scholarly journals Identification of Two Type V Myosins in Fission Yeast, One of Which Functions in Polarized Cell Growth and Moves Rapidly in the Cell

2001 ◽  
Vol 12 (5) ◽  
pp. 1367-1380 ◽  
Author(s):  
Fumio Motegi ◽  
Ritsuko Arai ◽  
Issei Mabuchi
Keyword(s):  
Cell Growth ◽  
Motor Activity ◽  
Fluorescent Protein ◽  
Secretory Vesicle ◽  
Cell Polarization ◽  
Wild Type ◽  
Myosin V ◽  
Growing Cell ◽  
Polarized Cell Growth ◽  
Actin Cables

We characterized the novel Schizosaccharomyces pombegenes myo4+andmyo5+, both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

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 Cooh-Terminal Domain of Myo2p, a Yeast Myosin V, Has a Direct Role in Secretory Vesicle Targeting

1999 ◽  
Vol 147 (4) ◽  
pp. 791-808 ◽  
Author(s):  
Daniel Schott ◽  
Jackson Ho ◽  
David Pruyne ◽  
Anthony Bretscher
Keyword(s):  
Secretory Vesicle ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Tail Domain ◽  
Myosin V ◽  
Direct Role ◽  
Rab Protein ◽  
Polarized Delivery ◽  
Actin Cables ◽  
Type V

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

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 Transport of Myosin II to the Equatorial Region without Its Own Motor Activity in Mitotic Dictyostelium Cells

1997 ◽  
Vol 8 (10) ◽  
pp. 2089-2099 ◽  
Author(s):  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Green Fluorescent Protein ◽  
Motor Activity ◽  
Transport Process ◽  
Time Course ◽  
Fluorescent Protein ◽  
Myosin Ii ◽  
Equatorial Region ◽  
Wild Type ◽  
Contractile Ring ◽  
Green Fluorescent

Fluorescently labeled myosin moved and accumulated circumferentially in the equatorial region of dividingDictyostelium cells within a time course of 4 min, followed by contraction of the contractile ring. To investigate the mechanism of this transport process, we have expressed three mutant myosins that cannot hydrolyze ATP in myosin null cells. Immunofluorescence staining showed that these mutant myosins were also correctly transported to the equatorial region, although no contraction followed. The rates of transport, measured using green fluorescent protein-fused myosins, were indistinguishable between wild-type and mutant myosins. These observations demonstrate that myosin is passively transported toward the equatorial region and incorporated into the forming contractile ring without its own motor activity.

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.

scholarly journals Regulation of the Formin for3p by cdc42p and bud6p

2007 ◽  
Vol 18 (10) ◽  
pp. 4155-4167 ◽  
Author(s):  
Sophie G. Martin ◽  
Sergio A. Rincón ◽  
Roshni Basu ◽  
Pilar Pérez ◽  
Fred Chang
Keyword(s):  
Cell Growth ◽  
Fission Yeast ◽  
Rho Gtpase ◽  
Intramolecular Interaction ◽  
Actin Cable ◽  
Polarized Cell Growth ◽  
N Terminus ◽  
Cable Assembly ◽  
Actin Cables

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Δ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition.

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.

Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis

2010 ◽  
Vol 429 (3) ◽  
pp. 497-504 ◽  
Author(s):  
Ryo Aoki ◽  
Tetsuya Kitaguchi ◽  
Manami Oya ◽  
Yu Yanagihara ◽  
Mai Sato ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Release Kinetics ◽  
Myosin Ii ◽  
Ph Sensor ◽  
Secretory Vesicle ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Wild Type ◽  
Pore Opening ◽  
Green Fluorescent

Since the fusion pore of the secretory vesicle is resealed before complete dilation during ‘kiss-and-run’ exocytosis, their cargoes are not completely released. Although the transient fusion pore is kept open for several seconds, the precise mechanisms that control fusion pore maintenance, and their physiological significance, are not well understood. Using dual-colour TIRF (total internal reflection fluorescence) microscopy in neuroendocrine PC12 cells, we show that myosin II regulates the fusion pore dynamics during kiss-and-run exocytosis. The release kinetics of mRFP (monomeric red fluorescent protein)-tagged tPA (tissue plasminogen activator) and Venus-tagged BDNF (brain-derived neurotrophic factor), which show slower release kinetics than NPY (neuropeptide Y)–mRFP and insulin–mRFP, were prolonged by the overexpression of a wild-type form of the RLC (myosin II regulatory light chain). In contrast, overexpression of a dominant-negative form of RLC shortened the release kinetics. Using spH (synapto-pHluorin), a green fluorescent protein-based pH sensor inside the vesicles, we confirmed that the modulation of the release kinetics by myosin II is due to changes in the duration of fusion pore opening. In addition, we revealed that the amount of hormone released into the extracellular space upon stimulation was increased by overexpression of wild-type RLC. We propose that the duration of fusion pore opening is regulated by myosin II to control the amount of hormone released from a single vesicle.

scholarly journals Schizosaccharomyces pombe Pmr1p Is Essential for Cell Wall Integrity and Is Required for Polarized Cell Growth and Cytokinesis

2004 ◽  
Vol 3 (5) ◽  
pp. 1124-1135 ◽  
Author(s):  
Juan Carlos G. Cortés ◽  
Reiko Katoh-Fukui ◽  
Kanako Moto ◽  
Juan Carlos Ribas ◽  
Junpei Ishiguro
Keyword(s):  
Cell Wall ◽  
Cell Growth ◽  
Fluorescent Protein ◽  
Mutant Cell ◽  
Optimal Growth ◽  
Single Copy ◽  
Protein Glycosylation ◽  
Cell Wall Integrity ◽  
Free Medium ◽  
Wild Type

ABSTRACT The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28°C in minimal medium and a lethal thermosensitive phenotype at 37°C. Cell growth is completely inhibited at 28°C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35°C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Δ strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-β-d-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.

scholarly journals Swf1p, a Member of the DHHC-CRD Family of Palmitoyltransferases, Regulates the Actin Cytoskeleton and Polarized Secretion Independently of Its DHHC Motif

2008 ◽  
Vol 19 (10) ◽  
pp. 4454-4468 ◽  
Author(s):  
Shubha A. Dighe ◽  
Keith G. Kozminski
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpase ◽  
Wild Type ◽  
Cortical Actin ◽  
Actin Organization ◽  
Bud Emergence ◽  
Polarized Secretion ◽  
Polarized Cell Growth ◽  
Actin Cables

Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton.

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