Translational diffusion in lipid membranes beyond the Saffman-Delbruck approximation

The Saffman-Delbrück approximation is commonly used in biophysics to relate the membrane inclusion size to its translational diffusion coefficient and membrane viscosity. However, this approximation has a restricted validity range, and its application to determination of inclusion sizes from diffusion data may in certain cases lead to unreliable results. At the same time, the model by Hughes et al. (Hughes, B. D., B. A. Pailthorpe, and C. R. White. 1981. J. Fluid Mech. 110:349-372.), providing diffusion coefficients of membrane inclusions for arbitrary inclusion sizes and viscosities of the membrane and surrounding fluids, involves substantial computational efforts, which prevents its use in practical data analysis. We develop a simple and accurate analytical approximation to the Hughes et al. model and demonstrate its performance and utility by applying it to the recently published experimental data on translational diffusion of micrometer-sized membrane domains.     

Contextual constraints on synonymous codon choice

We have studied the statistical constraints on synonymous codon choice to evaluate various proposals regarding the origin of the bias in synonymous codon usage observed by Fiers et al. (1975), Air et al. (1976), Grantham et al. (1980) and others. We have determined the statistical dependence of the degenerate third base on either of its nearest neighbors in mitochondrial, prokaryotic, and eukaryotic coding sequences. We noted an increasing dependence of the third base on its nearest neighbors in moving from mitochrondria to prokaryotes to eukaryotes.A statistical model assuming random equiprobable selection of synonymous codons was found grossly adequate for the mitochondria, but totally indequate for prokaryotes and eukaryotes. A model assuming selection of synonymous codons reflecting a genomic strategy, i.e. the genome hypothesis of Grantham et al. (1980), gave a good approximation of the mitochondrial sequences. A statistical model which exactly maintains codon frequency, but allows the position of corresponding synonymous codons to vary was only grossly adequate for prokaryotes and totally inadequate for eukaryotes. The results of these simulations are consistent with the measures on experimental sequences and suggest that a “frequency constraint” model such as that of Grantham et al. (1980) may be an adequate explanation of the codon usage in mitochondria. However, in addition to this frequency constraint, there may be constraints on synonymous codon choice in prokaryotes due to codon context. Furthermore, any proposal to explain codon usage in eukaryotes must involve a constraint on the context of a codon in the sequence.     

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.     

N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [¹⁴C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [¹⁴C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.NAPE is a widespread, albeit minor, membrane phospholipid in animal and plant tissues (Schmid et al., 1990; Chapman and Moore, 1993). Its unusual structural features (a third fatty acid moiety linked to the amino head group of PE) impart stabilizing properties to membrane bilayers (Domingo et al., 1994; LaFrance et al., 1997). NAPE and its hydrolysis products, NAEs, are known to accumulate in vertebrate tissues under pathological conditions (for review, see Schmid et al., 1990). Recently, there has been renewed interest in NAEs because of the contention that anandamide (N-arachidonylethanolamine) is an endogenous ligand for the cannabinoid receptor in mammalian brain (Devane et al., 1992; Fontana et al., 1995; Schmid et al., 1996). The likely route for NAE formation in neural and nonneural tissues, although the matter of some debate, is via the signal-mediated hydrolysis of NAPE (DiMarzo et al., 1994; Schmid et al., 1996; Sugiura, et al., 1996).In plants little is known regarding the catabolism of NAPE. In cottonseed microsomes NAPE was metabolized to NAE or NAlysoPE by PLD- or PLA-type activities, respectively (Chapman et al., 1995b). However, the metabolic fate of NAPE in vivo and the factors that regulate NAPE hydrolysis remain largely unknown. We previously noted that the biosynthesis of NAPE was increased in elicitor-treated cell suspensions of tobacco (Nicotiana tabacum L.). Here we extend our investigations with this model system to examine NAPE catabolism by plant cells in vivo. NAE was released from NAPE, and it accumulated extracellularly. We identified by GC-MS these tobacco NAEs as N-lauroylethanolamine and N-myristoylethanolamine. These NAEs were increased in elicitor-treated cell suspensions. Furthermore, we detected the enzymatic machinery capable of the release and the degradation of NAEs in tobacco cells. To our knowledge this represents the first identification of the NAE molecular species in plant cells. It is tempting to speculate that NAPE hydrolysis in elicitor-treated plant cells may be involved in a signaling pathway analogous to that found in mammalian cells.     

Microstructural Analysis of Peripheral Lung Tissue through CPMG Inter-Echo Time R2 Dispersion

Since changes in lung microstructure are important indicators for (early stage) lung pathology, there is a need for quantifiable information of diagnostically challenging cases in a clinical setting, e.g. to evaluate early emphysematous changes in peripheral lung tissue. Considering alveoli as spherical air-spaces surrounded by a thin film of lung tissue allows deriving an expression for Carr-Purcell-Meiboom-Gill transverse relaxation rates R ₂ with a dependence on inter-echo time, local air-tissue volume fraction, diffusion coefficient and alveolar diameter, within a weak field approximation. The model relaxation rate exhibits the same hyperbolic tangent dependency as seen in the Luz-Meiboom model and limiting cases agree with Brooks et al. and Jensen et al. In addition, the model is tested against experimental data for passively deflated rat lungs: the resulting mean alveolar radius of R _A = 31.46 ± 13.15 μm is very close to the literature value (∼34 μm). Also, modeled radii obtained from relaxometer measurements of ageing hydrogel foam (that mimics peripheral lung tissue) are in good agreement with those obtained from μCT images of the same foam (mean relative error: 0.06 ± 0.01). The model’s ability to determine the alveolar radius and/or air volume fraction will be useful in quantifying peripheral lung microstructure.     

Formulas predicting survival in patients with heart transplantation

Kirklin et al. (J Heart Transpl, 7 (1988) 331–336) reported survival data in 132 patients who underwent heart transplantation. Survival was evaluated by using the product-limit method of Kaplan-Meier and maximum likelihood method. In addition, the effect of pulmonary vascular resistance on survival was estimated by using multivariate analysis. A microcomputer program in BASIC for predicting the survival probability after transplantation in patients with heart transplantation is designed. The formula used in this program is derived from the survival data reported by Kirklin et al. (J Heart Transpl, 7 (1988) 331–336). A mathematical model of the ‘probacent’-probability equation and a computer program previously published by the author are employed in this study. Analysis of the computer-assisted predicted values and the data reported by Kirklin et al. (J Heart Transpl, 7 (1988) 331–336) indicates that the program is accurate and reliable with a complete agreement in expressing survival probability as a function of time after heart transplantation. The computer-assisted predictive formula can determine the relationship between the time and the survival probability and may be of value for prognostic evaluation of patients. The computer-assisted mathematical model of the ‘probacent’-probability equation may be proposed as a general approximation method to make useful predictions of probable outcomes in various biomedical phenomena.     

Computational studies of ligand diffusion in globins: I. Leghemoglobin

The thermally assisted diffusion of a small ligand (carbon monoxide) through a protein matrix (lupine leghemoglobin) is investigated computationally. The diffusion paths are calculated by a variant of the time-dependent Hartree approximation which we call LES (locally enhanced sampling). The variant which was recently introduced by Elber and Karplus is based on the classical TDSCF approximation of Gerber et al. The simulation enables more significant search for diffusion pathways than was possible before. This is done by increasing the number of ligand trajectories using a single trajectory for the protein. We compare qualitatively diffusion rates in leghemoglobin and in myoglobin. The calculation shows that the diffusion in leghemoglobin is much faster than the diffusion in myoglobin, in agreement with experiment. The gate in leghemoglobin is opened by fluctuations at a close contact between the B/C and the G helices. The most relevant fluctuation is the rigid shift of the C helix with respect to the G helix. This path is not observed in a comparable calculation for myoglobin. This finding is rationalized by the lack of the D helix in leghemoglobin and a significantly more flexible CE loop. Supporting experimental evidence for the importance of the CE loop in leghemoglobin can be found in the kinetics studies of Gibson et al.     

Model of the pathway of −1 frameshifting: Long pausing

It has been characterized that the programmed ribosomal ?1 frameshifting often occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot. In some prokaryotic cases such as the dnaX gene of Escherichia coli, an additional stimulatory signal—an upstream, internal Shine–Dalgarno (SD) sequence—is also necessary to stimulate the efficient ?1 frameshifting. However, the molecular and physical mechanism of the ?1 frameshifting is poorly understood. Here, we propose a model of the pathway of the ?1 translational frameshifting during ribosome translation of the dnaX ?1 frameshift mRNA. With the model, the single-molecule fluorescence data (Chen et al. (2014) [29]) on the dynamics of the shunt either to long pausing or to normal translation, the tRNA transit and sampling dynamics in the long-paused rotated state, the EF-G sampling dynamics, the mean rotated-state lifetimes, etc., are explained quantitatively. Moreover, the model is also consistent with the experimental data (Yan et al. (2015) [30]) on translocation excursions and broad branching of frameshifting pathways. In addition, we present some predicted results, which can be easily tested by future optical trapping experiments.     

Effect of aminoacylation on tRNA conformation

Translational diffusion coefficients have been simulated for various conformations of tRNA^Phe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes.     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.     

Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multibilayers.

A "membrane bilayer pathway" model, involving ligand partition into the bilayer, lateral diffusion, and receptor binding has been invoked to describe the 1,4-dihydropyridine (DHP) calcium channel antagonist receptor binding mechanism. In an earlier study (Chester et al. 1987. Biophys. J. 52:1021-1030), the diffusional component of this model was examined using an active fluorescence labeled DHP calcium channel antagonist, nisoldipine-lissamine rhodamine B (Ns-R), in purified cardiac sarcolemmal (CSL) lipid multibilayers. Diffusion coefficient measurements on membrane-bound drug and phospholipid at maximum bilayer hydration yielded similar values (3.8 x 10(-8) cm2/s). However, decreases in bilayer hydration resulted in dramatically reduced diffusion coefficient values for both probes with substantially greater impact on Ns-R diffusion. These data suggested that hydration dependent diffusional differences could be a function of relative probe location along the bilayer normal. In this communication, we have addressed the relative effect of the rhodamine substituent on Ns-R diffusion complex by examining the diffusional dynamics of free rhodamine B under the same conditions used to evaluate Ns-R complex and phospholipid diffusion. X-ray diffraction studies were performed to determine the Ns-R location in the membrane and model the CSL lipid bilayer profile structure to give a rationale for the differences in probe diffusional dynamics as a function of interbilayer water space.     

Obstructed metabolite diffusion within skeletal muscle cells in silico

Using a Monte Carlo simulation technique, we have modeled 3D diffusion of low molecular weight metabolites inside a skeletal muscle cell. The following structural elements are considered: (i) a regular lattice of actin and myosin filaments inside a myofibril, (ii) the membranes of sarcoplasmic reticulum and mitochondria surrounding the myofibrils, (iii) a set of myofibrils inside a skeletal muscle cell encircled by the outer cell membrane, and (iv) an additional set of regular intracellular structures ("macrocompartments") embedded into the cell interior. The macrocompartments are considered to simulate diffusion restrictions because of hypothetical cylindrical structures (16-22 μm in diameter) suggested earlier (de Graaf et al. Biophys J 78: 1657-1664, 2000). This model allowed us to calculate the apparent coefficients of particle diffusion in the radial and axial directions, D(app)(⊥) and D(app)(II), respectively. Particle movements in the axial direction are considered, at first approximation, as unrestricted diffusion (D(app)(II) = const). The apparent coefficient of radial diffusion, D(app)(⊥), decreases with time because of particle collisions with myofilaments and other rigid obstacles. Results of our random walk simulations are in fairly good agreement with experimental data on NMR measurements of restricted radial diffusion of phosphocreatine in white and red skeletal muscles of goldfish (Kinsey et al. NMR Biomed 12:1-7, 1999). Particle reflections from the low-permeable borders of macrocompartments (efficient diameter, D(eff)(MC) ≈ 9.2-10.4 μm) are the prerequisite for agreeing theoretical and experimental data. The low-permeable coverage of hypothetical macrocompartments (99.8% of coverage) provides the main contribution to time-dependent decrease in D(app)(⊥).     

Sec35p,a Novel Peripheral Membrane Protein,Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components (Rothman, 1994). The process can be divided into three stages: (a) vesicle budding, (b) vesicle docking, and (c) membrane fusion, with distinct sets of proteins mediating each phase. The budding step involves recruitment of coat proteins to the membrane and culminates with the release of coated vesicles (Schekman and Orci, 1996). The docking reaction is likely to require a set of integral membrane proteins on the vesicle and target membranes, termed v-SNAREs¹ and t-SNAREs (vesicle- and target membrane-soluble N-ethylmaleimide–sensitive fusion protein [NSF] attachment protein [SNAP] receptors, respectively), that are thought to confer specificity through their pair-wise interactions (Söllner et al., 1993b ). Small GTP-binding proteins of the rab family also assist in the docking process (Ferro-Novick and Novick, 1993), but their precise function is not known. The fusion step ensues after docking and results in the delivery of the vesicular cargo to the next compartment in the secretory pathway.Vesicular transport from the ER to the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively characterized. Transport vesicle budding involves the assembly of the COPII coat, composed of the Sec13p/Sec31p (Pryer et al., 1993; Salama et al., 1993; Barlowe et al., 1994) and Sec23p/Sec24p heterodimers (Hicke and Schekman, 1989; Hicke et al., 1992), under the direction of an integral membrane protein, Sec12p (Nakano et al., 1988; Barlowe and Schekman, 1993), a small GTPase, Sar1p (Nakano and Muramatsu, 1989), and a multidomain protein, Sec16p (Espenshade et al., 1995; Shaywitz et al., 1997). Docking is thought to require a tethering event mediated by Uso1p (Cao et al., 1998), the yeast homologue of mammalian p115 (Barroso et al., 1995; Sapperstein et al., 1995), followed by or concurrent with the interaction of a set of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p (Newman and Ferro-Novick, 1987; Newman et al., 1990; Ossig et al., 1991; Shim et al., 1991; Søgaard et al., 1994) and perhaps Ykt6p (Søgaard et al., 1994; McNew et al., 1997), with the cognate t-SNARE on the Golgi, Sed5p (Hardwick and Pelham, 1992). For some time it was thought that fusion may be initiated by disassembly of the v/t-SNARE complex (Söllner et al., 1993a ) by yeast SNAP, Sec17p, (Griff et al., 1992) and NSF, Sec18p (Eakle et al., 1988; Wilson et al., 1989). However, this concept has been challenged by studies with a yeast system that reconstitutes homotypic vacuolar fusion, which suggests the action of Sec18p is before vesicle docking (Mayer et al., 1996; Mayer and Wickner, 1997). In addition, a prefusion role for NSF has been supported by the recent finding that liposomes bearing SNAREs alone can fuse in the absence of NSF (Weber et al., 1998).Several proteins involved in the regulation of yeast ER to Golgi v/t-SNARE complex assembly have been identified, including Ypt1p, Uso1p, and Sly1p. Ypt1p is a member of the rab family of small GTP-binding proteins that have been identified as important components of almost every stage in the secretory pathway (Ferro-Novick and Novick, 1993). Hydrolysis of GTP by rab-like proteins has been hypothesized to provide the regulatory switch that controls the fidelity of vesicular transport (Bourne, 1988). A second protein, Uso1p (Nakajima et al., 1991), appears to function in the same pathway as Ypt1p (Sapperstein et al., 1996), and both proteins have been demonstrated to be essential for SNARE complex assembly (Søgaard et al., 1994; Sapperstein et al., 1996; Lupashin and Waters, 1997). The third protein, Sly1p, is associated with the t-SNARE Sed5p (Søgaard et al., 1994). SLY1 is an essential gene in yeast (Dascher et al., 1991; Ossig et al., 1991), and Sly1p is required for ER to Golgi transport in vitro (Lupashin et al., 1996) and in vivo (Ossig et al., 1991). However, several lines of evidence, particularly from Sly1p homologues in other organisms, indicate that Sly1p may also function as a negative regulator of v/t-SNARE complex assembly, perhaps by preventing the association of the v- and t-SNAREs (Hosono et al., 1992; Pevsner et al., 1994; Schulze et al., 1994). A dominant allele of SLY1, termed SLY1-20, is capable of suppressing mutations in YPT1 and USO1, including complete deletions (Dascher et al., 1991; Sapperstein et al., 1996). Thus, in the presence of Sly1-20p, two components required for SNARE complex assembly are no longer essential. We have proposed a model (Sapperstein et al., 1996; Lupashin and Waters, 1997) in which Ypt1p and Uso1p function to relieve the inhibitory action of Sly1p on SNARE complex assembly. In this model Sly1-20p can be thought of as a noninhibitory form of SLY1 that renders Ypt1p and Uso1p superfluous.We believe that the ability of SLY1-20 to suppress defects in upstream docking regulators can be used to identify additional components involved in the regulation of vesicular docking. We have undertaken a genetic screen (to be presented elsewhere) to isolate novel components in this pathway which, when mutated, depend upon Sly1-20p for viability. In the course of this work, we discovered that two recently identified mutants, sec34 and sec35, can be suppressed by SLY1-20 and thus satisfy the criterion of our screen. These mutants were isolated in a novel screen to identify components involved in transport at any step between the ER and the trans-Golgi network (i.e., the Kex2p compartment) in yeast (Wuestehube et al., 1996). Both sec34 and sec35 accumulate the core-glycosylated form of secretory proteins at the nonpermissive temperature, indicating a block in ER to Golgi transport. Furthermore, electron microscopy indicated that both sec34 and sec35 accumulate numerous vesicles upon shift to the restrictive temperature (Wuestehube et al., 1996), a hallmark of genes whose protein products are involved in the docking or fusion phase of transport (Kaiser and Schekman, 1990). In this report we describe the cloning of SEC35 and analysis of its genetic interactions with other secretory genes. Strong genetic interaction between SEC35 and SLY1, YPT1, and USO1 suggests that Sec35p may function in vesicle docking. To test this possibility, we devised an in vitro transport assay that depends on the addition of purified Sec35p and Uso1p. Vesicles synthesized in the absence of functional Sec35p do not fuse with the Golgi compartment and remain as freely diffusible intermediates. Upon addition of Sec35p and Uso1p, vesicles dock to the Golgi and proceed to membrane fusion. Requirements for Sec35p at the vesicle docking step correlates our genetic experiments with the biochemically distinguishable steps of vesicle docking and membrane fusion.     

Cholesterol's decoupling effect on membrane partitioning and permeability revisited: Is there anything beyond Fick's law of diffusion?

In general, Fick's law of diffusion describes membrane permeation of hydrophobic or amphiphilic molecules. In contrast to this, Thomae et al. recently identified the volume ratio between barrier and aqueous compartments as important additional determinants of membrane permeability (P_m) [A.V. Thomae, T. Koch, C. Panse, H. Wunderli-Allenspach, and S.D. Kramer, Comparing the lipid membrane affinity and permeation of drug-like acids: the intriguing effects of cholesterol and charged lipids, Pharm. Res. 24 (2007) 1457-1472.]. This new theory was supported by the striking observation that low concentrations of cholesterol increased P_m of salicylic acid. As Fick's law is of fundamental importance to all membrane transport processes, we reinvestigated this phenomenon. We measured the electrophoretic mobility of vesicles and used electrochemical scanning microscopy to study the adsorption of the SA anion to lipid vesicular bilayers and SA transport through planar lipid bilayers, respectively. As predicted by Fick's law, P_m of SA decreased continuously with increasing cholesterol content. Thomae et al. made the contrasting artifactual observation because their kinetic approach lacked the required time resolution and led to an underestimation of P_m by five orders of magnitude. We conclude that there is nothing beyond Fick's law of diffusion. It is still valid.     

Interaction of an ion beam with the target in electron microscope applications

When based on the power-potential law of Lindhard et al. (Mat. Fys. Dan. Vid. Selsk, 33: 1–42, 1963) for ionic impact phenomena on the surfaces of a target, the universal curves of nuclear and electronic energy loss-energy, their resulting yield-energy relationships of sputtering and secondary electron emission yield-energy and range-energy have consistently been derived.According to the results obtained from the above experimental data, a diffusion model of an ion beam penetrating a target is proposed, which takes place throughout a hemisphere with its centre located at half the diffusion depth, and which is found to agree well with the empirical data of ion beam penetration, energy-dissipation profiles and the backscattering coefficient as a function of the reduced depth.Owing to the diffusion model's data, the total secondary electron emission yield due to both primary and backscattering ions is obtained. More importantly, radiation damage in ion beam applications is consistently evaluated as a function of the reduced energy ratio.     

Slow Sedimentation and Deformability of Charged Lipid Vesicles

The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity.