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Summary: Background: During endocytosis in yeast, a choreographed series of discrete local events at the plasma membrane lead to a rapid burst of actin polymerization and the subsequent internalization of an endocytic vesicle. What initiates Arp2/3-dependent actin polymerization in this process is not well understood. Results: The Schizosaccharomyces pombe WISH/DIP/SPIN90 ortholog dip1p is an actin-patch protein that regulates the temporal sequence of endocytic events. dip1Δ mutants exhibit a novel phenotype in which early events such as WASp localization occur normally but arrival of Arp2/3, actin polymerization, and subsequent steps are delayed and occur with apparently random timing. In studying this mutant, we demonstrate that positive feedback loops of WASp, rapid actin assembly, and Arp2/3 contribute to switch-like behavior that initiates actin polymerization. In the absence of dip1p, a subset of patches is activated concurrently with the “touch” of a neighboring endocytic vesicle. Conclusions: These studies reveal a switch-like mechanism responsible for the initiation of actin assembly during endocytosis. This switch may be activated in at least two ways, through a dip1p-dependent mechanism and through contact with another endocytic vesicle. [Copyright &y& Elsevier]

Summary: Electric signals surround tissues and cells and have been proposed to participate in directing cell polarity in processes such as development, wound healing, and host invasion []. The application of exogenous electric fields (EFs) can direct cell polarization in cell types ranging from bacteria and fungi to neurons and neutrophils []. The mechanisms by which EFs modulate cell polarity, however, remain poorly understood. Here we introduce the fission yeast Schizosaccharomyces pombe as a model organism to elucidate the mechanisms underlying this process. In these rod-shaped cells, an exogenous EF reorients cell growth in a direction orthogonal to the field, producing cells with a bent morphology. A candidate genetic screen identifies conserved factors involved in this process: an integral membrane proton ATPase pma1p that regulates intracellular pH, the small GTPase cdc42p, and the formin for3p that assembles actin cables. Interestingly, mutants in these genes still respond to the EF but orient in a different direction, toward the anode. In addition, EFs also cause electrophoretic movement of cell wall synthase complex proteins toward the anode. These data suggest molecular models for how the EF reorients cell polarization by modulating intracellular pH and steering cell polarity factors in multiple directions. [Copyright &y& Elsevier]

Summary: For successful cell division, cytokinesis must be properly timed to occur only after the segregation of chromosomes during mitosis. In the fission yeast Schizosaccharomyces pombe, contractile ring assembly initiates at the onset of mitosis, and ring contraction occurs concomitant with septation at the end of anaphase. Although many of the conserved factors necessary for ring assembly and regulation of cytokinesis have been characterized [], still little is known about cell-cycle regulation of events that initiate cytokinesis. The formin cdc12p is an essential ring component with a well-characterized function in F-actin assembly []. Here we show that overexpression of a cdc12p fragment bypasses normal cell-cycle controls and induces contractile ring assembly and sometimes even ring contraction and septation, all during interphase. Activation of cytokinesis occurs without the apparent activation of cell-cycle regulators such as polo kinase or the septation initiation network []. For this effect, cdc12p contributes at least two separable activities: actin assembly and one or more additional functions in cytokinesis initiation. These observations suggest that the formin cdc12p participates downstream of cell-cycle regulators in a network that drives the initiation of cytokinesis. [Copyright &y& Elsevier]

Summary: Background: Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. In the fission yeast Schizosaccharomyces pombe, the formin for3p is necessary for the formation of actin cables, which are bundles of short parallel actin filaments that regulate cell polarity. These filaments are largely organized with their barbed ends facing the cell tip, where for3p is thought to function in their assembly. Results: Here, using a functional for3p-3GFP fusion expressed at endogenous levels, we find that for3p localizes to small dots that appear transiently at cell tips and then move away on actin cables at a rate of 0.3 μm/s. These movements were dependent on the continuous assembly of actin in cables, on the ability of for3p to bind actin within its FH2 domain, and on profilin and bud6p, two formin binding proteins that promote formin activity. Bud6p transiently colocalizes with for3p at the cell tip and stays behind at the cell tip when for3p detaches. Conclusions: These findings suggest a new model for actin cable assembly: a for3p particle is activated and promotes the assembly of a short actin filament at the cell tip for only seconds. For3p and the actin filament may then be released from the cell tip and carried passively into the cell interior by retrograde flow of actin filaments in the cable. These studies reveal a complex and dynamic cycle of formin regulation and actin cable assembly in vivo. [Copyright &y& Elsevier]

Summary: Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking [1, 2]. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells [3]. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips [4]. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains. [Copyright &y& Elsevier]

Investigates how fission yeast divide in the middle. Diagram illustrating cell division in fission yeast; Molecular pathway controlling contractile ring formulation and cell division; Models for how the division site is positioned in fission yeast.

Summary: Cells often grow to a certain cell size before entering mitosis and dividing. Two recent articles suggest that fission yeast cells sense their own size through the action of an intracellular gradient emanating from cell tips and a sensor at the cell middle. [Copyright &y& Elsevier]

Microtubules play a central role in the establishment of cell polarity by directing the transport of polarity determinants to their site of action. Recent work has revealed a novel membrane-anchoring mechanism which complements the microtubule transport of the fission yeast polarity determinant tea1p to ensure its retention at the cell tip. [Copyright &y& Elsevier]

Summary: The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces. [ABSTRACT FROM AUTHOR]

Summary: Mechanical properties contribute to the control of cell size, morphogenesis, development, and lifestyle of fungal cells . Tip growth can be understood by a viscoplastic model, in which growth is derived by high internal turgor pressure and cell-wall elasticity . To understand how these properties regulate growth in the rod-shaped fission yeast Schizosaccaromyces pombe, we devised femtoliter cylindrical polydimethylsiloxane (PDMS) microchambers with varying elasticity as force sensors for single cells. By buckling cells in these chambers, we determine the elastic surface modulus of the cell wall to be 20.2 ± 6.1 N.m−1. By analyzing the growth of the cells as they push against the walls of the chamber, we derive force-velocity relationships and values for internal effective turgor pressure of 0.85 ± 0.15 MPa and a growth-stalling force of 11 ± 3 μN. The behavior of cells buckling under the force of their own growth provides an independent test of this model and parameters. Force generation is dependent on turgor pressure and a glycerol synthesis gene, gpd1 + (glycerol-3-phosphate dehydrogenase), and is independent of actin cables. This study develops a quantitative framework for tip cell growth and characterizes mechanisms of force generation that contribute to fungal invasion into host tissues. [Copyright &y& Elsevier]

Summary: Dynamic properties of microtubules contribute to the establishment of spatial order within cells. In the fission yeast Schizosaccharomyces pombe, interphase cytoplasmic microtubules are organized into antiparallel bundles that attach to the nuclear envelope and are needed to position the nucleus at the geometric center of the cell . Here, we show that after the nucleus is displaced by cell centrifugation, these microtubule bundles efficiently push the nucleus back to the center. Asymmetry in microtubule number, length, and dynamics contributes to the generation of force responsible for this unidirectional movement. Notably, microtubules facing the distal cell tip are destabilized when the microtubules in the same bundle are pushing from the proximal cell tip. The CLIP-170-like protein tip1p and the microtubule-bundling protein ase1p are required for this asymmetric regulation of microtubule dynamics, indicating contributions of factors both at microtubule plus ends and within the microtubule bundle. Mutants in these factors are defective in nuclear movement. Thus, cells possess an efficient microtubule-based engine that produces and senses forces for centering the nucleus. These studies may provide insights into mechanisms of asymmetric microtubule behaviors and force sensing in other processes such as chromosome segregation and cell polarization. [Copyright &y& Elsevier]

Introduction: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe.Results: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles.Conclusions: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast. [Copyright &y& Elsevier]

Summary The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro , it is not yet clear whether these proteins participate in MT nucleation in vivo . Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe , the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo . In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo . We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT. [ABSTRACT FROM AUTHOR]

Summary The shape of walled cells such as fungi, bacteria, and plants are determined by the cell wall. Models for cell morphogenesis postulate that the effects of turgor pressure and mechanical properties of the cell wall can explain the shapes of these diverse cell types [ 1–6 ]. However, in general, these models await validation through quantitative experiments. Fission yeast Schizosaccharomyces pombe are rod-shaped cells that grow by tip extension and then divide medially through formation of a cell wall septum. Upon cell separation after cytokinesis, the new cell ends adopt a rounded morphology. Here, we show that this shape is generated by a very simple mechanical-based mechanism in which turgor pressure inflates the elastic cell wall in the absence of cell growth. This process is independent of actin and new cell wall synthesis. To model this morphological change, we first estimate the mechanical properties of the cell wall using several approaches. The lateral cell wall behaves as an isotropic elastic material with a Young’s modulus of 50 ± 10 MPa inflated by a turgor pressure estimated to be 1.5 ± 0.2 MPa. Based upon these parameters, we develop a quantitative mechanical-based model for new end formation that reveals that the cell wall at the new end expands into its characteristic rounded shape in part because it is softer than the mature lateral wall. These studies provide a simple example of how turgor pressure expands the elastic cell wall to generate a particular cell shape. [ABSTRACT FROM AUTHOR]

Summary: A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2–4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7–9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum. [ABSTRACT FROM AUTHOR]

Summary: Background: Microtubules (MTs) participate in the spatial regulation of actin-based processes such as cytokinesis and cell polarization . The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that exhibits polarized cell growth at cell tips. MT plus ends contact and shrink from the cell tips and contribute to polarity regulation. Results: Here, we investigate the effects of changing cell shape on MTs and cell-polarization machinery. We physically bend fission yeast cells by forcing them into microfabricated femtoliter chambers. In these bent cells, MTs maintain a straight axis and contact and shrink from cortical sites at the sides of cells. At these ectopic sites, polarity factors such as bud6p, for3p (formin), and cdc42p are recruited and assemble actin cables in a MT-dependent manner. MT contact at the cortex induces the appearance of a bud6p dot within seconds. The accumulation of polarity factors leads to cell growth at these sites, when the MT-associated polarity factor tea1p is absent. This process is dependent on MTs, mal3p (EB1), moe1p (an EB1-binding protein), and for3p but, surprisingly, is independent of the tea1p-tea4p pathway. Conclusions: These studies provide a direct demonstration for how MTs induce actin assembly at specific locations on the cell cortex and begin to identify a new pathway involved in this process. MT interactions with the cortex may be regulated by cortical-attachment sites. These findings highlight the crosstalk between cell shape, polarity mechanisms, and MTs responsible for cell morphogenesis. [Copyright &y& Elsevier]

Summary: Intrinsic spatial cues ensure the proper placement of the cell division plane. In the fission yeast Schizosaccharomyces pombe, the position of the nucleus helps to direct the medial positioning of contractile-ring assembly and subsequent cell division . An important factor in this process is mid1p (anillin-like protein), which is a peripheral-membrane protein that forms a broad cortical band of dots overlying the nucleus in interphase and recruits myosin in early mitosis . How mid1p localizes to this cortical band and tracks the nucleus is not clear, especially because its localization is independent of the cytoskeleton . Here, we used a combination of experimental and computational approaches to test mid1p localization mechanisms. We provide evidence that pom1p, a DYRK-family protein kinase that forms a concentration gradient emanating from the nongrowing cell end, inhibits mid1p. In pom1 mutants, mid1p is distributed over half of the cell, covering the nongrowing cell end. This abnormal distribution is established in a dynamic manner in interphase and leads to the formation of misplaced or multiple contractile rings. Our computational and experimental results support a model in which both positive cues from the medial nucleus and negative cues from the cell tips specify the position of the division plane. [Copyright &y& Elsevier]

Summary How cell size is determined and maintained remains unclear, even in simple model organisms. In proliferating cells, cell size is regulated by coordinating growth and division through sizer, adder, or timer mechanisms or through some combination [ 1, 2 ]. Currently, the best-characterized example of sizer behavior is in fission yeast, Schizosaccharomyces pombe , which enters mitosis at a minimal cell size threshold. The peripheral membrane kinase Cdr2 localizes in clusters (nodes) on the medial plasma membrane and promotes mitotic entry [ 3 ]. Here, we show that the Cdr2 nodal density, which scales with cell size, is used by the cell to sense and control its size. By analyzing cells of different widths, we first show that cdr2 + cells divide at a fixed cell surface area. However, division in the cdr2 Δ mutant is more closely specified by cell volume, suggesting that Cdr2 is essential for area sensing and supporting the existence of a Cdr2-independent secondary sizer mechanism more closely based on volume. To investigate how Cdr2 nodes may sense area, we derive a minimal mathematical model that incorporates the cytoplasmic kinase Ssp1 as a Cdr2 activator. The model predicts that a cdr2 mutant in an Ssp1 phosphorylation site (cdr2-T166A) [ 4 ] should form nodes whose density registers cell length. We confirm this prediction experimentally and find that thin cells now follow this new scaling by dividing at constant length instead of area. This work supports the role of Cdr2 as a sizer factor and highlights the importance of studying geometrical aspects of size control. Graphical Abstract Highlights • Cdr2 nodal density mediates surface-area-based size control in fission yeast • Mathematical modeling predicts length scaling for nodal mutant Cdr2-T166A density • Thin cdr2-T166A cells divide using length, demonstrating size control reprogramming • A secondary sizer mechanism based more closely on volume is active in a cdr2 mutant By using cells of different widths, Facchetti et al. show that fission yeast size homeostasis is based on cell surface area as registered by Cdr2 nodal density. A mathematical model allows reprogramming of this Cdr2-based size regulation from area to length. Secondary size control in a cdr2 mutant more closely based on volume is also uncovered. [ABSTRACT FROM AUTHOR]

The cytoplasm is a complex, crowded, actively driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40 nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of individual fission yeast (Schizosaccharomyces pombe) cellscells. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak nonergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, the actin and microtubule cytoskeletons, cell-cyle stage, and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40 nm size scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis or the consequences when this homeostasis is perturbed. Here, we identify conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to general accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of "supergrowth" wherein rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a mechanism for global proteome homeostasis based on modulation of volume growth and dilution., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fundamental mechanisms governing cell size control and homeostasis are still poorly understood. The relationship between sizes at division and birth in single cells is used as a metric to categorize the basis of size homeostasis. Cells dividing at a fixed size regardless of birth size (sizer) are expected to show a division-birth slope of zero, whereas cells dividing after growing for a fixed size increment (adder) have an expected slope of +1. These two theoretical values are, however, rarely experimentally observed. For example, rod-shaped fission yeast Schizosaccharomyces pombe cells, which divide at a fixed surface area, exhibit a division-birth slope for cell lengths of 0.25 ± 0.02, significantly different from the expected sizer value of zero. Here, we investigate possible reasons for this discrepancy by developing a mathematical model of sizer control including the relevant sources of variation. Our results support pure sizer control and show that deviation from zero slope is exaggerated by measurement of an inappropriate geometrical quantity (e.g., length instead of area), combined with cell-to-cell radius variability. The model predicts that mutants with greater errors in size sensing or septum positioning paradoxically appear to behave as better sizers. Furthermore, accounting for cell width variability, we show that pure sizer control can in some circumstances reproduce the apparent adder behavior observed in Escherichia coli. These findings demonstrate that analysis of geometric variation can lead to new insights into cell size control., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chemical gradients can generate pattern formation in biological systems. In the fission yeast Schizosaccharomyces pombe, a cortical gradient of pom1p (a DYRK-type protein kinase) functions to position sites of cytokinesis and cell polarity and to control cell length. Here, using quantitative imaging, fluorescence correlation spectroscopy, and mathematical modeling, we study how its gradient distribution is formed. Pom1p gradients exhibit large cell-to-cell variability, as well as dynamic fluctuations in each individual gradient. Our data lead to a two-state model for gradient formation in which pom1p molecules associate with the plasma membrane at cell tips and then diffuse on the membrane while aggregating into and fragmenting from clusters, before disassociating from the membrane. In contrast to a classical one-component gradient, this two-state gradient buffers against cell-to-cell variations in protein concentration. This buffering mechanism, together with time averaging to reduce intrinsic noise, allows the pom1p gradient to specify positional information in a robust manner., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an alphabeta-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer., (2010 Elsevier Inc. All rights reserved.)

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.

Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside G(M1) and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergent-resistant membranes contain Src and PLCgamma, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to cytokinesis.

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.

Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.