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

2010 ◽  
Vol 21 (7) ◽  
pp. 1237-1252 ◽  
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.

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

2006 ◽  
Vol 175 (6) ◽  
pp. 957-969 ◽  
Author(s):  
Thomas M. Huckaba ◽  
Thomas Lipkin ◽  
Liza A. Pon
Keyword(s):  
Actin Polymerization ◽  
Budding Yeast ◽  
Type Ii ◽  
Retrograde Flow ◽  
Cortical Actin ◽  
Actin Networks ◽  
Actin Cable ◽  
Intracellular Movement ◽  
Actin Cables ◽  
Type Ii Myosin

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.

scholarly journals Role of Actin and Myo2p in Polarized Secretion and Growth ofSaccharomyces cerevisiae

2000 ◽  
Vol 11 (5) ◽  
pp. 1727-1737 ◽  
Author(s):  
Tatiana S. Karpova ◽  
Samara L. Reck-Peterson ◽  
N. Barry Elkind ◽  
Mark S. Mooseker ◽  
Peter J. Novick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Growth ◽  
Polarized Growth ◽  
Tail Domain ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Polarized Secretion ◽  
Actin Patch ◽  
Actin Cables

We examined the role of the actin cytoskeleton in secretion inSaccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant,bee1Δ(las17Δ), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Δ, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p,myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

scholarly journals Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables

2018 ◽  
Vol 29 (8) ◽  
pp. 923-936 ◽  
Author(s):  
Myungjoo Shin ◽  
Jolanda van Leeuwen ◽  
Charles Boone ◽  
Anthony Bretscher
Keyword(s):  
Budding Yeast ◽  
Negative Regulator ◽  
Pivotal Role ◽  
Cortical Region ◽  
Actin Assembly ◽  
Actin Monomer ◽  
Filamentous Actin ◽  
Capping Protein ◽  
End Capping ◽  
Actin Cables

How cells balance the incorporation of actin into diverse structures is poorly understood. In budding yeast, a single actin monomer pool is used to build both actin cables involved in polarized growth and actin cortical patches involved in endocytosis. Here we report how Aim21/Tda2 is recruited to the cortical region of actin patches, where it negatively regulates actin assembly to elevate the available actin monomer pool. Aim21 has four polyproline regions and is recruited by two SH3-containing patch proteins, Bbc1 and Abp1. The C-terminal region, which is required for its function, binds Tda2. Cell biological and biochemical data reveal that Aim21/Tda2 is a negative regulator of barbed end filamentous actin (F-actin) assembly, and this activity is necessary for efficient endocytosis and plays a pivotal role in balancing the distribution of actin between cables and patches. Aim21/Tda2 also forms a complex with the F-actin barbed end capping protein Cap1/Cap2, revealing an interplay between regulators and showing the complexity of regulation of barbed end assembly.

scholarly journals Tropomyosin-containing Actin Cables Direct the Myo2p-dependent Polarized Delivery of Secretory Vesicles in Budding Yeast

1998 ◽  
Vol 143 (7) ◽  
pp. 1931-1945 ◽  
Author(s):  
David W. Pruyne ◽  
Daniel H. Schott ◽  
Anthony Bretscher
Keyword(s):  
Budding Yeast ◽  
Secretory Vesicle ◽  
Rab Gtpase ◽  
Secretory Vesicles ◽  
Class V ◽  
Bud Emergence ◽  
Isotropic Distribution ◽  
Bud Growth ◽  
Polarized Delivery ◽  
Actin Cables

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle–associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p-dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.

Confocal microscopy of F-actin distribution in Xenopus oocytes

Zygote ◽  
1994 ◽  
Vol 2 (2) ◽  
pp. 111-124 ◽  
Author(s):  
Amy D. Roeder ◽  
David L. Gard
Keyword(s):  
Confocal Microscopy ◽  
Three Dimensional ◽  
Germinal Vesicle ◽  
Stage I ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Mitochondrial Mass ◽  
Cytoplasmic Actin ◽  
Dimensional Network ◽  
Actin Cables

SummaryWe have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 μm in diameter. In oocytes with diameters of 35–50 μm F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.

scholarly journals Opposing Roles for Actin in Cdc42p Polarization

2005 ◽  
Vol 16 (3) ◽  
pp. 1296-1304 ◽  
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.

scholarly journals Actin cable distribution and dynamics arising from cross-linking, motor pulling, and filament turnover

2014 ◽  
Vol 25 (19) ◽  
pp. 3006-3016 ◽  
Author(s):  
Haosu Tang ◽  
Damien Laporte ◽  
Dimitrios Vavylonis
Keyword(s):  
Actin Filaments ◽  
Three Dimensions ◽  
Cross Linking ◽  
Semiflexible Polymers ◽  
Myosin V ◽  
Cable Structures ◽  
Actin Cable ◽  
Quantitative Understanding ◽  
Actin Cables ◽  
Type V

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.

scholarly journals Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

1992 ◽  
Vol 118 (3) ◽  
pp. 561-571 ◽  
Author(s):  
S Chowdhury ◽  
K W Smith ◽  
M C Gustin
Keyword(s):  
Osmotic Stress ◽  
Actin Filament ◽  
Physiological Process ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhodamine Phalloidin ◽  
Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

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.

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