Biology subjects

Martin, S. G.

Publications and source records attributed to Martin, S. G..

At least 19 recordsLinked to original sources

bioRxiv2026

PAK-family kinases promote cell fusion irreversibility by preventing cell wall repair

Cell-cell fusion is critical for the sexual life cycle, as it drives the unidirectional transition between haploid and diploid phases. In fungi, fusion requires not only plasma membrane merging but also local cell wall removal. However, the integrity of the cell wall, which resists internal turgor pressure and is essential for survival, is normally monitored by a cell wall integrity pathway (CIP) involving Rho1 GTPase, protein kinase C (PKC) and MAPK signaling, which promotes damage repair. How the CIP allows localized cell wall degradation is not known. In the fission yeast Schizosaccharomyces pombe, we previously identified pak2{Delta} mutants that exhibit transient fusion: cells briefly fuse and exchange cytoplasmic contents, which signal catastrophic haploid meiosis, but then reseal their fusion pore. Here, we show that PAK-family kinase activity is essential for cell-cell fusion, with major and minor contribution for Pak2 and the essential Pak1, respectively. Strikingly, osmotic stabilization largely suppresses all PAK-associated fusion defects, pointing to a role in cell-wall remodeling. Pak2 accumulates at the fusion site, where it promotes fusion pore opening and expansion. In its absence, Rho1-GTP, PKC and beta-glucan synthase Bgs4 show elevated levels at the fusion site, and their partial loss-of-function rescues fusion. Pak1 mutants have elevated MAPK activity. Thus, Pak2 and Pak1 both antagonize the CIP. Furthermore, correlative light and cryo-soft-X-ray tomography reveals that resealed pores in PAK mutants rebuilt an intact cell wall. We conclude that PAKs enforce fusion directionality by antagonizing cell-wall repair mechanisms that otherwise restore separation between mating partners.

cell biology↗

bioRxiv2026

Love-thy-neighbor: Neural networks for tracking and lineage tracing in budding yeast

Tracking and lineage tracing are widely needed tasks in biological image analysis. For cells that grow and divide, tracking is challenging because cells change in number, shape, and size throughout a recording. As the time interval between images increases, it becomes more difficult to establish correspondences between cells across timepoints. Consequently, tracking has to be performed between consecutive or temporally close images, which leads to exponentially decreasing tracking accuracy and thus high sensitivity to error rates. For budding yeast, this challenge is further heightened by the similarity of cells in colonies, their dense packing, the asymmetric nature of cell divisions, and movement due to growth of the colony. A related task, lineage tracing, is similarly challenging without fluorescent markers due to multiple potential mother cells surrounding a new daughter cell. Here, we present neural networks for budding yeast tracking and lineage tracing, named LYN-track and LYN-trace, respectively. These methods leverage fine geometric features of cells and their neighborhoods. To train and test the algorithms, we recorded and annotated new budding and fission yeast microscopy movies (78,852 frame-to-frame tracklets, 2,512 images), which we make freely available. On these and existing datasets, our neural network-based methods demonstrate robust, above state-of-the-art performance. Both tools have been integrated into graphical user interfaces (GUIs), available on Github, and can be straightforwardly retrained with custom data if desired.

bioinformatics↗

bioRxiv2025

Genomic and Epigenetic Interplay Drives Adaptive Fusion via Reproduction Trade-Off

Investment in sexual reproduction has long been seen as a fitness cost for asexual proliferation, raising the question of how cells evolve to accommodate cyclical demands of sexual and asexual cycles. We addressed this question by evolving Schizosaccharomyces pombe lacking the critical fusion gene Prm1 through 18 cycles of alternating meiosis and mitosis. Initially, transcriptional tweaks offered a fragile means to fuse despite Prm1s absence. However, a telomeric 1% genome deletion that epigenetically silenced Clr5 ultimately locked in a latent, Prm1-independent fusion pathway involving the mating regulator Ste11 and the pheromone-MAPK scaffold Sms1. This adaptation, though efficient at restoring sexual reproduction, came at the cost of mitotic growth. By contrast, rationally constructing mutations in a "synthetic evolution" approach bypassed the trade-off, enabling simultaneous high fusion efficiency and near-wild-type proliferation. Our findings illustrate how epigenetic de-repression and genetic assimilation converge to create innovative solutions under cyclical selection pressures with trade-offs.

evolutionary biology↗

bioRxiv2025

Negative feedback regulation of the hemi-arrestin MAPK scaffold Sms1 prevents untimely mating

Mitogen-activated protein kinases (MAPK) are ancestral kinases that form essential signalling cascades. However, scaffolds that recruit kinases to subcellular locations and promote signal transduction have only been described in few species. Notably, no scaffold was thought necessary for the MAPK cascade promoting sexual differentiation in fission yeast. Here, we identify the hemi-arrestin protein Sms1 as a structurally novel scaffold of this MAPK cascade. Interactions with PIP2 and the pheromone receptor-coupled G subunit target Sms1 to plasma membrane patches, where it assembles the active cascade by binding each MAP kinase. These interactions are essential for signal transduction and local signal interpretation for polarised growth. Phosphorylation, including by the MAPK itself, antagonises Sms1 membrane translocation, establishing a negative feedback that underlies polarity patch turnover and prevents untimely mating attempts. Thus, Sms1 is a MAPK scaffold with canonical functions despite its distinct structural fold, highlighting convergent evolution of MAPK scaffolds across eukaryotes.

cell biology↗

bioRxiv2025

Spatial control of secretory vesicle targeting by the Ync13-Rga7-Rng10 complex during cytokinesis

Cytokinesis requires precise coordination of contractile-ring constriction, vesicle trafficking and fusion to the plasma membrane, and extracellular matrix assembly/remodeling at the cleavage furrow to ensure faithful cell division and maintain cell integrity. These processes and proteins involved are broadly conserved across eukaryotes, yet molecular mechanisms controlling the spatiotemporal pathways of membrane trafficking remain poorly understood. Here, using fission yeast genetics, microscopy, and in vitro binding assays, we identify a conserved module including the Munc13 protein Ync13, F-BAR protein Rga7, and coiled-coil protein Rng10 to be critical for precise and selective vesicle targeting during cytokinesis. The module specifically recruit the TRAPP-II but not exocyst complex to tether vesicles containing the glucan synthases Bgs4 and Ags1 along the cleavage furrow. Ync13 subsequently interacts with the SM protein Sec1 for vesicle fusion. Mutations in this pathway disrupt septum integrity and lead to cell lysis. Our work provides key insights into how membrane trafficking is tightly controlled to maintain cell integrity during cytokinesis.

cell biology↗

bioRxiv2025

High levels of Cdc42 GTPase underlie an all-or-none decision to fuse

Cdc42 is a Rho-family GTPase conserved across eukaryotes, where it plays essential roles in cell polarization. In single-celled yeast systems, Cdc42 is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit eRectors to organize the cytoskeleton and polarize secretion. Here we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae. Notably, the cdc42-mCherrySW allele (but not the cdc42-sfGFPSW allele), which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherrySW and demonstrate that cell fusion requires higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that polarized growth responds linearly to Cdc42 protein levels, whereas sexual reproduction exhibits a sharp switch-like response. Thus, the topology of the Cdc42 regulatory network is distinct for its polarization and mating functions.

cell biology↗

bioRxiv2025

High resolution mapping of the actin fusion focus reveals myosin V-dependent transport of formin for actin aster compaction

Many cellular processes such as polarized growth and secretion require the formation of specific actin networks. In fungi, the morphogenetic process of cell-cell fusion requires cell wall digestion mediated by the local secretion of lytic enzymes at the site of cell-cell contact. In Schizosaccharomyces pombe, lytic enzyme-containing secretory vesicles are transported by the myosin V Myo52 on the actin fusion focus, an aster-like actin network assembled by the condensate-forming formin Fus1. The fusion focus also concentrates proteins regulating cell polarity, cell-cell communication, actin cytoskeleton, exocytosis and membrane merging, but their precise position from the time of focus formation to cell fusion is unknown. Here, using centroid tracking and averaging, we present a first spatiotemporal map of the fusion site with a precision of 8 nm. We show that the bulk of secretory vesicles remains at constant distance from the plasma membrane as the actin structure condenses. Notably, though necessary to transport vesicles, Myo52 detaches from the vesicle pool and colocalizes with Fus1 in a more membrane-proximal position. We show that Myo52 physically interacts with Fus1 and transports it along actin filaments, and that Myo52 and Fus1 actin assembly activity contribute to focus compaction. Thus, myosin V-driven transport of the formin Fus1 along actin filaments nucleated by other Fus1 molecules underlies a positive feedback mechanism for actin aster formation.

cell biology↗

bioRxiv2024

TORC1 reactivation by pheromone signaling revealed by phosphoproteomics of fission yeast sexual reproduction

Starvation, which is associated with inactivation of the growth-promoting TOR complex 1 (TORC1), is a strong environmental signal for cell differentiation. In the fission yeast Schizosaccharomyces pombe, nitrogen starvation has distinct physiological consequences depending on the presence of mating partners. In their absence, cells enter quiescence, and TORC1 inactivation prolongs their life. In presence of compatible mates, TORC1 inactivation is essential for sexual differentiation. Gametes engage in paracrine pheromone signaling, grow towards each other, fuse to form the diploid zygote, and form resistant, haploid spore progenies. To understand the signaling changes in the proteome and phospho-proteome during sexual reproduction, we developed cell synchronization strategies and present (phospho-)proteomic datasets that dissect pheromone from starvation signals over the sexual differentiation and cell-cell fusion processes. Unexpectedly, these datasets reveal phosphorylation of ribosomal protein S6 during sexual development, which we establish requires TORC1 activity. We demonstrate that TORC1 is re-activated by pheromone signaling, in a manner that does not require autophagy. Mutants with low TORC1 re-activation exhibit compromised mating and poorly viable spores. Thus, while inactivated to initiate the mating process, TORC1 is reactivated by pheromone signaling in starved cells to support sexual reproduction.

cell biology↗

bioRxiv2024

Arp2/3-dependent actin assembly shapes endosomes and promotes intracellular trafficking in fission yeast

Endosomes serve as crucial sorting centres that streamline the distribution of cell surface proteins. The early endosome receives traffic from both the plasma membrane (PM) and the Golgi, and orchestrates the redistribution of cargoes for recycling to the PM or through retrograde movement to the Golgi, and for degradation to late endosomes and lysosomes 1. In animal cells and amoebae, Arp2/3-mediated F-actin assembly plays critical roles in many aspects of endosome function, from capturing cargo to generating the forces required for membrane deformation and creating cargo-enriched transport carriers, thus promoting both recycling and degradative trafficking routes 2. Yeast models, which allowed dissection of the major membrane trafficking routes 3, exhibit highly simplified endosomes, as shown in Saccharomyces cerevisiae, where the trans-Golgi network (TGN) functions as recycling endosome 4. Furthermore, there is no reported role for Arp2/3 or F-actin in endomembrane remodelling; in fact, the Arp2/3 activators on animal endosomes - the WASH complex promoting recycling of transmembrane receptors towards the PM or TGN 5,6, and Annexin A2 necessary for endosome maturation and the movement of cargoes towards the degradative pathway 7 - do not exist in yeast cells 8,9. Here, we report that Arp2/3 and F-actin are present on fission yeast endosomes. Through live-imaging and correlative light-electron tomography, we demonstrate that Arp2/3 activity controls endosomal morphology and that this activity is essential for trafficking from the endosome to the degradative vacuole. Thus, Arp2/3-dependent actin assembly has a deeply conserved role in shaping and promoting the function of the endomembrane trafficking system.

cell biology↗

bioRxiv2023

Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival

Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeast Schizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation. Significance StatementThe delivery of new membrane from internal pools at zones of polarized secretion induces in-plane plasma membrane flows that displace slowly mobile membrane-associated proteins from the zone of secretion. However, zones of polarized secretion are themselves specified by the activity of membrane-associated polarity factors, such as the small GTPase Cdc42. Through combined modelling and experimental approaches, this work demonstrates that the fast mobility of the Cdc42 GTPase is critical to allow the establishment and maintenance of a polarity patch, which is reinforced by flow-mediated displacement of a negative regulator.

cell biology↗

bioRxiv2022

mNeonGreen-tagged fusion proteins and nanobodies reveal localization of tropomyosin to patches, cables, and contractile actomyosin rings in live yeast cells

Tropomyosins are structurally conserved -helical coiled-coil dimeric proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments in non-muscle cells and are essential for calcium regulation of myosin II contractility in the muscle. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live cell imaging tools currently available in many organisms. Here, we report mNeon-Green (mNG) tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin localization and dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8), and Saccharomyces cerevisiae (Tpm1 and Tpm2), in vivo and in isolated S. pombe cell division apparatuses. We extended this approach to also visualize the mammalian TPM2 isoform. Finally, we generated a camelid-nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo without significantly influencing cell growth and dynamics of actin cytoskeleton. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring, and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and actin cables in vivo.

cell biology↗

bioRxiv2022

Condensation of the fusion focus by the intrinsically disordered region of the formin Fus1 is essential for cell-cell fusion

Spatial accumulation of secretory vesicles underlies various cellular processes, such as neurotransmitter release at neuronal synapses [1], hyphal steering in filamentous fungi [2, 3], and local cell wall digestion preceding the fusion of yeast gametes [4]. Secretory vesicles transported on actin filaments by myosin V motors form clusters that serve as pool for local content release. During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes [5-7]. Focus position and coalescence are controlled by local signalling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis [6, 8-10], but the mechanisms of focusing have been elusive. Here, we show that the regulatory N-terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude other macromolecules. Fus1 lacking its IDR fails to condense in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, replacement of Fus1 IDR with a heterologous low-complexity region that forms liquid condensates fully restores Fus1 condensation and function. By contrast, replacement of Fus1 IDR with a domain that forms more stable oligomers restores condensation but poorly supports cell fusion, suggesting that condensation is tuned to yield a structure selectively permeable for secretory vesicles. We propose that condensation of actin structures by an intrinsically disordered region may be a general mechanism for actin network organisation and the selective local concentration of secretory vesicles.

cell biology↗

bioRxiv2022

The actin assembly requirements of the formin Fus1 to build the fusion focus

Formins form the largest family of actin filament nucleators and elongators, involved in the assembly of diverse actin structures. Actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, common to all formins. However, the rate of these reactions varies between formins by at least 20-fold. Typically, each cell expresses several distinct formins, each contributing to the assembly of one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in the fission yeast Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates for function in vivo. Fus1 assembles the actin fusion focus, an aster-like structure of actin filaments at the contact site between gametes, necessary for the process of cell fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on the architecture of the fusion focus, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in the Fus1 FH2 domain that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional property of the FH2 domain. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

cell biology↗

bioRxiv2021

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo-to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P-than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

cell biology↗

bioRxiv2020

Cell patterning by secretion-induced plasma membrane flows

Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity. One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

cell biology↗

bioRxiv2020

Fungal zygote survival and ploidy maintenance rely on cell cycle-dependent and independent mating blocks

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly two haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins post-fusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least two mechanisms where the zygotic fate imposed by Mei2 and the cell cycle re-entry triggered by Mei3 synergize to prevent zygotic mating.

cell biology↗

bioRxiv2020

Direct and indirect regulation of Pom1 cell size pathway by the protein phosphatase 2C Ptc1

The fission yeast cells Schizosaccharomyces pombe divide at constant cell size regulated by environmental stimuli. An important pathway of cell size control involves the membrane-associated DYRK-family kinase Pom1, which forms decreasing concentration gradients from cell poles and inhibits mitotic inducers at mid-cell. Here, we identify the phosphatase 2C Ptc1 as negative regulator of Pom1. Ptc1 localizes to cell poles in a manner dependent on polarity and cell-wall integrity factors. We show that Ptc1 directly binds Pom1 and can dephosphorylate it in vitro but modulates Pom1 localization indirectly upon growth in low glucose conditions by influencing microtubule stability. Thus, Ptc1 phosphatase plays both direct and indirect roles in the Pom1 cell size control pathway.

cell biology↗

bioRxiv2020

Clustering-mediated activation of Cdc42 GTPase antagonized by GAPs in fission yeast

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2 and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 allele at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, activation of CRY2-Cdc42 does not individually depend on Scd1 or the GEF Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4 on cell sides. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

cell biology↗