Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.

Download Full-text

Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e09-05-0412 ◽

2010 ◽

Vol 21 (7) ◽

pp. 1237-1252 ◽

Cited By ~ 20

Author(s):

Takaharu Yamamoto ◽

Junko Mochida ◽

Jun Kadota ◽

Miyoko Takeda ◽

Erfei Bi ◽

...

Keyword(s):

Budding Yeast ◽

Vesicle Transport ◽

Filamentous Actin ◽

Cortical Actin ◽

Endocytic Recycling ◽

Actin Cable ◽

Bud Growth ◽

Actin Cables ◽

Type V ◽

Endocytic Vesicles

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.

Download Full-text

Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth

Journal of Cell Science ◽

10.1242/jcs.247916 ◽

2021 ◽

Vol 134 (1) ◽

pp. jcs247916

Author(s):

Ying Xie ◽

Yansong Miao

Keyword(s):

Actin Polymerization ◽

Budding Yeast ◽

Fungal Growth ◽

System Response ◽

Macromolecular Complex ◽

Cell Axis ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Dynamic Assembly ◽

Actin Cables

ABSTRACTDynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs) orchestrate the condensation of the polarisome components and the dynamic assembly of the complex. In addition, we address how these components are dynamically distributed in and out of the assembly zone, thereby regulating polarized growth. We also discuss the potential mechanical feedback mechanisms by which the force-induced actin polymerization at the tip of the budding yeast regulates the assembly and function of the polarisome.

Download Full-text

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease

Science ◽

10.1126/science.aay5663 ◽

2020 ◽

Vol 369 (6500) ◽

pp. 202-207 ◽

Cited By ~ 3

Author(s):

Sarah A. Cook ◽

William A. Comrie ◽

M. Cecilia Poli ◽

Morgan Similuk ◽

Andrew J. Oler ◽

...

Keyword(s):

Actin Polymerization ◽

Synapse Formation ◽

Immune Cell ◽

Filamentous Actin ◽

Cortical Actin ◽

Akt Phosphorylation ◽

Actin Networks ◽

Evolutionarily Conserved ◽

Regulatory Complex ◽

Immune Cell Migration

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Download Full-text

Septin Ring Assembly Requires Concerted Action of Polarisome Components, a PAK Kinase Cla4p, and the Actin Cytoskeleton inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0254 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5329-5345 ◽

Cited By ~ 82

Author(s):

Jun Kadota ◽

Takaharu Yamamoto ◽

Shiro Yoshiuchi ◽

Erfei Bi ◽

Kazuma Tanaka

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

Small Gtpase ◽

Septin Ring ◽

Ring Assembly ◽

Actin Cable ◽

Family Protein ◽

Latrunculin A ◽

Actin Cables ◽

Initial Assembly

Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Δ cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Δ cla4-75-td, bud6Δ cla4-75-td, and pea2Δ cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Δ mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.

Download Full-text

Myosin Vs organize actin cables in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e12-07-0499 ◽

2012 ◽

Vol 23 (23) ◽

pp. 4579-4591 ◽

Cited By ~ 34

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.

Download Full-text

Live cell imaging of the assembly, disassembly, and actin cable–dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.200404173 ◽

2004 ◽

Vol 167 (3) ◽

pp. 519-530 ◽

Cited By ~ 135

Author(s):

Thomas M. Huckaba ◽

Anna Card Gay ◽

Luiz Fernando Pantalena ◽

Hyeong-Cheol Yang ◽

Liza A. Pon

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Retrograde Flow ◽

Yeast Saccharomyces Cerevisiae ◽

Actin Cable ◽

Complex Mutation ◽

Conveyor Belts ◽

Actin Patch ◽

Mother Cells ◽

Actin Cables

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.

Download Full-text

Opposing Roles for Actin in Cdc42p Polarization

Molecular Biology of the Cell ◽

10.1091/mbc.e04-05-0430 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1296-1304 ◽

Cited By ~ 56

Author(s):

Javier E. Irazoqui ◽

Audrey S. Howell ◽

Chandra L. Theesfeld ◽

Daniel J. Lew

Keyword(s):

Actin Cytoskeleton ◽

Cell Polarity ◽

Budding Yeast ◽

Cortical Actin ◽

Independent Manner ◽

Actin Depolymerization ◽

Cell Biol ◽

Actin Cables ◽

Unexpected Difference

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399–416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.

Download Full-text

EXO1 Plays a Role in Generating Type I and Type II Survivors in Budding Yeast

Genetics ◽

10.1093/genetics/166.4.1641 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1641-1649

Author(s):

Laura Maringele ◽

David Lydall

Keyword(s):

Homologous Recombination ◽

Budding Yeast ◽

Human Cells ◽

Telomere Maintenance ◽

Recombination Process ◽

Yeast Cells ◽

Type I ◽

Type Ii ◽

Recombination Proteins

Abstract Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination. Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase. We show that EXO1 is important for generating type I survivors in yku70Δ mre11Δ cells and type II survivors in tlc1Δ cells. Moreover, in tlc1Δ cells, EXO1 seems to contribute to the senescence process itself.

Download Full-text

Reconstitution of contractile actomyosin rings in vesicles

Nature Communications ◽

10.1038/s41467-021-22422-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Thomas Litschel ◽

Charlotte F. Kelley ◽

Danielle Holz ◽

Maral Adeli Koudehi ◽

Sven K. Vogel ◽

...

Keyword(s):

Large Scale ◽

Actin Polymerization ◽

Living Cells ◽

Actin Ring ◽

Actin Networks ◽

Mechanical Transformation ◽

Myosin Motors ◽

Spherical Vesicles ◽

Theoretical Considerations ◽

Encapsulation Methods

AbstractOne of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

Download Full-text

Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

Journal of Experimental Medicine ◽

10.1084/jem.20101125 ◽

2011 ◽

Vol 208 (5) ◽

pp. 1055-1068 ◽

Cited By ~ 123

Author(s):

Bebhinn Treanor ◽

David Depoil ◽

Andreas Bruckbauer ◽

Facundo D. Batista

Keyword(s):

Actin Cytoskeleton ◽

B Cell ◽

Protein Function ◽

Actin Polymerization ◽

Lymphocyte Activation ◽

Dominant Negative ◽

Temporary Increase ◽

Cortical Actin ◽

Erm Proteins ◽

Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

Download Full-text