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.     

Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.During cell division, the correct organization of microtubules in bipolar spindles is necessary to distribute chromosomes to the daughter cells. The slow growing or minus ends of the microtubules are focused at each pole, while the plus ends interact with the chromosomes in the center of the spindle (Telzer and Haimo, 1981; McIntosh and Euteneuer, 1984). Current concepts of spindle assembly are based primarily on mitotic spindles of animal cells, which contain centrosomes. Centrosomes are thought to be instrumental for organization of the spindle poles and for determining both microtubule polarity and the spindle axis. In the prevailing model, termed “Search and Capture,” dynamic microtubules growing from two focal points, the centrosomes, are captured and stabilized by chromosomes, generating a bipolar array (Kirschner and Mitchison, 1986). However, while centrosomes are required for spindle assembly in some systems (Sluder and Rieder, 1985; Rieder and Alexander, 1990; Zhang and Nicklas, 1995a ,b), in other systems they appear to be dispensable (Steffen et al., 1986; Heald et al., 1996). Furthermore, centrosomes are not present in higher plant cells and in female meiosis of most animal species (Bajer and Mole, 1982; Gard, 1992; Theurkauf and Hawley, 1992; Albertson and Thomson, 1993; Lambert and Lloyd, 1994). In the absence of centrosomes, bipolar spindle assembly seems to occur through the self-organization of microtubules around mitotic chromatin (McKim and Hawley, 1995; Heald et al., 1996; Waters and Salmon, 1997). The observation of apparently different spindle assembly pathways raises several questions: Do different types of spindles share common mechanisms of organization? How do centrosomes influence spindle assembly? In the absence of centrosomes, what aspects of microtubule self-organization promote spindle bipolarity?To begin to address these questions, we have used Xenopus egg extracts, which can be used to reconstitute different types of spindle assembly. Spindle assembly around Xenopus sperm nuclei is directed by centrosomes (Sawin and Mitchison, 1991). Like other meiotic systems (Bastmeyer et al., 1986; Steffen et al., 1986), Xenopus extracts also support spindle assembly around chromatin in the absence of centrosomes through the movement and sorting of randomly nucleated microtubules into a bipolar structure (Heald et al., 1996). In this process, the microtubule-based motor cytoplasmic dynein forms spindle poles by cross-linking and sliding microtubule minus ends together. Increasing evidence suggests that the function of dynein in spindle assembly depends on its interaction with other proteins, including dynactin, a dynein-binding complex, and NuMA¹ (nuclear protein that associates with the mitotic apparatus) (Merdes et al., 1996; Echeverri et al., 1996; Gaglio et al., 1996). In this paper, we demonstrate that both in the presence and absence of centrosomes, spindle pole assembly occurs by a common dynein-dependent mechanism. We show that when centrosomes are present, they are tethered to spindle poles by dynein. In the absence of dynein function, microtubules are still sorted into an antiparallel array, indicating that other aspects of microtubule self-assembly independent of pole formation promote spindle bipolarity around mitotic chromatin. Since centrosomes are dispensable for pole formation in this system, what is their function? We show here that if only one centrosome is present, it acts as a dominant site for microtubule nucleation and focal organization, resulting in a monopolar spindle. Therefore, although centrosomes are not required in this system, they can influence spindle pole formation and bipolarity.     

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.     

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.     

PRC1 Cooperates with CLASP1 to Organize Central Spindle Plasticity in Mitosis

During cell division, chromosome segregation is governed by the interaction of spindle microtubules with the kinetochore. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, microtubule-bundling protein PRC1, and Aurora B kinase complex. However, the precise role of PRC1 in central spindle organization has remained elusive. Here we show that PRC1 recruits CLASP1 to the central spindle at early anaphase onset. CLASP1 belongs to a conserved microtubule-binding protein family that mediates the stabilization of overlapping microtubules of the central spindle. PRC1 physically interacts with CLASP1 and specifies its localization to the central spindle. Repression of CLASP1 leads to sister-chromatid bridges and depolymerization of spindle midzone microtubules. Disruption of PRC1-CLASP1 interaction by a membrane-permeable peptide abrogates accurate chromosome segregation, resulting in sister chromatid bridges. These findings reveal a key role for the PRC1-CLASP1 interaction in achieving a stable anti-parallel microtubule organization essential for faithful chromosome segregation. We propose that PRC1 forms a link between stabilization of CLASP1 association with central spindle microtubules and anti-parallel microtubule elongation.To ensure that each daughter cell receives the full complement of the genome in each cell division, chromosomes move poleward, and non-kinetochore fibers become bundled at the onset of anaphase, initiating assembly of the central spindle, a set of anti-parallel microtubules that serves to concentrate key regulators of cytokinesis (1–3). Chromosomal passengers are a group of evolutionarily conserved proteins that orchestrates chromosome segregation and central spindle plasticity (4, 5). This protein complex containing Aurora B, Survivin, INCENP, and Borealin is relocated from the kinetochore to the central spindle upon anaphase onset (5–9). Perturbation of their function results in defects in metaphase chromosome alignment, chromosome segregation, and cytokinesis (10).Among the central spindle maintenance components, only two have been reported to mediate the microtubule bundling in the central spindle. One is centralspindlin, a heterotetramer containing CeMKLP1/ZEN-4 and RhoGAP/CYK-4 (11), and the other one is an evolutionarily conserved protein, PRC1 (also named Feo in fruit fry, Ase1 in yeast, and MAP65 in plant cells). PRC1 is a non-motor microtubule-binding and -bundling protein in human cells originally identified as a Cdc2 substrate essential for cytokinesis (12, 13). Similar microtubule regulatory activities have been reported in yeast, fruit fly, and plant cells. It is well known that overexpression of wild type PRC1 in HeLa cells can result in thick microtubule bundles in cells at interphase (13). Bundling activity of PRC1, as well as centralspindlin, is required for the organization of the central spindle as well as for the successful progression of cytokinesis. PRC1 molecules accumulate on the midline of a central spindle with the cell cycle progression to anaphase. As a non-motor microtubule-binding protein, transportation of PRC1 to the midline is promoted by its association to kinesin, KIF4A, and timing of this progression is controlled by the dephosphorylation of Thr-481 on PRC1 when the cell exits metaphase by phosphatase Cdc14 (14). Our recent study shows that prevention of the phosphorylation of PRC1 at Thr-470 causes an inhibition in PRC1 oligomerization in vitro and an aberrant organization of central spindle in vivo, suggesting that this phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle (15).Spatiotemporal regulation of microtubule organization and dynamics is responsible for the mitotic apparatus such as the central spindle. However, it has remained elusive as to how the central spindle microtubule organization and dynamics are regulated. There are large varieties of microtubule-associated proteins responsible for regulation of the dynamic behavior of microtubules and microtubule-mediated transport. Among these, proteins that associate with the tips of microtubules are called +TIPs, for “plus-end tracking proteins.” These proteins have been shown to be important in different organisms and cellular systems (16). Using yeast two-hybrid assay, CLASPs were identified as interacting partners of the CLIPs and characterized as new +TIP proteins (17).The microtubule-binding protein CLASP is emerging as an important microtubule regulator in the formation of the mitotic apparatus (18–22). CLASP is required for promoting plus-end growth of spindle microtubules in prometaphase (23). Although the molecular mechanisms underlying its regulation of microtubule dynamics remain elusive, it is generally believed that CLASP orchestrates microtubule dynamics via its physical interacting with EB1, CLIP170, and microtubules (17, 24).To delineate the molecular function of PRC1 in central spindle organization and spatiotemporal regulation, we carried out a new search for PRC1-interacting proteins. Our studies show that PRC1 physically interacts with CLASP1, and the two proteins cooperate in the organization of the central spindle. Our studies provide a novel regulatory mechanism in which the PRC1 complex operates central spindle organization in mitosis.     

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.