The core spindle pole body scaffold Ppc89 links the pericentrin orthologue Pcp1 to the fission yeast spindle pole body via an evolutionarily conserved interface.

Centrosomes and spindle pole bodies (SPBs) are important for mitotic spindle formation and serve as cellular signaling platforms. Although centrosomes and SPBs differ in morphology, many mechanistic insights into centrosome function have been gleaned from SPB studies. In the fission yeast Schizosaccharomyces pombe, the α-helical protein Ppc89, identified based on its interaction with the septation initiation network scaffold Sid4, comprises the SPB core. High-resolution imaging has suggested that SPB proteins assemble on the Ppc89 core during SPB duplication, but such interactions are undefined. Here, we define a connection between Ppc89 and the essential pericentrin Pcp1. Specifically, we found that a predicted third helix within Ppc89 binds the Pcp1 pericentrin-AKAP450 centrosomal targeting (PACT) domain complexed with calmodulin. Ppc89 helix 3 contains similarity to present in the N-terminus of Cep57 (PINC) motifs found in the centrosomal proteins fly SAS-6 and human Cep57 and also to the S. cerevisiae SPB protein Spc42. These motifs bind pericentrin-calmodulin complexes and AlphaFold2 models suggest a homologous complex assembles in all four organisms. Mutational analysis of the S. pombe complex supports the importance of Ppc89-Pcp1 binding interface in vivo. Our studies provide insight into the core architecture of the S. pombe SPB and suggest an evolutionarily conserved mechanism of scaffolding pericentrin-calmodulin complexes for mitotic spindle formation.

Hybrid assemblies of microbiome Blastocystis protists reveal evolutionary diversification reflecting host ecology.

The most prevalent microbial eukaryote in the human gut is Blastocystis, an obligate commensal protist also common in many other vertebrates. Blastocystis is descended from free-living stramenopile ancestors; how it has adapted to thrive within humans and a wide range of hosts is unclear. Here, we cultivated six Blastocystis strains spanning the diversity of the genus and generated highly contiguous, annotated genomes with long-read DNA-seq, Hi-C, and RNA-seq. Comparative genomics between these strains and two closely related stramenopiles with different lifestyles, the lizard gut symbiont Proteromonas lacertae and the free-living marine flagellate Cafeteria burkhardae, reveal the evolutionary history of the Blastocystis genus. We find substantial gene content variability between Blastocystis strains. Blastocystis isolated from an herbivorous tortoise has many plant carbohydrate metabolizing enzymes, some horizontally acquired from bacteria, likely reflecting fermentation within the host gut. In contrast, human-isolated Blastocystis have gained many heat shock proteins, and we find numerous subtype-specific expansions of host-interfacing genes, including cell adhesion and cell surface glycan genes. In addition, we observe that human-isolated Blastocystis have substantial changes in gene structure, including shortened introns and intergenic regions, as well as genes lacking canonical termination codons. Finally, our data indicate that the common ancestor of Blastocystis lost nearly all ancestral genes for heterokont flagella morphology, including cilia proteins, microtubule motor proteins, and ion channel proteins. Together, these findings underscore the huge functional variability within the Blastocystis genus and provide candidate genes for the adaptations these lineages have undergone to thrive in the gut microbiomes of diverse vertebrates.

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition.

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through liquid-liquid phase separation. Here, we find that the phase separation of Caenorhabditis elegans SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation. We identify the SAD-1 kinase as a regulator of SYD-2 phase separation and determine presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. Using phosphoproteomics, we find SAD-1 phosphorylates SYD-2 on 3 sites that are critical to activate phase separation. Mechanistically, SAD-1 phosphorylation relieves a binding interaction between 2 folded domains in SYD-2 that inhibits phase separation by an intrinsically disordered region (IDR). We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, activating its phase separation and active zone assembly.

SAD-1 kinase controls presynaptic phase separation by relieving SYD-2/Liprin-α autoinhibition.

Neuronal development orchestrates the formation of an enormous number of synapses that connect the nervous system. In developing presynapses, the core active zone structure has been found to assemble through a liquid-liquid phase separation. Here, we find that the phase separation of SYD-2/Liprin-α, a key active zone scaffold, is controlled by phosphorylation. Using phosphoproteomics, we identify the SAD-1 kinase to phosphorylate SYD-2 and a number of other substrates. Presynaptic assembly is impaired in sad-1 mutants and increased by overactivation of SAD-1. We determine SAD-1 phosphorylation of SYD-2 at three sites is critical to activate its phase separation. Mechanistically, phosphorylation relieves a binding interaction between two folded SYD-2 domains that inhibits phase separation by an intrinsically disordered region. We find synaptic cell adhesion molecules localize SAD-1 to nascent synapses upstream of active zone formation. We conclude that SAD-1 phosphorylates SYD-2 at developing synapses, enabling its phase separation and active zone assembly.

Finding functions of phase separation in the presynapse.

Synapses are the basic units of neuronal communication. Understanding how synapses assemble and function is therefore essential to understanding nervous systems. Decades of study have identified many molecular components and functional mechanisms of synapses. Recently, an additional level of synaptic protein organization has been identified: phase separation. In the presynapse, components of the central active zone and a synaptic vesicle-clustering factor have been shown to form liquid-liquid phase-separated condensates or hydrogels. New in vivo functional studies have directly tested how phase separation impacts both synapse formation and function. Here, we review this emerging evidence for in vivo functional roles of phase separation at the presynapse and discuss future functional studies necessary to understand its complexity.

Opposite Surfaces of the Cdc15 F-BAR Domain Create a Membrane Platform That Coordinates Cytoskeletal and Signaling Components for Cytokinesis.

Many eukaryotes assemble an actin- and myosin-based cytokinetic ring (CR) on the plasma membrane (PM) for cell division, but how it is anchored there remains unclear. In Schizosaccharomyces pombe, the F-BAR protein Cdc15 links the PM via its F-BAR domain to proteins in the CR's interior via its SH3 domain. However, Cdc15's F-BAR domain also directly binds formin Cdc12, suggesting that Cdc15 may polymerize a protein network directly adjacent to the membrane. Here, we determine that the F-BAR domain binds Cdc12 using residues on the face opposite its membrane-binding surface. These residues also bind paxillin-like Pxl1, promoting its recruitment with calcineurin to the CR. Mutation of these F-BAR domain residues results in a shallower CR, with components localizing ∼35% closer to the PM than in wild type, and aberrant CR constriction. Thus, F-BAR domains serve as oligomeric membrane-bound platforms that can modulate the architecture of an entire actin structure.

Assembly of synaptic active zones requires phase separation of scaffold molecules.

The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system1. Every presynapse builds a core 'active zone' structure, where ion channels cluster and synaptic vesicles release their neurotransmitters2. Although the composition of active zones is well characterized2,3, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-α) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.

DYRK kinase Pom1 drives F-BAR protein Cdc15 from the membrane to promote medial division.

In many organisms, positive and negative signals cooperate to position the division site for cytokinesis. In the rod-shaped fission yeast Schizosaccharomyces pombe, symmetric division is achieved through anillin/Mid1-dependent positive cues released from the central nucleus and negative signals from the DYRK-family polarity kinase Pom1 at cell tips. Here we establish that Pom1's kinase activity prevents septation at cell tips even if Mid1 is absent or mislocalized. We also find that Pom1 phosphorylation of F-BAR protein Cdc15, a major scaffold of the division apparatus, disrupts Cdc15's ability to bind membranes and paxillin, Pxl1, thereby inhibiting Cdc15's function in cytokinesis. A Cdc15 mutant carrying phosphomimetic versions of Pom1 sites or deletion of Cdc15 binding partners suppresses division at cell tips in cells lacking both Mid1 and Pom1 signals. Thus, inhibition of Cdc15-scaffolded septum formation at cell poles is a key Pom1 mechanism that ensures medial division.

The F-BAR Domain of Rga7 Relies on a Cooperative Mechanism of Membrane Binding with a Partner Protein during Fission Yeast Cytokinesis.

F-BAR proteins bind the plasma membrane (PM) to scaffold and organize the actin cytoskeleton. To understand how F-BAR proteins achieve their PM association, we studied the localization of a Schizosaccharomyces pombe F-BAR protein Rga7, which requires the coiled-coil protein Rng10 for targeting to the division site during cytokinesis. We find that the Rga7 F-BAR domain directly binds a motif in Rng10 simultaneously with the PM, and that an adjacent Rng10 motif independently binds the PM. Together, these multivalent interactions significantly enhance Rga7 F-BAR avidity for membranes at physiological protein concentrations, ensuring the division site localization of Rga7. Moreover, the requirement for the F-BAR domain in Rga7 localization and function in cytokinesis is bypassed by tethering an Rga7 construct lacking its F-BAR to Rng10, indicating that at least some F-BAR domains are necessary but not sufficient for PM targeting and are stably localized to specific cortical positions through adaptor proteins.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring.

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Linking up at the BAR: Oligomerization and F-BAR protein function.

As cells grow, move, and divide, they must reorganize and rearrange their membranes and cytoskeleton. The F-BAR protein family links cellular membranes with actin cytoskeletal rearrangements in processes including endocytosis, cytokinesis, and cell motility. Here we review emerging information on mechanisms of F-BAR domain oligomerization and membrane binding, and how these activities are coordinated with additional domains to accomplish scaffolding and signaling functions.

Structural organization of membrane-inserted hexamers formed by Helicobacter pylori VacA toxin.

Helicobacter pylori colonizes the human stomach and is a potential cause of peptic ulceration or gastric adenocarcinoma. H. pylori secretes a pore-forming toxin known as vacuolating cytotoxin A (VacA). The 88 kDa secreted VacA protein, composed of an N-terminal p33 domain and a C-terminal p55 domain, assembles into water-soluble oligomers. The structural organization of membrane-bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. We show that membrane-bound VacA organizes into hexameric oligomers. Comparison of the two-dimensional averages of membrane-bound and soluble VacA hexamers generated using single particle electron microscopy reveals a structural difference in the central region of the oligomers (corresponding to the p33 domain), suggesting that membrane association triggers a structural change in the p33 domain. Analyses of the isolated p55 domain and VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.

The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis.

F-BAR proteins link cellular membranes to the actin cytoskeleton in many biological processes. Here we investigated the function of the Schizosaccharomyces pombe Imp2 F-BAR domain in cytokinesis and find that it is critical for Imp2's role in contractile ring constriction and disassembly. To understand mechanistically how the F-BAR domain functions, we determined its structure, elucidated how it interacts with membranes, and identified an interaction between dimers that allows helical oligomerization and membrane tubulation. Using mutations that block either membrane binding or tubulation, we find that membrane binding is required for Imp2's cytokinetic function but that oligomerization and tubulation, activities often deemed central to F-BAR protein function, are dispensable. Accordingly, F-BARs that do not have the capacity to tubulate membranes functionally substitute for the Imp2 F-BAR, establishing that its major role is as a cell-cycle-regulated bridge between the membrane and Imp2 protein partners, rather than as a driver of membrane curvature.

Characterization of Cytokinetic F-BARs and Other Membrane-Binding Proteins.

Multiple membrane-binding proteins are key players in cytokinesis in yeast and other organisms. In vivo techniques for analyzing protein-membrane interactions are currently limited. In vitro assays allow characterization of the biochemical properties of these proteins to build a mechanistic understanding of protein-membrane interactions during cytokinesis. Here, we describe two in vitro assays to characterize FCH-Bin/Amphyphysin/RVS (F-BAR) domains and other protein's interactions with membranes: liposome co-pelleting and giant unilamellar vesicle fluorescent binding.

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis.

F-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains-including those from Fer and RhoGAP4-share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending.

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles.

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.

Regulation of contractile ring formation and septation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has become a powerful model organism for cytokinesis studies, propelled by pioneering genetic screens in the 1980s and 1990s. S. pombe cells are rod-shaped and divide similarly to mammalian cells, utilizing a medially-placed actin-and myosin-based contractile ring. A cell wall division septum is deposited behind the constricting ring, forming the new ends of each daughter cell. Here we discuss recent advances in our understanding of the regulation of contractile ring formation through formin proteins and the role of the division septum in S. pombe cell division.