scholarly journals Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

2005 ◽  
Vol 16 (1) ◽  
pp. 372-384 ◽  
Author(s):  
Avital A. Rodal ◽  
Lukasz Kozubowski ◽  
Bruce L. Goode ◽  
David G. Drubin ◽  
John H. Hartwig
Keyword(s):  
Cell Division ◽  
High Resolution ◽  
Actin Cytoskeleton ◽  
Yeast Cell ◽  
Budding Yeast ◽  
Model Organism ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Actin Patch ◽  
Order Structures

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

2002 ◽  
Vol 115 (8) ◽  
pp. 1703-1715 ◽  
Author(s):  
Derek T. Warren ◽  
Paul D. Andrews ◽  
Campbell W. Gourlay ◽  
Kathryn R. Ayscough
Keyword(s):  
Immunofluorescence Microscopy ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Binding Studies ◽  
Endocytic Machinery ◽  
Single Complex ◽  
Actin Patch ◽  
First Time

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p(YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

scholarly journals Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sofia Duarte ◽  
Álvaro Viedma-Poyatos ◽  
Elena Navarro-Carrasco ◽  
Alma E. Martínez ◽  
María A. Pajares ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Types ◽  
Hiv Protease ◽  
Cell Cortex ◽  
Tail Domain ◽  
Cellular Functions ◽  
Cortical Actin ◽  
Intrinsically Disordered ◽  
Actin Cortex ◽  
Vimentin Filaments

Abstract The vimentin network displays remarkable plasticity to support basic cellular functions and reorganizes during cell division. Here, we show that in several cell types vimentin filaments redistribute to the cell cortex during mitosis, forming a robust framework interwoven with cortical actin and affecting its organization. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of vimentin tail domain gradually impair cortical association and mitosis progression. Disruption of f-actin, but not of microtubules, causes vimentin bundling near the chromosomes. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, full filament formation is dispensable for cortical association, which also occurs in vimentin particles. These results unveil implications of vimentin dynamics in cell division through its interplay with the actin cortex.

scholarly journals Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveal coordinate control of lipid metabolism

2020 ◽  
Vol 31 (10) ◽  
pp. 1069-1084 ◽  
Author(s):  
Heidi M. Blank ◽  
Ophelia Papoulas ◽  
Nairita Maitra ◽  
Riddhiman Garge ◽  
Brian K. Kennedy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Lipid Metabolism ◽  
Cell Division ◽  
Yeast Cell ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Dynamic Changes ◽  
Coordinate Control ◽  
Cycle Dependent ◽  
Budding Yeast Cell Cycle

In several systems, including budding yeast, cell cycle-dependent changes in the transcriptome are well studied. In contrast, few studies queried the proteome during cell division. There is also little information about dynamic changes in metabolites and lipids in the cell cycle. Here, the authors present such information for dividing yeast cells.

scholarly journals EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (8) ◽  
pp. 4294-4304 ◽  
Author(s):  
H Y Tang ◽  
A Munn ◽  
M Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Factor Receptor ◽  
Cell Cortex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cortical Actin ◽  
Kinase Substrate ◽  
Physiological Relevance ◽  
Eh Domain

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

scholarly journals The Septation Apparatus, an Autonomous System in Budding Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2747-2759 ◽  
Author(s):  
Dong-Hyun Roh ◽  
Blair Bowers ◽  
Martin Schmidt ◽  
Enrico Cabib
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Nuclear Division ◽  
Fluorescent Proteins ◽  
Budding Yeast ◽  
Cell Cortex ◽  
Nonpermissive Temperature ◽  
Contractile Ring ◽  
Primary Septum ◽  
Endocytic Vesicles

Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal. To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature. With the fluorescent fusion proteins mentioned above, we observed that incdc3 at 37°C both Myo1p and Chs2p colocalize at different spots of the cell cortex. A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles. Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations. The formation of delocalized septa did not require nuclear division. We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa.

scholarly journals Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

1997 ◽  
Vol 136 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rong Li
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Function ◽  
Actin Filaments ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Yeast Protein ◽  
Wiskott Aldrich Syndrome ◽  
Striking Change ◽  
And Function ◽  
Syndrome Protein

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.

scholarly journals Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins.

1993 ◽  
Vol 120 (6) ◽  
pp. 1305-1320 ◽  
Author(s):  
D J Lew ◽  
S I Reed
Keyword(s):  
Cell Cycle ◽  
Actin Cytoskeleton ◽  
Yeast Cell ◽  
Cell Cycle Regulation ◽  
Regulatory Protein ◽  
Yeast Cell Cycle ◽  
Actin Binding ◽  
Cortical Actin ◽  
Bud Site ◽  
Cycle Regulation

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.

scholarly journals Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anan Chen ◽  
Luisa Ulloa Severino ◽  
Thomas C. Panagiotou ◽  
Trevor F. Moraes ◽  
Darren A. Yuen ◽  
...  
Keyword(s):  
Cell Division ◽  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Actin Network ◽  
Contractile Ring ◽  
Actin Networks ◽  
Membrane Blebbing ◽  
Actin Isoform

AbstractDuring cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

scholarly journals Polarization of cell growth in yeast

2000 ◽  
Vol 113 (4) ◽  
pp. 571-585 ◽  
Author(s):  
D. Pruyne ◽  
A. Bretscher
Keyword(s):  
Actin Cytoskeleton ◽  
Structural Basis ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Class V ◽  
Precise Control ◽  
Dependent Protein ◽  
Actin Cables ◽  
Protein Kinase Signaling ◽  
Structural Determinant

The actin cytoskeleton provides the structural basis for cell polarity in Saccharomyces cerevisiae as well as most other eukaryotes. In Part I of this two-part commentary, presented in the previous issue of Journal of Cell Science, we discussed the basis by which yeast establishes and maintains different states of polarity through Ρ GTPases and cyclin-dependent protein kinase signaling. Here we discuss how, in response to those signals, the actin cytoskeleton guides growth of the yeast cell. A polarized array of actin cables at the cell cortex is the primary structural determinant of polarity. Motors such as class V myosins use this array to transport secretory vesicles, mRNA and organelles towards growth sites, where they are anchored by a cap of cytoskeletal and regulatory proteins. Cortical actin patches enhance and maintain this polarity, probably through endocytic recycling, which allows reuse of materials and prevents continued growth at old sites. The dynamic arrangement of targeting and recycling provides flexibility for the precise control of morphogenesis.

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