The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

The structure of a tetrameric septin complex reveals a hydrophobic element essential for NC-interface integrity.

The septins of the yeast Saccharomyces cerevisiae assemble into hetero-octameric rods by alternating interactions between neighboring G-domains or N- and C-termini, respectively. These rods polymerize end to end into apolar filaments, forming a ring beneath the prospective new bud that expands during the cell cycle into an hourglass structure. The hourglass finally splits during cytokinesis into a double ring. Understanding these transitions as well as the plasticity of the higher order assemblies requires a detailed knowledge of the underlying structures. Here we present the first X-ray crystal structure of a tetrameric Shs1-Cdc12-Cdc3-Cdc10 complex at a resolution of 3.2 Å. Close inspection of the NC-interfaces of this and other septin structures reveals a conserved contact motif that is essential for NC-interface integrity of yeast and human septins in vivo and in vitro. Using the tetrameric structure in combination with AlphaFold-Multimer allowed us to propose a model of the octameric septin rod.

Transient septin sumoylation steers a Fir1-Skt5 protein complex between the split septin ring.

Ubiquitylation and phosphorylation control composition and architecture of the cell separation machinery in yeast and other eukaryotes. The significance of septin sumoylation on cell separation remained an enigma. Septins form an hourglass structure at the bud neck of yeast cells that transforms into a split septin double ring during mitosis. We discovered that sumoylated septins recruit the cytokinesis checkpoint protein Fir1 to the peripheral side of the septin hourglass just before its transformation into the double-ring configuration. As this transition occurs, Fir1 is released from the septins and seamlessly relocates between the split septin rings through synchronized binding to the scaffold Spa2. Fir1 binds and carries the membrane-bound Skt5 on its route to the division plane where the Fir1-Skt5 complex serves as receptor for chitin synthase III.

Biochemical Characterization of a Human Septin Octamer.

Septins are part of the cytoskeleton and polymerize into non-polar filaments of heteromeric hexamers or octamers. They belong to the class of P-loop GTPases but the roles of GTP binding and hydrolysis on filament formation and dynamics are not well understood. The basic human septin building block is the septin rod, a hetero-octamer composed of SEPT2, SEPT6, SEPT7, and SEPT9 with a stoichiometry of 2:2:2:2 (2-6-7-9-9-7-6-2). Septin rods polymerize by end-to-end and lateral joining into linear filaments and higher ordered structures such as rings, sheets, and gauzes. We purified a recombinant human septin octamer from E. coli for in vitro experimentation that is able to polymerize into filaments. We could show that the C-terminal region of the central SEPT9 subunit contributes to filament formation and that the human septin rod decreases the rate of in vitro actin polymerization. We provide further first kinetic data on the nucleotide uptake- and exchange properties of human hexameric and octameric septin rods. We could show that nucleotide uptake prior to hydrolysis is a dynamic process and that a bound nucleotide is exchangeable. However, the hydrolyzed γ-phosphate is not released from the native protein complex. We consequently propose that GTP hydrolysis in human septins does not follow the typical mechanism known from other small GTPases.

Essential role of the endocytic site-associated protein Ecm25 in stress-induced cell elongation.

How cells adopt a different morphology to cope with stress is not well understood. Here, we show that budding yeast Ecm25 associates with polarized endocytic sites and interacts with the polarity regulator Cdc42 and several late-stage endocytic proteins via distinct regions, including an actin filament-binding motif. Deletion of ECM25 does not affect Cdc42 activity or cause any strong defects in fluid-phase and clathrin-mediated endocytosis but completely abolishes hydroxyurea-induced cell elongation. This phenotype is accompanied by depolarization of the spatiotemporally coupled exo-endocytosis in the bud cortex while maintaining the overall mother-bud polarity. These data suggest that Ecm25 provides an essential link between the polarization signal and the endocytic machinery to enable adaptive morphogenesis under stress conditions.

Type V myosin focuses the polarisome and shapes the tip of yeast cells.

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.

A time-resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth.

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle-specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations.

Cdc24 interacts with septins to create a positive feedback loop during bud site assembly in yeast.

Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. During recruitment, septin rods follow the high concentration of Cdc42GTP that is generated by the focused localization of the Cdc42 guanine-nucleotide-exchange factor Cdc24. We show that, shortly before budding, Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 that reduce affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize to form the septin ring, Cdc24 is found at the cortex of the bud and directs further outgrowth from this position.

The cell polarity proteins Boi1 and Boi2 direct an actin nucleation complex to sites of exocytosis in Saccharomyces cerevisiae.

Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane.

YFR016c/Aip5 is part of an actin nucleation complex in yeast.

The polarisome comprises a network of proteins that organizes polar growth in yeast and filamentous fungi. The yeast formin Bni1 and the actin nucleation-promoting factor Bud6 are subunits of the polarisome that together catalyze the formation of actin cables below the tip of yeast cells. We identified YFR016c (Aip5) as an interaction partner of Bud6 and the polarisome scaffold Spa2. Yeast cells lacking Aip5 display a reduced number of actin cables. Aip5 binds with its N-terminal region to Spa2 and with its C-terminal region to Bud6. Both interactions collaborate to localize Aip5 at bud tip and neck, and are required to stimulate the formation of actin cables. Our experiments characterize Aip5 as a novel subunit of a complex that regulates the number of actin filaments at sites of polar growth.

An Interaction Network of the Human SEPT9 Established by Quantitative Mass Spectrometry.

Septins regulate the organization of the actin cytoskeleton, vesicle transport and fusion, chromosome alignment and segregation, and cytokinesis in mammalian cells. SEPT9 is part of the core septin hetero-octamer in human cells which is composed of SEPT2, SEPT6, SEPT7, and SEPT9. SEPT9 has been linked to a variety of intracellular functions as well as to diseases and diverse types of cancer. A targeted high-throughput approach to systematically identify the interaction partners of SEPT9 has not yet been performed. We applied a quantitative proteomics approach to establish an interactome of SEPT9 in human fibroblast cells. Among the newly identified interaction partners were members of the myosin family and LIM domain containing proteins. Fluorescence microscopy of SEPT9 and its interaction partners provides additional evidence that SEPT9 might participate in vesicle transport from and to the plasma membrane as well as in the attachment of actin stress fibers to cellular adhesions.

Dissecting the nucleotide binding properties of the septins from S. cerevisiae.

Septins are a conserved family of guanosine triphosphate (GTP)-binding proteins that assemble into an ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. They are present in all higher eukaryotes except plants. Septins belong structurally to the P-Loop nucleoside triphosphatase (NTPases) like Rab and Ras. However, unlike other small guanosine triphosphatase (GTPases) septins are supposed to act as scaffolds rather than signalling mediators. This is why they are considered as the fourth class of cytoskeletal proteins. It is assumed that septins fulfil their functions independently of the bound nucleotide. The role of guanosine diphosphosphate (GDP) and GTP binding and subsequent hydrolysis was controversial debated in the last couple of years. Lack of crystal structures of yeast septin subunits or rods and difficulties to isolate single monomeric septin subunits often hindered the correlation of results obtained from in vivo studies with biochemical data. Recently, nucleotide binding and hydrolysis was connected to the formation of septin rods from its subunits. However, the evidence was only indirectly obtained through the use of septin mutants in the context of intact cells. We provide here mechanistic insight into the nucleotide binding of the yeast septins by in vitro assays using purified septin rods and building blocks, thereby adding further insights to the already available models on septin filament formation.

The cell polarity proteins Boi1p and Boi2p stimulate vesicle fusion at the plasma membrane of yeast cells.

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These regions are distinguished by an elaborate architecture of proteins and lipids that are specialized to capture and fuse post-Golgi vesicles. Here, we show that the proteins Boi1p and Boi2p are important elements of this area of active exocytosis at the tip of growing yeast cells. Cells lacking Boi1p and Boi2p accumulate secretory vesicles in their buds. The essential PH domains of Boi1p and Boi2p interact with Sec1p, a protein required for SNARE complex formation and vesicle fusion. Sec1p loses its tip localization in cells depleted of Boi1p and Boi2p but overexpression of Sec1p can partially compensate for their loss. The capacity to simultaneously bind phospholipids, Sec1p, multiple subunits of the exocyst, Cdc42p and the module for generating active Cdc42p identify Boi1p and Boi2p as essential mediators between exocytosis and polar growth.

A Split-Ubiquitin Based Strategy Selecting for Protein Complex-Interfering Mutations.

Understanding the topologies and functions of protein interaction networks requires the selective removal of single interactions. We introduce a selection strategy that enriches among a random library of alleles for mutations that impair the binding to a given partner protein. The selection makes use of a split-ubiquitin based protein interaction assay. This assay provides yeast cells that carry protein complex disturbing mutations with the advantage of being able to survive on uracil-lacking media. Applied to the exemplary interaction between the PB domains of the yeast proteins Bem1 and Cdc24, we performed two independent selections. The selections were either analyzed by Sanger sequencing of isolated clones or by next generation sequencing (NGS) of pools of clones. Both screens enriched for the same mutation in position 833 of Cdc24. Biochemical analysis confirmed that this mutation disturbs the interaction with Bem1 but not the fold of the protein. The larger dataset obtained by NGS achieved a more complete representation of the bipartite interaction interface of Cdc24.

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry.

The septins are a conserved family of GTP-binding proteins that, in the baker's yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified. Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.

Crystal structure of Cdc11, a septin subunit from Saccharomyces cerevisiae.

Septins are a conserved family of GTP-binding proteins that assemble into a highly ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. Many molecular functions and mechanisms of the septins in S. cerevisiae were already uncovered. However, structural information is only available from modeling the crystallized subunits of the human septins into the EM cryomicroscopy data of the yeast hetero-octameric septin rod. Octameric rods are the building block of septin filaments in yeast. We present here the first crystal structure of Cdc11, the terminal subunit of the octameric rod and discuss its structure in relation to its human homologues. Size exclusion chromatography analysis revealed that Cdc11 forms homodimers through its C-terminal coiled coil tail.

SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space.

Protein interactions occur at certain times and at specific cellular places. The past years have seen a massive accumulation of binary protein-protein interaction data. The rapid increase of this context-free information necessitates robust methods to monitor protein interactions with temporal and spatial resolution in single cells. We have developed a simple split-ubiquitin-based method (SPLIFF) that uses the ratio of two fluorescent reporters as a signal for protein-protein interactions. One protein of the pair of interest is attached to the linear fusion of mCherry, the C-terminal half of ubiquitin, and GFP (mCherry-Cub-GFP). The other potential binding partner is expressed as a C-terminal fusion to the N-terminal half of ubiquitin (Nub). Upon co-expression the interaction between the two proteins of interest induces the reassociation of Nub and Cub to the native-like ubiquitin. GFP is subsequently cleaved from the C-terminus of Cub and degraded whereas the red-fluorescent mCherry stays attached to the Cub-fusion protein. We first implemented this method in the model yeast Saccharomyces cerevisiae. One fusion protein is expressed in cells of the a-mating type and the complementary fusion protein in cells of the α-mating type. Upon mixing, both cell types fuse and the Nub- and Cub-fusion proteins are free to interact. The red and green fluorescence is monitored by two-channel fluorescence time-lapse microcopy. The moment of cell fusion defines the start of the analysis. The calculated ratio of green to red fluorescence allows mapping the spatiotemporal interaction profiles of the investigated proteins in single cells.

Evaluation of Genetically Encoded Chemical Tags as Orthogonal Fluorophore Labeling Tools for Single-Molecule FRET Applications.

Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations on the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths, and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores.

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip.

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase-activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.