scholarly journals Myosin Vs organize actin cables in fission yeast

2012 ◽  
Vol 23 (23) ◽  
pp. 4579-4591 ◽  
Author(s):  
Libera Lo Presti ◽  
Fred Chang ◽  
Sophie G. Martin
Keyword(s):  
Flexible Structures ◽  
Cell Polarization ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Myosin V ◽  
Actin Cable ◽  
Dense Cytoplasm ◽  
Pulling Forces ◽  
Actin Cables

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.

scholarly journals Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor

2011 ◽  
Vol 22 (21) ◽  
pp. 4016-4028 ◽  
Author(s):  
Brian R. Graziano ◽  
Amy Grace DuPage ◽  
Alphee Michelot ◽  
Dennis Breitsprecher ◽  
James B. Moseley ◽  
...  
Keyword(s):  
Actin Assembly ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
Cable Assembly ◽  
Nucleation Promoting Factor ◽  
Actin Cables ◽  
Elongation Phase

Formins are a conserved family of actin assembly–promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.

scholarly journals Yeast Formins Bni1 and Bnr1 Utilize Different Modes of Cortical Interaction during the Assembly of Actin Cables

2007 ◽  
Vol 18 (5) ◽  
pp. 1826-1838 ◽  
Author(s):  
Shawnna M. Buttery ◽  
Satoshi Yoshida ◽  
David Pellman
Keyword(s):  
Mother Cell ◽  
Fluorescence Recovery After Photobleaching ◽  
Actin Assembly ◽  
Actin Cable ◽  
Bud Neck ◽  
Cable Assembly ◽  
Different Populations ◽  
Actin Cables ◽  
Assembly Activity

The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.

scholarly journals Regulation of a formin complex by the microtubule plus end protein tea1p

2004 ◽  
Vol 165 (5) ◽  
pp. 697-707 ◽  
Author(s):  
Becket Feierbach ◽  
Fulvia Verde ◽  
Fred Chang
Keyword(s):  
Cell Growth ◽  
Cell Polarization ◽  
Actin Assembly ◽  
Triple Mutant ◽  
Spatial Regulation ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
Cable Assembly ◽  
Actin Cables ◽  
Mutant Cells

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large “polarisome” complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end–binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Δbud6Δtea1Δ triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.

scholarly journals The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

2014 ◽  
Vol 205 (3) ◽  
pp. 357-375 ◽  
Author(s):  
Ning Wang ◽  
Libera Lo Presti ◽  
Yi-Hua Zhu ◽  
Minhee Kang ◽  
Zhengrong Wu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Molecular Motors ◽  
Coiled Coil ◽  
Myosin V ◽  
Contractile Ring ◽  
Novel Proteins ◽  
Ring Assembly ◽  
Actin Cables ◽  
Order Complexes

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8+ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

scholarly journals Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

2014 ◽  
Vol 25 (1) ◽  
pp. 66-75 ◽  
Author(s):  
Joseph E. Clayton ◽  
Luther W. Pollard ◽  
Maria Sckolnick ◽  
Carol S. Bookwalter ◽  
Alex R. Hodges ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Myosin V ◽  
Class V ◽  
Physiological Relevance ◽  
Actin Cables

A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.

scholarly journals Aip1 and Cofilin Promote Rapid Turnover of Yeast Actin Patches and Cables: A Coordinated Mechanism for Severing and Capping Filaments

2006 ◽  
Vol 17 (7) ◽  
pp. 2855-2868 ◽  
Author(s):  
Kyoko Okada ◽  
Harini Ravi ◽  
Ellen M. Smith ◽  
Bruce L. Goode
Keyword(s):  
Close Correlation ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Interacting Protein ◽  
Rapid Turnover ◽  
Actin Cable ◽  
Actin Turnover ◽  
Actin Cables

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin–binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to “prune” tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Δ mutants.

scholarly journals Actin-based Motility during Endocytosis in Budding Yeast

2006 ◽  
Vol 17 (3) ◽  
pp. 1354-1363 ◽  
Author(s):  
Kyoungtae Kim ◽  
Brian J. Galletta ◽  
Kevin O. Schmidt ◽  
Fanny S. Chang ◽  
Kendall J. Blumer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Force Production ◽  
Actin Assembly ◽  
Capping Protein ◽  
Actin Cables ◽  
Endocytic Vesicles ◽  
Initial Movement ◽  
Fluorescence Markers

Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility.

scholarly journals Microtubule-independent movement of the fission yeast nucleus

2020 ◽  
Author(s):  
Sanju Ashraf ◽  
David A. Kelly ◽  
Kenneth E. Sawin
Keyword(s):  
Fission Yeast ◽  
Actin Polymerization ◽  
Nuclear Movement ◽  
Myosin V ◽  
Nuclear Positioning ◽  
Membrane Contact Sites ◽  
Growing Cell ◽  
Pulling Forces ◽  
Associated Proteins ◽  
Actin Cables

ABSTRACTMovement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

scholarly journals Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ying Xie ◽  
Jialin Sun ◽  
Xiao Han ◽  
Alma Turšić-Wunder ◽  
Joel D. W. Toh ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Growth ◽  
Actin Polymerization ◽  
Stress Conditions ◽  
Scaffolding Protein ◽  
Actin Assembly ◽  
Intrinsically Disordered ◽  
Actin Cables

Abstract A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation.

scholarly journals Microtubule-independent movement of the fission yeast nucleus

2021 ◽  
pp. jcs.253021
Author(s):  
Sanju Ashraf ◽  
Ye Dee Tay ◽  
David A. Kelly ◽  
Kenneth E. Sawin
Keyword(s):  
Fission Yeast ◽  
Actin Polymerization ◽  
Nuclear Movement ◽  
Myosin V ◽  
Nuclear Positioning ◽  
Membrane Contact Sites ◽  
Growing Cell ◽  
Pulling Forces ◽  
Associated Proteins ◽  
Actin Cables

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

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