysis favoring septin homodimerization and the recent reordering of the SEPT2/6/7/9 hetero-octamer, it is plausible that paralogues with faster GTPase activities (e.g., SEPT9) and in excess of their cognate partners evade heteromerization into typical complexes. More studies are needed in cell types and disease states, in which certain septin paralogues or isoforms are disproportionally expressed and may take unique or pathogenic functions. Similarly, studies of septins in polarized cell types and stem cell systems, where asymmetry is key for cell fate and renewal, are lacking, and so is our understanding of septin functions in cell regeneration and repair. With many more open questions than answers, the next 50 years of septin research promises important breakthroughs in our basic understanding of cellular asymmetry and potentially the development of septin-based therapies in regenerative medicine and beyond.     

Fifty years of cycling

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.

The cell cycle is a process that orders a number of cellular processes to ensure the accurate duplication of the cell. It was hoped that a genetic analysis would reveal how the events were integrated. The inspiration for this was the work of Bob Edgar and Bill Wood on bacteriophage morphogenesis, which revealed the ordered steps by which phage parts were assembled and then put together (Wood and Edgar, 1967). The major questions were how DNA replication and spindle morphogenesis were integrated to achieve accurate chromosome segregation; how cell division was integrated with mitosis to ensure that both daughter cells received a full chromosome complement; and how growth and division were integrated to maintain a constant cell size.Mutants that block cell cycle progression were identified by screening collections of randomly generated temperature sensitive mutants (Hartwell et al., 1970a). Each mutant was screened individually by time-lapse photomicroscopy to identify cell division control (CDC) mutants that caused all cells in the population to arrest at the same point in the cell cycle at the restrictive temperature. The use of budding yeast was critical because the presence and size of the daughter bud provided a simple readout of where cells were in the cell cycle. The first collection of CDC mutants was derived from screening 1500 temperature-sensitive mutants and identified a total of 147 mutants, which fell into 32 complementation groups (Hartwell et al., 1973). An example of one of the first mutants identified is shown in Figure 1. Wild-type cells are found at all stages of the cell cycle at the restrictive temperature (panel A), whereas the CDC mutant cells arrest in late in the cell cycle with large daughter buds (panel B).Open in a separate windowFIGURE 1:An example of one of the first CDC mutants isolated in budding yeast. Wild-type cells and temperature-sensitive mutant cells were grown at the permissive temperature and then shifted to the restrictive temperature, and CDCs were followed by photomicroscopy. (A) Wild-type cells, which are found at all stages of the cell cycle at the restrictive temperature, as indicated by the presence of cells at all stages of the daughter cell budding cycle. (B) A CDC mutant in which all cells have arrested at a cell cycle stage with large daughter buds.The phenotypes of the mutants revealed some preliminary answers to the major questions (Hartwell et al., 1970a, b). Assuming that the primary biochemical defect in a mutant was the process that stopped first, the following conclusions could be drawn. The elongation of the spindle was dependent on prior duplication of the spindle poles and the completion of DNA replication. Cell division and mitosis were coordinated because the formation of the daughter bud was dependent on spindle pole duplication in the previous cycle and cytokinesis was dependent on prior elongation of the spindle. Growth and division were coordinated because the CDC28 (CDK1) function at Start required sufficient growth to initiate all the events of the cell cycle. Cell fusion during mating of haploid cells was coordinated with cell division because mating hormones arrested the cell cycle at the CDC28 step and fusion was restricted to that step in the cell cycle.These observations raised the question of how the dependence of events on one another was controlled. Two models were considered. One, named substrate–product, proposed that a late step was dependent on an early step because the latter was the substrate for the former (e.g., replicated DNA was a substrate for the spindle). The other was regulation, meaning either that signals from completion of an early event induced a late event or that an incomplete early event inhibited a late event. In one example, regulation was evident when a genetic analysis of how damaged DNA arrested nuclear division revealed a signaling pathway (the DNA damage checkpoint) that also accounted for why incomplete DNA replication prevented mitosis (Weinert and Hartwell, 1988).The mechanisms underlying the dependence of cell cycle events upon one another have now been defined in considerable molecular detail. By way of illustration, we will briefly summarize what is known about the dependence of mitosis on replicated chromosomes and the dependence of cell division on mitosis. In some cases, the cdc mutants contributed to this work as a means to identify the relevant genes. However, in many cases, important components were not isolated as cdc mutants. Some of these have since been identified through biochemistry and subsequently shown to have Cdc phenotypes after mutants were created by in vitro mutagenesis. Additional cell cycle components were identified in other genetic screens or isolated using the original cdc mutants as starting points to search for genetic interactors. For example, the cyclins from yeast (which are redundant and nonessential), and even humans, were isolated, in part, as high-copy suppressors of the yeast cdc28 mutant (Hadwiger et al., 1989).One prominent class of cdc mutants affected DNA replication. Mutants targeting two of the three essential replicative polymerases were isolated, as were DNA ligase, a gene required for replication near telomeres, and genes required for the production of deoxyribonucleotides. We now know that these mutations resulted in robust arrest phenotypes because they led to the accumulation of significant amounts of ssDNA, the signal recognized by the replication checkpoint pathway (Zou and Elledge, 2003). This checkpoint signaling pathway both blocks mitosis and feeds back to replication. This feedback to replication helps stalled forks progress and also blocks origins that have not yet fired from doing so. The critical checkpoint phosphorylation events that effect these goals are well understood. Mitotic arrest is largely achieved by phosphorylation and stabilization of Pds1 (Cohen-Fix and Koshland, 1997), an event that blocks sister chromatid separation. Blocking origin firing is mediated by the phosphorylation of two proteins required for origin firing (Lopez-Mosqueda et al., 2010; Zegerman and Diffley, 2010). Finally, the restart of replication forks stalled by either mutational disruption or exogenous agents is promoted by the phosphorylation of several critical targets that increase nucleotide levels and modify the activity of proteins that act at the fork (Ciccia and Elledge, 2010). If measured by viability after fork arrest, this last function is by far the most significant role of this checkpoint pathway, although mitotic arrest and blocking origin firing are also important for preserving genome integrity.While the cdc screen was effective in identifying genes involved in the mechanics of DNA replication, it was less effective in identifying genes that function exclusively to establish origins of replication. An exception to this, CDC6, sheds some light on why this may be. cdc6 mutants brought to a fully nonpermissive temperature do not form replication forks, and thus do not activate the replication checkpoint, although they eventually arrest in mitosis due to the formation of an aberrant spindle that triggers the spindle assembly checkpoint (Piatti et al., 1995; Stern and Murray, 2001). Temperature-sensitive alleles of polymerase or ligases are likely to generate a few nonfunctional replication forks even when the alleles are weak, thus activating the checkpoint and providing a clear Cdc phenotype. Of course, it could not have been foreseen at the time of this screen that mutations in some cell cycle processes might eliminate the very signals for arrest that the screen was designed to identify.Major progress has also been made in understanding the coordination of events needed to ensure successful chromosome segregation to daughter cells during mitosis. The spindle poles duplicate and separate to form a microtubule-based spindle. During DNA replication, the cohesin complex is loaded onto sister chromatids to keep them paired until the metaphase to anaphase transition. At the same time, the kinetochores that mediate attachment of chromosomes to the spindle microtubules assemble on centromeres. These are examples of substrate–product relationships where cohesin and kinetochores will assemble once the chromatin templates are available. Similarly, kinetochores make attachments to the spindle microtubules as soon as they are assembled. The spindle assembly checkpoint, a regulatory pathway, monitors kinetochore–microtubule interactions and halts the metaphase-to-anaphase transition until all chromosomes are properly attached (Hoyt et al., 1991; Li and Murray, 1991). Once the checkpoint is satisfied, cells activate the anaphase-promoting complex to release the linkage between sister chromatids and allow the spindle to elongate and pull chromosomes to opposite poles. As the spindle elongates into the daughter cell, the cell reverses Cdc28 substrate phosphorylations to promote mitotic exit and cytokinesis.How are all of these events coordinated? Surprisingly, although corresponding temperature-sensitive mutants exist for most mitotic genes, the cdc screen did not isolate the structural components of the yeast spindle, spindle pole, or kinetochore, with the exception of one pole mutant, cdc31. In contrast, the screen identified many signaling molecules that regulate the metaphase-to-anaphase transition. The cdc screen identified five subunits of the anaphase-promoting complex, which coordinates this transition by ensuring the degradation of proteins that lead to the removal of cohesion from chromosomes and the down-regulation of Cdc28 activity (King et al., 1995; Sudakin et al., 1995). One of the substrates that must be degraded is Pds1, the same protein that is the target of the DNA checkpoint (Cohen-Fix et al., 1996). Pds1 inhibits the enzyme separase that releases cohesin to ensure the timely separation of sister chromatids (Ciosk et al., 1998; Uhlmann et al., 2000). The targeted degradation of Pds1 and cyclins by a single complex couples spindle elongation and chromosome segregation to Cdc28 inactivation. The spindle assembly checkpoint inhibits the anaphase-promoting complex, reinforcing the coordination between proper spindle attachment to chromosomes and anaphase progression (Hwang et al., 1998; Kim et al., 1998). After chromosome segregation, many Cdc28 substrates must also be dephosphorylated to exit from mitosis. Each of the essential kinases and phosphatases in this control system, called the mitotic exit network, were found in the cdc screen (Shou et al., 1999; Visintin et al., 1999). The mitotic exit network coordinates cytokinesis with the spindle delivering chromosomes to the daughter cell (Bardin et al., 2000; Pereira et al., 2000). Finally, several members of the septin ring that ensures cytokinesis, the last event in the cell cycle, were identified as cdc mutants. The septin mutants continue to bud, replicate DNA, and undergo mitosis in the next cell cycle, showing that completion of all events in the prior cell cycle is not necessarily required for progression. However, looking back, most of the key mitotic events are coordinated by regulatory events that reinforce the dependence of one event on the next, as opposed to the substrate–product relationship. Even in cases where there are clear substrate–product dependencies, such as spindle attachment to kinetochores, the cell has multiple regulatory mechanisms in place to halt the cell cycle until errors are detected and corrected, thus ensuring the proper execution of mitosis.What do the next 50 years hold? The short examples above illustrate the tremendous progress that has been made in understanding the molecular mechanisms that ensure the coordination of cell cycle events. However, there are still major questions about its specificity, accuracy, and complexity, as well as how it is altered in disease. Specialized cell divisions such as meiosis and asymmetric cell division or modified cell cycle states such as quiescence require modifications to the cell cycle. The reconstitution of molecular events has helped to identify the minimal components and regulation required, but this has not accounted for the exquisite precision of these processes in the cell. The complexity of how individual cell cycle events integrate with other cellular processes such as metabolism is still in the early stages. Uncontrolled cell division is the root of cancer, so identifying therapeutic targets that specifically cause cancer vulnerabilities and avoid toxicity to normal cell divisions is still very much needed. In sum, many of the principles gained from cell cycle research have guided our thinking about biological processes; further elucidating the underlying mechanisms of the cell cycle will continue to influence fundamental biology and disease research for decades to come.     

Mitotic spindle: lessons from theoretical modeling

Cell biology is immensely complex. To understand how cells work, we try to find patterns and suggest hypotheses to identify underlying mechanisms. However, it is not always easy to create a coherent picture from a huge amount of experimental data on biological systems, where the main players have multiple interactions or act in redundant pathways. In such situations, when a hypothesis does not lead to a conclusion in a direct way, theoretical modeling is a powerful tool because it allows us to formulate hypotheses in a quantitative manner and understand their consequences. A successful model should not only reproduce the basic features of the system but also provide exciting predictions, motivating new experiments. Much is learned when a model based on generally accepted knowledge cannot explain experiments of interest, as this indicates that the original hypothesis needs to be revised. In this Perspective, we discuss these points using our experiences in combining experiments with theory in the field of mitotic spindle mechanics.

The goal of anyone studying biology is to learn how life works, but for many students the choice of biology is reinforced by a desire to escape mathematics, physics, complex equations, and theoretical work. Yet research in biology often needs theoretical analysis. Theoretical modeling is valuable because it allows us to formulate our hypotheses in a rigorous manner and recognize their implications. Theory in cell biology has been the subject of thought-provoking reviews discussing different types of models as well as why and how to do theoretical modeling (Mogilner et al., 2006; Gunawardena, 2014; Möbius and Laan, 2015; Phillips, 2015; Tyson and Novák, 2015). In this essay, we illustrate the lessons that emerge from the interplay of theory and experiments using examples from spindle mechanics, emphasizing how theory is useful also when it cannot explain experiments and how it becomes especially valuable when it predicts unexpected behavior.The mitotic spindle is a marvelous microtubule-based micromachine that segregates the genome from one cell into two equal parts destined to the future daughter cells (McIntosh, 2016). Spindle microtubules can be divided into three main classes according to their localization and function: kinetochore microtubules that bind the kinetochore, a protein complex at the centromere of each chromosome; overlap microtubules, which extend from the opposite spindle halves and overlap in the middle; and astral microtubules, which grow from the spindle pole toward the cell cortex. Nucleation, dynamics, and forces exerted by spindle microtubules are regulated by hundreds of microtubule-binding and other mitotic proteins, which have multiple mutual interactions. These complex biochemical interactions drive self-organization, a process where order arises from local interactions between initially disordered components, into a molecular machine that can generate large-scale forces to move the chromosomes (Pavin and Tolic´, 2016). Yet, despite the great amount of knowledge about the spindle, this complexity of interactions makes the mechanisms of spindle functioning still largely unclear. Precisely because of the complexity, theoretical modeling is helpful in testing hypotheses and identifying key mechanisms.     

Training matters! Narrative from a Black scientist

As STEM (Science, Technology, Engineering, and Math) professionals, we are tasked with increasing our understanding of the universe and generating discoveries that advance our society. An essential aspect is the training of the next generation of scientists, including concerted efforts to increase diversity within the scientific field. Despite these efforts, there remains disproportional underrepresentation of Black scientists in STEM. Further, efforts to recruit and hire Black faculty and researchers have been largely unsuccessful, in part due to a lack of minority candidates. Several factors contribute to this including access to opportunities, negative training experiences, lack of effective mentoring, and other more lucrative career options. This is a narrative of a Black male scientist to illustrate some of the issues in retaining Black students in STEM and to highlight the impact of toxic training environments that exists at many institutions. To increase Black participation in STEM careers, we must first acknowledge, then address, the problems that exist within our STEM training environments in hopes to inspire and retain Black students at every level of training.

I write this today as the curtain of systemic racism and oppression has lifted on our nation. I write this today knowing that difficult conversations about race are happening all across America. As a result of tremendous sacrifices and lives lost, there have been demonstrations and rallies internationally demanding change, prompting governments, organizations, and companies to issue statements claiming that Black Lives Matter (Asmelash, 2020). While the rage has sparked the demand for equity in our society, what does this mean for science?My heart is heavy with these discussions as I have reflected on my own journey in science and revisit the toxic environment that often makes up our science culture. The journey has been long and brutal. It has taken me from first realizing that I wanted to become a scientist, to having this dream deferred by racism, to adopting a persona of persistence and resilience, and finally becoming a professor and cell biologist. This trek through science is one that is not traversed by many Black people (Graf et al., 2018).When confronted by the pervasiveness of racism in science, I remember surviving the assault by learning about the resilience story of Carl Brashear (Robbins, 2000). In 1970, Master Chief Petty Officer Brashear became the first African American master diver in the Navy, and he showed unwavering strength and persistence in the face of racism. Brashear faced an onslaught of racism during his training that endangered his life countless times, but he persisted and eventually won the admiration of his fellow divers. Upon reflection, his story has many signs of an abusive hazing relationship. However, at the time, I thought emulating his behaviors of persistence was the answer to success in science. I thought, “All you have to do is not give up.” I focused on what I thought I could control and kept the Japanese proverb, “Fall down seven, stand up eight” above my bench. I worked long hours, made many mistakes, but always got right back up to the bench to try again. I never saw myself as the brightest or smartest, but I would tell myself “I will be the one who does not give up.” When I recall these stories and talk to students about my journey, I would always say I wanted to be like the cockroach. Because, as is commonly known, you can never get rid of the cockroach. What I never realized with this persistence or “grit” mentality was that it never addressed the problems of systemic racism within the culture of science (Das, 2020). This message of persistence is akin to blaming the victim and not dealing with the root problems in science, including the lack of mentoring, implicit bias, and hostile teaching and training environments (Barber et al., 2020; Team, 2020).In her book, We Want to Do More Than Survive, Bettina Love talks about the idea of teaching persistence or “grit” to African American students as the educational equivalent to the Hunger Games, a fictional competition where participants battle to the death until there is only one victor (Love, 2019). Instead of addressing institutional barriers to success for African Americans in science (i.e., dismantling the Hunger Games arena), we prepare them to survive in a toxic environment. We tell African American students at a young age that the system is structured against them and that they have to be twice as good and work twice as hard as white students (Thomas and Wetlaufer, 1997; Cavounidis and Lang, 2015; Danielle, 2015). We heap a tremendous amount of pressure and responsibility on their shoulders without ever addressing the question, why is it like this? We are in effect training them for the Hunger Games. As they enter college as science majors, they are pitted against each other, and the few victors move into science careers.This Hunger Games analogy (Love, 2019) is reflective of my thinking early on in my science career. As a freshman marine biology major, I imagined myself, like Brashear, a soldier during basic training. I was a member of the “people of color” (POC) squad that was given the least amount of resources and the most dangerous duties. As part of the POC squad, we moved forward through our college years. I saw many fellow soldiers drop from science, and there were only a handful of us left when I reached my junior year (Koenig, 2009).Recently, Michael Eisen, Editor-in-Chief of eLife, authored an opinion article entitled “Racism in Science: We need to act now” (Eisen, 2020). In this article, he reflected on the current racial climate in science and examined his role as both a principle investigator (PI) of a research laboratory and an editor of a prestigious journal. Of note, he highlighted the dire lack of African Americans he had worked with over his career, including the number of researchers he trained in his laboratory, senior editors, and even reviewers for the articles sent for publication to eLife. I appreciated his honesty in shedding light on the issue that so many people whisper about in department hallways or during coffee breaks at national conferences. Based on my journey, I truly understand this lack of diversity, as so few of us are victors in the scientific Hunger Games.As we struggle as a nation with the role of policing within our society, I find similarities between aggressive policing in the Black community and training of Black and Brown students (North, 2020). There are strong implicit biases that we hold within our training environment, and Black students usually find themselves very quickly judged (or prejudged) for a perceived lack of commitment, motivation, or focus (Park et al., 2020). They are also stereotyped as lacking in quantitative abilities (especially the ability to do math) (McClain, 2014). Taken together, these biased judgements result in a lack of trust regarding their data (Steele, 1997). In other words, research supervisors may implicitly expect Black students to be untrustworthy. This is extremely problematic because educational research shows that one of the greatest determinants of students’ success is their teachers’ expectations (Boser et al., 2014). Consequently, it is predictable that if research supervisors expect Black students to be untrustworthy, they will fail.As PIs, we must trust our research students because they are extensions of ourselves in the laboratory. Due to our inability to spend significant amounts of time at the bench, we must trust our students to figure it out and get the work done. Inevitably, experimental approaches will fail; however, based on my experiences in science, Black students are often not given the benefit of the doubt. Instead, I have seen mis/distrust of their commitment, values, and abilities that creates the narrative that they are not motivated, do not care about science, and/or are unable to get the work done, resulting in a broken trainer/trainee relationship. I have witnessed too many Black students fall victim to a “one strike” policy. This was true of me in my early training in marine biology, where I was asked to leave after only 6 months of working in a laboratory. The professor suggested that I had a lack of commitment to my project and was told by other lab members that they collected “my” data, thus providing justification to ask me not to continue. However, what the professor did not know (or care to ask about) was that the other lab members deemed me as someone who did not belong. Consequently, without my knowledge, they collected data on my project and sent it to the PI, thereby working to reinforce the narrative of my lack of commitment. This experience significantly hindered my access to research opportunities and blacklisted me from any other marine biology labs at my university because I was labeled as uncommitted to science. This ended my career in marine biology. I lost the Hunger Games.As a graduate student, I found another opportunity in a cell biology laboratory, and I tried to apply lessons learned from my earlier participation in the Games. I overcommitted to lab work, blocking out any activities related to my culture or personal life. Instead, I dedicated myself completely to the lab. Working 12-h days, I found that my research was progressing, but I was burning out and losing any desire toward a research career. In particular, my burnout was connected to the perception that any interest in my culture and community would not be allowed or accepted or would signal a lack of adequate commitment to science. In effect, I was learning that being a scientist meant that I could not be Black. This, coupled with the constant microaggressions that I faced from professors in classes, among my graduate cohort, and my laboratory colleagues, broadcasted the message that I was an intruder in science. Luckily, I received good mentoring and advice on how to succeed in my graduate program, learning that it was not a sprint, but a marathon. I learned how to balance my personal and professional life, and I always kept them separate. Additionally, the mental image of the resilient cockroach helped me repeatedly during my graduate training, from failing my qualifying exams and failed experiments at the bench to rejections of papers and fellowship applications. While all scientists know that being a scientist means accepting significant amounts of failure, I could not help but feel that the failures I experienced were more frequent, more recognized by others, and even expected by some. This culture of expected failure for people of color (i.e., presumed incompetence), combined with implicit biases and microaggressions, can establish significant barriers for entering and staying in STEM training environments (Smith et al., 2007).To overcome barriers to success in STEM, I worked hard to become a professor in cell biology. I believed that as a professor, I could make a difference, change the environment, and contribute to the change that is so desperately needed. However, I have discovered that the current science culture is just as toxic as when I was a student. Yes, there are programs targeting the inclusion of historically underrepresented groups. There are also a growing number of institutions that are adopting inclusive teaching strategies. Further, we are seeing hiring committees require diversity statements from their applicants as well as receiving implicit bias trainings (Wood, 2019). However, there remains nearly a complete lack of Black faculty members at universities and colleges (Jayakumar et al., 2009; Garrison, 2013; Li and Koedel, 2017). This is, in part, because we have not changed the systemic racism that exists within our training environments. In fact, this racism comes from our noninclusive faculty bodies (Hardy, 2020). In essence, we have nearly a complete absence of Black faculty in STEM because so few Black trainees survive the Hunger Games. More troubling, if they survive, they may be found otherwise unacceptable.Changing the system starts with the belief that Black students can be scientists, followed by acting to proactively encourage and support Black students in STEM. As Eisen states, “This is a solvable problem, we have chosen not to solve it” (Eisen, 2020). Recruiting Black students and scientists at every level is a good start, but without changing the scientific environment to be more welcoming and affirming, those recruited to science will continue to be traumatized. In other words, while increasing access to science is required, it is not sufficient. The dominant majority in science also needs to identify and address their own biases to create antiracist environments. This will only happen when scientists from all groups recognize our convergent interests to advance our universal missions, which is to increase our understanding of the world around us and to solve research questions that will benefit our communities. This is best achieved by a diverse and inclusive scientific workforce for greater knowledge, discovery, and innovation.     

Engineering therapeutic protein disaggregases

Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD.We urgently need to pioneer game-changing solutions to remedy a number of increasingly prevalent and fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD; Cushman et al., 2010 ; Jackrel and Shorter, 2015 ). These disorders relentlessly erode our morale and economic resources. Aging is the major risk factor for all of these diseases, which threaten public health on a global scale and represent a severe impediment to living longer lives. A number of promising drugs have emerged to treat cancer and heart disease, but, distressingly, this is not the case for these and other neurodegenerative diseases, for which drug pipelines lie dormant and empty. This situation is unacceptable, and an impending healthcare crisis looms worldwide as population demographics inexorably shift toward older age groups.ALS, PD, AD, and related neurodegenerative disorders are unified by a common underlying theme: the misfolding and aggregation of specific proteins (characteristic for each disease) in the CNS (Cushman et al., 2010 ; Eisele et al., 2015 ). Thus, in ALS, typically an RNA-binding protein with a prion-like domain, TDP-43, mislocalizes from the nucleus to cytoplasmic inclusions in degenerating motor neurons (Neumann et al., 2006 ; Gitler and Shorter, 2011 ; King et al., 2012 ; Robberecht and Philips, 2013 ; March et al., 2016 ). In PD, α-synuclein forms toxic soluble oligomers and cytoplasmic aggregates, termed Lewy bodies, in degenerating dopaminergic neurons (Dehay et al., 2015 ). By contrast, in AD, amyloid-β (Aβ) peptides form extracellular plaques and the microtubule-binding protein, tau, forms cytoplasmic neurofibrillary tangles in afflicted brain regions (Jucker and Walker, 2011 ). Typically, these disorders are categorized into ∼80–90% sporadic cases and ∼10–20% familial cases. Familial forms of disease often have clear genetic causes, which might one day be amenable to gene editing via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 therapeutics if critical safety and ethical concerns can be successfully addressed and respected (Doudna and Charpentier, 2014 ; Baltimore et al., 2015 ; Rahdar et al., 2015 ; Callaway, 2016 ). However, the more common sporadic forms of disease often have no clear underlying genetics, and wild-type proteins misfold (Cushman et al., 2010 ; Jucker and Walker, 2011 ; Robberecht and Philips, 2013 ; Dehay et al., 2015 ). Consequently, therapeutic agents that directly target and safely reverse deleterious protein misfolding are likely to have broad utility (Eisele et al., 2015 ).There are no treatments that directly target the reversal of the protein-misfolding phenomena that underlie these disorders (Jackrel and Shorter, 2015 ). Strategies that directly reverse protein misfolding and restore proteins to native form and function could, in principle, eradicate any severely damaging loss-of-function or toxic gain-of-function phenotypes caused by misfolded conformers (Figure 1; Jackrel and Shorter, 2015 ). Moreover, therapeutic disaggregases would dismantle self-templating amyloid or prion structures, which spread pathology and nucleate formation of neurotoxic, soluble oligomers (Figure 1; Cushman et al., 2010 ; Cohen et al., 2013 ; Guo and Lee, 2014 ; Jackrel and Shorter, 2015 ). My group has endeavored to engineer and evolve Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast (DeSantis and Shorter, 2012 ; Sweeny and Shorter, 2015 ), to more effectively disaggregate misfolded proteins connected with various neurodegenerative disorders, including ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). Although wild-type Hsp104 can disaggregate diverse amyloid and prion conformers, as well as toxic soluble oligomers (Lo Bianco et al., 2008 ; DeSantis et al., 2012 ), its activity against human neurodegenerative disease proteins is suboptimal. Is it even possible to improve on existing Hsp104 disaggregase activity, which has been wrought over the course of millions of years of evolution?Open in a separate windowFIGURE 1:Therapeutic protein disaggregases. Two malicious problems are commonly associated with protein misfolding into disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils: 1) a toxic gain of function of the protein in various misfolded states; and 2) a loss of function of the protein in the various misfolded states. These problems can contribute to the etiology of diverse neurodegenerative diseases in a combinatorial or mutually exclusive manner. A therapeutic protein disaggregase would reverse protein misfolding and recover natively folded functional proteins from disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils. In this way, any toxic gain of function or toxic loss of function caused by protein misfolding would be simultaneously reversed. Ideally, all toxic misfolded conformers would be purged. Therapeutic protein disaggregases could thus have broad utility for various fatal neurodegenerative diseases.Remarkably, the answer to this question is yes! We used nimble yeast models of neurodegenerative proteinopathies (Outeiro and Lindquist, 2003 ; Gitler, 2008 ; Johnson et al., 2008 ; Sun et al., 2011 ; Khurana et al., 2015 ) as a platform to isolate enhanced disaggregases from large libraries of Hsp104 variants generated by error-prone PCR (Jackrel et al., 2014b ). In this way, we reprogrammed Hsp104 to yield the first disaggregases that reverse TDP-43, FUS (another RNA-binding protein with a prion-like domain connected to ALS), and α-synuclein (connected to PD) aggregation and proteotoxicity (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ; Torrente et al., 2016 ). Remarkably, a therapeutic gain of Hsp104 function could be elicited by a single missense mutation (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Under conditions in which Hsp104 was ineffective, potentiated Hsp104 variants dissolved protein inclusions, restored protein localization (e.g., TDP-43 returned to the nucleus from cytoplasmic inclusions), suppressed proteotoxicity, and attenuated dopaminergic neurodegeneration in a Caenorhabditis elegans PD model (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Remarkably, these therapeutic modalities originated from degenerate loss of amino acid identity at select positions of Hsp104 rather than specific mutation (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Some of these changes were remarkably small (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, potentiated Hsp104 variants could be generated by removal of a methyl group from a single side chain or addition or removal of a single methylene bridge from a single side chain (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, small molecules that bind in accessible regions of Hsp104 rich in potentiating mutations might also be able to enhance activity. However, a small-scale screen for small-molecule modulators of Hsp104 revealed only inhibitors (Torrente et al., 2014 ). Nonetheless, our work has established that disease-associated aggregates and amyloid are tractable targets and that enhanced artificial disaggregases can restore proteostasis and mitigate neurodegeneration (Jackrel and Shorter, 2015 ).One surprising aspect of this work is just how many Hsp104 variants we could isolate with potentiated activity. We now have hundreds (Jackrel et al., 2014a ; Jackrel et al., 2015 ). Typically, potentiated Hsp104 variants displayed enhanced activity against several neurodegenerative disease proteins. For example, Hsp104^A503S rescued the aggregation and toxicity of TDP-43, FUS, TAF15, and α-synuclein (Jackrel et al., 2014a ; Jackrel and Shorter, 2014 ). By contrast, some potentiated Hsp104 variants rescued only the aggregation and toxicity of a subset of disease proteins. For example, Hsp104^D498V rescued only the aggregation and toxicity of FUS and α-synuclein (Jackrel et al., 2014a ). A challenge that lies ahead is to engineer potentiated Hsp104 variants that are highly substrate specific to mitigate any potential off-target effects, should they arise (Jackrel and Shorter, 2015 ).Very small changes in primary sequence yield potentiated Hsp104 variants. However, Hsp104 has no metazoan homologue (Erives and Fassler, 2015 ). Now comes the important point. Neuroprotection could be broadly achieved by making very subtle modifications to existing human chaperones with newly appreciated disaggregase activity—for example, Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and HtrA1 (Poepsel et al., 2015 ).Whether Metazoa even possess a powerful protein disaggregation and reactivation machinery had been a long-standing enigma (Torrente and Shorter, 2013 ). However, it has recently emerged that two metazoan chaperone systems—1) Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and 2) HtrA1 (Poepsel et al., 2015 )—possess disaggregase activity that could be therapeutically harnessed or stimulated to reverse deleterious protein misfolding in neurodegenerative disease. I suspect that Metazoa harbor additional disaggregase systems that remain to be identified (Guo et al., 2014 ). Whether due to vicissitudes of aging, environment, or genetic background, these disaggregase systems fail in the context of ALS, PD, and AD. Based on the surprising precedent of our potentiated versions of Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ), I hypothesize that it is possible to engineer and evolve potentiated variants of these human protein disaggregases to more effectively counter deleterious misfolding events in ALS, PD, and AD (Torrente and Shorter, 2013 ; Mack and Shorter, 2016 ).Using classical biochemical reconstitution, it was discovered that one mammalian protein-disaggregase system comprises three molecular chaperones—Hsp110, Hsp70, and Hsp40—which synergize to dissolve and reactivate model proteins trapped in disordered aggregates and can even depolymerize amyloid fibrils formed by α-synuclein from their ends (Shorter, 2011 ; Duennwald et al., 2012 ; Torrente and Shorter, 2013 ). Hsp110, Hsp70, and Hsp40 isoforms are found in the cytoplasm, nucleus, and endoplasmic reticulum, which suggest that protein disaggregation and reactivation can occur in several compartments (Finka et al., 2015 ). Subsequent studies suggest that this system may be more powerful than initially anticipated (Rampelt et al., 2012 ; Mattoo et al., 2013 ; Gao et al., 2015 ; Nillegoda et al., 2015 ). Nonetheless, this system must become overwhelmed in neurodegenerative disorders. Perhaps selectively vulnerable neurons display particular deficits in this machinery. Hence, potentiating the activity of this system via engineering could be extremely valuable. It is promising that directed evolution studies yielded DnaK (Hsp70 in Escherichia coli) variants with improved ability to refold specific substrates (Aponte et al., 2010 ; Schweizer et al., 2011 ; Mack and Shorter, 2016 ), but whether this can be extended to human Hsp70 and the disaggregation of neurodegenerative disease proteins is uncertain.It is exciting that recent studies have revealed that HtrA1, a homo-oligomeric PDZ serine protease, can dissolve and degrade AD-linked tau and Aβ42 fibrils in an ATP-independent manner (Tennstaedt et al., 2012 ; Poepsel et al., 2015 ). HtrA1 first dissolves tau and Aβ42 fibrils and then degrades them, as protease-defective HtrA1 variants dissolve fibrils without degrading them (Poepsel et al., 2015 ). HtrA1 is found in the cytoplasm (∼30%) but is also secreted (∼70%; Poepsel et al., 2015 ). Indeed, HtrA1 is known to degrade substrates in both the extracellular space and the cytoplasm (Chien et al., 2009 ; Campioni et al., 2010 ; Tiaden and Richards, 2013 ). Thus HtrA1 could dissolve Aβ42 fibrils in the extracellular space and tau fibrils in the cytoplasm and simultaneously destroy the two cardinal features of AD (Poepsel et al., 2015 ). I suspect that this system becomes overwhelmed or is insufficient in AD, and thus potentiating and tailoring HtrA1 disaggregase activity could be a valuable therapeutic strategy. For example, it might be advantageous to simply degrade Aβ42 after disaggregation if the peptide has no beneficial function. Thus HtrA1 variants with enhanced disaggregation and degradation activity against Aβ42 could be extremely useful. However, Aβ42 (and related Aβ peptides) may have physiological functions that are presently underappreciated (Soscia et al., 2010 ; Fedele et al., 2015 ), in which case HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity could be vital. HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity may also be particularly important to recover functional tau from neurofibrillary tangles to reverse loss of tau function in AD and various tauopathies (Santacruz et al., 2005 ; Trojanowski and Lee, 2005 ; Dixit et al., 2008 ).I suggest that relatively small changes in primary sequence will yield large increases in disaggregase activity for these systems as they do for Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). If true, this would further suggest that small molecules that bind in the appropriate regions of Hsp110, Hsp70, Hsp40, or HtrA1 might also enhance disaggregase activity. Thus, isolating small-molecule enhancers of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase systems could yield important therapeutics. Indeed, I hypothesize that enhancing the activity of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase system with specific small molecules will enable dissolution of toxic oligomeric and amyloid forms of various disease proteins and confer therapeutic benefits in ALS, PD, AD, and potentially other neurodegenerative disorders.It is intriguing that several small molecules are already known to enhance various aspects of Hsp70 chaperone activity (Pratt et al., 2015 ; Shrestha et al., 2016 ). These include MKT-077, JG-98, YM-1, YM-8, and 115-7c (Wisen et al., 2010 ; Pratt et al., 2015 ). It is not known whether any of these stimulates the disaggregase activity of the Hsp110, Hsp70, and Hsp40 system. MKT-077, JG-98, YM-1, and YM-8 are rhodocyanines that bind with low micromolar affinity to the nucleotide-binding domain of ADP- but not ATP-bound Hsp70, stabilizing the ADP-bound state (Pratt et al., 2015 ). The ADP-bound state of Hsp70 engages clients with higher affinity, and consequently MKT-077, JG-98, and YM-1 activate binding of Hsp70 to misfolded proteins (Wang et al., 2013 ; Pratt et al., 2015 ). Thus, under some conditions, these small molecules can promote folding of certain Hsp70 clients (Morishima et al., 2011 ; Pratt et al., 2015 ). However, prolonged interaction of clients with Hsp70 promotes their CHIP-dependent ubiquitylation and degradation in vivo (Morishima et al., 2011 ; Wang et al., 2013 ; Pratt et al., 2015 ). Intriguingly, YM-1 promotes clearance of polyglutamine oligomers and aggregates in cells (Wang et al., 2013 ; Pratt et al., 2015 ). MKT-0777, YM-1, JG-98, and YM-8 also promote clearance of tau and confer therapeutic benefit in tauopathy models (Abisambra et al., 2013 ; Miyata et al., 2013 ; Fontaine et al., 2015 ). Of importance, YM-8 is long lived in vivo and crosses the blood–brain barrier (Miyata et al., 2013 ). The dihydropyrimidine 115-7c activates Hsp70 ATPase turnover rate, promotes Hsp70 substrate refolding, and reduces α-synuclein aggregation in cell culture (Wisen et al., 2010 ; Kilpatrick et al., 2013 ). It binds to the IIA subdomain of Hsp70 and promotes the active Hsp70–Hsp40 complex (Wisen et al., 2010 ). Small-molecule enhancers of HtrA1 protease activity have also emerged (Jo et al., 2014 ). Thus it will be important to assess whether these small molecules enhance the activity of their respective disaggregases against various neurodegenerative substrates.Although small molecules that enhance disaggregase activity of endogenous human proteins might be the most immediately translatable, gene-, mRNA-, or protein-based therapies can also be envisioned. For example, adeno-associated viruses expressing enhanced disaggregases might be used to target degenerating neurons (Dong et al., 2005 ; Lo Bianco et al., 2008 ; Deverman et al., 2016 ). Alternatively, if viral vectors are undesirable, modified mRNAs might serve as an alternative to DNA-based gene therapy (Kormann et al., 2011 ). Protein-based therapeutics could also be explored. For example, intraperitoneal injection of human Hsp70 increased lifespan, delayed symptom onset, preserved motor function, and prolonged motor neuron viability in a mouse model of ALS (Gifondorwa et al., 2007 ; Gifondorwa et al., 2012 ). Several other studies suggest that exogenous delivery of Hsp70 can have beneficial, neuroprotective effects in mice (Nagel et al., 2008 ; Bobkova et al., 2014 ; Bobkova et al., 2015 ).Ultimately, if safety and ethical concerns can be overcome in a circumspect, risk-averse manner, CRISPR-Cas9–based therapeutics might even be used to genetically alter the underlying disaggregase to a potentiated form in selectively vulnerable neuronal populations. This approach might be particularly valuable if enhanced disaggregase activity is not detrimental in the long term. Moreover, stem cell–based therapies for replacing lost neurons could also be fortified to express enhanced disaggregase systems. Thus they would be endowed with resistance to potential infection by prion-like conformers that might have accumulated during disease progression (Cushman et al., 2010 ).Enhanced disaggregase activity is likely to be highly advantageous to neurons under circumstances in which protein misfolding has overwhelmed the system (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). However, inappropriate hyperactivity of protein disaggregases might also have detrimental, off-target effects under regular conditions in which protein misfolding is not an overwhelming issue (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Thus it may be advantageous to engineer enhanced protein disaggregases to be highly substrate specific. In this way, off-target effects would be readily avoided. There is strong precedent for directed evolution or engineering of specialized chaperone or protein activity from a generalist antecedent (Wang et al., 2002 ; Farrell et al., 2007 ; Smith et al., 2015 ). Thus, engineering specialist disaggregases for each disease substrate could be achieved. Alternatively, transient or intermittent doses of enhanced disaggregases at specific times or places where they are most needed would also minimize potentially toxic side effects. For example, enhanced disaggregase activity might be applied ephemerally to clear existing misfolded conformers and then be withdrawn once the endogenous proteostasis network regains control. Similarly, it is straightforward to envision administration of small-molecule enhancers of disaggregase activity in intermittent protocols that enable facile recovery from potential side effects (Fontaine et al., 2015 ). In this way, any adverse effects of enhanced protein-disaggregase activity under normal physiological conditions would be avoided. Many barriers will need to be safely overcome to implement a successful therapeutic disaggregase, including how to deliver enhanced disaggregase activity to exactly where it is needed. However, these obstacles are not a reason to be pessimistic. On the contrary, the isolation of engineered disaggregases that efficaciously reverse deleterious misfolding of neurodegenerative disease proteins directs our attention to considerably expand the environs in which they should be sought. My closing sentences, therefore, are intended to be provocative.I suspect that neuroprotection could be broadly actualized via precise but subtle alterations to existing protein-disaggregase modalities. The engineering and evolution of protein disaggregases could yield important solutions to avert an imminent plague of neurodegenerative disorders that promises to devastate our society. I strongly suspect that cures for various neurodegenerative disorders will be realized by pioneering as-yet-uncharted regions of disaggregase sequence space or chemical space to elucidate small-molecule enhancers of disaggregase activity.     

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.     

Mapping the electrostatic profiles of cellular membranes

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately –35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Lipids and (glyco)proteins are the main constituents of biological membranes. Sugar moieties of glycoproteins, glycolipids, and adherent glycocalyx components such as hyaluronic acid can bear ionizable groups that confer a net negative charge on the outer surface of the plasma membrane. The aggregate surface charge of the outer membrane has been estimated indirectly by measuring the ζ potential—the potential at the slipping plane—by electrophoretic means (e.g., Tippe, 1981; Silva Filho et al., 1987) or by measuring streaming potentials (Vandrangi et al., 2012). The plasma membrane, however, is highly asymmetric; its inner (cytosolic) aspect is virtually devoid of carbohydrate moieties. Nevertheless, the cytosolic leaflet is also thought to be negatively charged, due primarily to the accumulation of anionic phospholipids, namely phosphoinositides and phosphatidylserine (PtdSer). Based on biochemical determinations of its lipid composition, the net negative charge of the plasmalemmal inner leaflet is estimated to generate an electrical field of 10⁵ V/cm (Olivotto et al., 1996). The membranes of intracellular organelles can also contain anionic lipids, but their precise lipid composition and topology have been difficult to assess and hence their surface charge has not been estimated.The surface potentials of biological membranes have important functional implications: they can alter the disposition of charged regions of transmembrane proteins, cause local accumulation of soluble counterions in the vicinity—altering the local pH as well as the concentration of physiologically important ions such as calcium—and serve to recruit extrinsic cationic proteins (McLaughlin, 1989). It is therefore important to determine the electrostatic properties of each of the organellar membranes. In principle, this could be accomplished by measuring the ζ potentials of isolated organelles. However, the purity of such preparations is imperfect, changes in lipid composition (particularly phosphoinositide degradation) and sidedness cannot be avoided, and loosely adherent components that may alter the surface charge can be removed during the isolation process. Alternative approaches to estimating the surface potential are therefore required.Here we used recombinant and synthetic polycationic peptides to obtain a quantitative estimate of the surface potential of the inner leaflet of the plasma membrane and to establish a hierarchical map of the potentials of the cytosolic surfaces of the major intracellular organelles in live cells.     

A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

How a first research experience had an impact on my scientific journey

As I look back to my scientific trajectory on the occasion of being the recipient of the E. B. Wilson Medal of the American Society for Cell Biology, I realize how much an early scientific experience had an impact on my research many years later. The major influence that the first scientific encounters can have in defining a scientist’s path makes the choice of the training environment so important for a future career.

During my scientific career I have worked on different topics. However, my scientific journey represents a progression of steps that build onto each other. Each experience, no matter how successful, had an impact on how I interpreted subsequent results, on how I assessed their implications, and on how I made subsequent programmatic decisions. My research at the interface of cell biology and neuroscience has spanned from membrane traffic and membrane remodeling, to phosphoinositide signaling, and more recently also to the role of membrane contact sites in the control of membrane lipid homeostasis in physiology and disease. While only partially related, all these topics are interconnected by an intellectual thread. As an example—which shows how defining our training years can be—I will summarize here how an early scientific experience in medical school contributed to shape in unexpected ways my research directions many years later.From the day I enrolled in medical school at the University of Milano I knew I wanted to be a scientist. While a medical student, I explored several research environments that would expose me to leading-edge science. Eventually I encountered a young scientist, Jacopo Meldolesi, who had just returned to Italy after studying the secretory pathway as a postdoc with Jim Jamieson and George Palade at Rockefeller University, and I decided to join his lab to carry out the required short medical school thesis project. Jacopo was interested in the regulation of secretion and proposed that I explore mechanisms underlying the so-called “PI (Phosphatidyl Inositol) effect” described by Hokin and Hokin in the 1950s (Hokin and Hokin, 1953). These investigators had found that stimulation of secretory cells resulted in the rapid cleavage of PI immediately followed by its resynthesis. It remained unclear whether these changes reflected a signaling reaction elicited by secretagogues or metabolic changes associated with membrane traffic reactions triggered by the stimulus. So, the idea was to determine in which membrane compartment this turnover occurred: selectively at the plasma membrane (thus supporting a role in signaling) or in membranes of the secretory pathway. We were puzzled by finding that at least under the rather poor time resolution of our experiments—incubation of pancreatic lobules for tens of minutes with radioactive inositol following stimulation with acetylcholine—newly synthesized PI was found at similar concentrations in all membranes. But then we became aware of the recently described proteins that transport lipids between membranes through the cytosol and independent of membrane traffic (Wirtz and Zilversmit, 1969). We thought that perhaps these proteins could contribute to rapidly equilibrating PI pools in different compartments, questioning the validity of our experimental approach to identify the site of PI resynthesis. I moved on to other projects, and I thought I would never again work with lipids.In the following years the work of several labs established that the PI effect discovered by Hokin and Hokin mainly reflected agonist-stimulated phospholipase C (PLC)-dependent cleavage of the phosphoinositide PI(4,5)P₂ at the plasma membrane to generate the second messengers diacylglycerol (DAG) and IP3 (phosphoinositides are the phosphorylated products of PI) (Lapetina and Michell, 1973; Inoue et al., 1977; Berridge et al., 1983). These findings became textbook knowledge, and the idea that PI(4,5)P₂ had primarily a function in signal transduction at the cell surface became mainstream. The additional discovery of the PI(3,4)P₂ and PI(3,4,5)P₃ as a mediators of growth factor actions added a new twist to the field by showing that not only metabolites generated by PI(4,5)P₂ cleavage, but also phosphoinositides themselves, could have a signaling role (Traynor-Kaplan et al., 1988; Whitman et al., 1988; Auger et al., 1989). However, once again, this signaling appeared to be implicated in classical signal transduction, not in membrane traffic.In the meantime, I had moved to Yale, first as a postdoc with Paul Greengard and then as a faculty in the Section of Cell Biology (which subsequently became the Department of Cell Biology). Following my postdoc with Greengard, I had become interested in the cell biology of synapses and in the molecular machinery underlying the exo-endocytic recycling of synaptic vesicles. In what turned out to be a seminal finding, in the mid-1990s Peter McPherson, a postdoc in my laboratory, identified a nerve terminal–enriched protein that shared some interactions with the GTPase dynamin, another abundant presynaptic protein studied in our lab (McPherson et al., 1994). As dynamin mediates the fission reaction of endocytosis by cutting the neck of endocytic buds to generate free vesicles (Takei et al., 1995; Roux et al., 2006; Ferguson and De Camilli, 2012), we speculated that this new protein would also have a role in endocytic traffic. Peter set out to clone it, and I vividly recall my surprise when, upon my returning from a summer vacation in Italy he reported to me that a domain of this protein, which we called synaptojanin, had homology to an inositol 5-phosphatase that dephosphorylates PI(4,5)P₂ (McPherson et al., 1996). A second domain of this protein was subsequently found to have inositol 4-phosphatase activity (Guo et al., 1999), so that synaptojanin can dephosphorylate PI(4,5)P₂ all the way to PI. Thus, I was back to lipids, specifically to inositol phospholipids, and to a potential role of these lipids not in the transduction of extracellular signals, but in membrane traffic. With a mind primed by my project in medical school, I delved with enthusiasm into this topic. PI(4,5)P₂ had been shown to interact with clathrin adaptors (Beck and Keen, 1991), but the physiological significance of these findings had remained unclear. Our studies led to a model in which the selective concentration of PI(4,5)P₂ in the plasma membrane is required for the recruitment of clathrin adaptors and other endocytic factors to this membrane, while the tight coupling of PI(4,5)P₂ dephosphorylation by synaptojanin to the dynamin-dependent fission reaction of endocytosis is required to allow the shedding of such factors once the vesicle has undergone separation from the plasma membrane (Cremona et al., 1999; Cremona and De Camilli, 2001; Wenk et al., 2001). We also discovered curvature-generating/sensing proteins (BAR domain–containing proteins, primarily endophilin) that are responsible for achieving this coupling by coordinating the formation of the narrow neck of endocytic buds with the recruitment of synaptojanin (Takei et al., 1999; Farsad et al., 2001; Frost et al., 2009; Wu et al., 2010; Milosevic et al., 2011). This model, which is encapsulated in Figure 1 and which is supported by genetic studies in mice and other model organisms, posits that a cycle of PI phosphorylation and dephosphorylation is nested within the synaptic vesicle cycle. The implication of a phosphoinositide phosphatase in membrane transport converged with the identification of a PI kinase (the PI 3-kinase Vps34) involved in “vacuolar protein sorting” in yeast (Schu et al., 1993). This raised the possibility that the phosphorylation and dephosphorylation of inositol phospholipids could have a general significance in membrane traffic (De Camilli et al., 1996).Open in a separate windowFIGURE 1:Schematic cartoon depicting the occurrence of a cycle of phosphatidylinositol phosphorylation–dephosphorylation nested within the exo-endocytic recycling of synaptic vesicles at synapses. PI(4,5)P₂ in the plasma membrane helps define this membrane as the acceptor for the fusion of synaptic vesicles and functions as a coreceptor for endocytic factors responsible for their reinternalization. PI(4,5)P₂ dephosphorylation by synaptojanin allows the shedding of endocytic factors and the reutilization of vesicles for a new cycle of secretion. Recruitment of synaptojanin is coupled to dynamin-dependent fission via its binding to the BAR domain–containing protein endophilin, a curvature-generating/sensing protein that also binds dynamin.The field grew very rapidly. It was found that reversible phosphorylation of phosphatidylinositol at the 3,4 and 5 positions of the inositol ring by a multiplicity of enzymes generates seven phosphoinositide species whose differential protein-binding specificities help control, often via a dual key mechanism (Wenk and De Camilli, 2004), membrane cytosol interfaces (Di Paolo and De Camilli, 2006; Vicinanza et al., 2008; Balla, 2013). Along with small GTPases (Behnia and Munro, 2005), phosphoinositides are key determinants of the identity of membranes or membrane subdomains and thus control the multiplicity of reactions that occur at membrane–cytosol interfaces, such as the recruitment and shedding of trafficking proteins, the function of integral membrane proteins, and the assembly and disassembly of signaling and cytoskeleton complexes. Importantly, it became clear that, as we had found for synaptojanin, interconversion of phosphoinositide species along the secretory and endocytic pathways functions as a switch to release membrane-associated factors that define one compartment and to recruit factors that define the next compartment (Odorizzi et al., 2000; Di Paolo and De Camilli, 2006; Zoncu et al., 2009). I think I would not have immediately grasped the significance of the identification of synaptojanin and then invested much of our work in this field had it not been for my early research experience in medical school.The roles of phosphoinositides in membrane identity imply the existence of mechanisms to tightly control their localizations and also to ensure availability of PI in membranes—primarily the plasma membrane—where the backbone of this lipid is consumed by PLC. Surprisingly, genetic evidence suggested that one mechanism to control PI4P levels in membranes is via their direct contacts with the endoplasmic reticulum (ER), where a main PI4P phosphatase is localized (Foti et al., 2001; Stefan et al., 2011; Mesmin et al., 2013). Concerning availability of PI, DAG generated by PLC and its phosphorylated downstream product phosphatidic acid must be returned to the ER for metabolic recycling by the ER-localized phosphatidylinositol synthase, and newly synthesized PI then needs to be rapidly transported to the plasma membrane to replace its depleted pool. Strong evidence indicates that these reactions are mediated at least in part by lipid transfer proteins, and we have now learned that much of this protein-mediated lipid transport occurs at membrane contact sites (Mesmin et al., 2013; Wong et al., 2017; Cockcroft and Raghu, 2018; Pemberton et al., 2020). This is what motivated my lab to enter the young field of membrane contact sites. Here, once again, my early appreciation of the importance of lipid transfer proteins during medical school had an impact on my decision to invest in this rapidly developing area of cell biology.Not only has it been rewarding to help expand the inventory of proteins known to function both as membrane tethers and as lipid transporters, and to elucidate their function and regulation (Giordano et al., 2013; Schauder et al., 2014; Chung et al., 2015; Dong et al., 2016; Saheki et al., 2016), but this has been an opportunity, in collaboration with my Yale colleague Karin Reinisch, to participate in the discovery of something unexpected and new in the biology of eukaryotic cells. Until recently, it was thought that nonvesicular lipid transport, including the transport occurring at membrane contact sites, occurred only via protein modules that function as shuttles between two bilayers, often acting as countertransporters, delivering different cargoes as they move back and forth between two closely apposed membranes. However, our investigations of VPS13, a protein originally identified in yeast for its function in membrane transport and subsequently linked to lipid dynamics (Lang et al., 2015; Park et al., 2016), raised the possibility that this protein, and thus also the closely related autophagy factor ATG2, may be the founding members of a protein superfamily with bulk lipid transport properties, that is, proteins that could facilitate the net fluxes of lipids between adjacent membranes, and thus in membrane expansion (Kumar et al., 2018). As was subsequently shown by structural studies (Valverde et al., 2019; Li et al., 2020), these rod-like proteins harbor a hydrophobic groove that spans their length, thus supporting a bridge-like model of lipid transport to support bulk lipid flow (Li et al., 2020; Guillen-Samander et al., 2021; Leonzino et al., 2021). Inspection of protein sequence and structure databases suggests the existence of several other proteins with these properties, implying that this mode of lipid transport (a bridge-like mechanism), previously described for the transport of lipids from the inner to the outer membrane of Gram-negative bacteria (Bishop, 2019), may be of broad relevance in cell biology. As mutations in VPS13 family proteins result in neurodegenerative diseases, including a Huntington-like disease and Parkinson’s disease (Ugur et al., 2020), this is a field rich in medical implications. In fact, my curiosity about disease mechanisms stemming from my medical school training contributed to making me specially interested in studying these proteins.It has been a wonderful journey, rich with twists and turns, exploring new territories and continuously discovering that there are untapped frontiers whose existence we still do not know about. During the course of my career, I found especially rewarding bringing together different fields and finding new connections between fundamental cell biology and medicine, more so in recent years, where genetic studies of diseases often provide the first clues to molecular and cellular mechanisms. As I have emphasized in this essay, each of our experiences, no matter how successful, becomes part of the body of knowledge that defines us and has an impact on our research trajectory. I typically encourage students to search for training opportunities in different settings rather than follow a linear path, as this will enable them to bring together in their independent careers the expertise and knowledge of different worlds and thus to carry out original science. Our specific and unique paths are what make our research innovative and special.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.