scholarly journals Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

2019 ◽  
Author(s):  
Yidi Sun ◽  
Johnannes Schoeneberg ◽  
Shirley Chen ◽  
Tommy Jiang ◽  
Charlotte Kaplan ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Budding Yeast ◽  
Protein Abundance ◽  
Actin Assembly ◽  
Specific Mechanism ◽  
Membrane Invagination ◽  
Endocytic Protein ◽  
Nucleation Activity ◽  
Species Specific

AbstractConserved proteins drive clathrin-mediated endocytosis (CME), which universally involves a burst of actin assembly. To gain fundamental mechanistic insights into this process, a side-by-side quantitative comparison of CME was performed on two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae are more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast than in budding yeast. In both yeasts, Arp2/3 complex activation drives membrane invagination when triggered by the accumulation of ∼70 WASP molecules. In contrast to budding yeast, WASP-mediated actin nucleation activity plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, though two-zone actin assembly is a fission yeast-specific mechanism, which is not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations and have broad implications that extend from yeast to humans.

scholarly journals Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yidi Sun ◽  
Johannes Schöneberg ◽  
Xuyan Chen ◽  
Tommy Jiang ◽  
Charlotte Kaplan ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Yeast Species ◽  
Quantitative Comparison ◽  
Protein Abundance ◽  
Actin Assembly ◽  
Membrane Invagination ◽  
Endocytic Protein ◽  
Species Specific

Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.

scholarly journals Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

2010 ◽  
Vol 21 (16) ◽  
pp. 2894-2904 ◽  
Author(s):  
Vladimir Sirotkin ◽  
Julien Berro ◽  
Keely Macmillan ◽  
Lindsey Zhao ◽  
Thomas D. Pollard
Keyword(s):  
Mathematical Modeling ◽  
Fission Yeast ◽  
Time Course ◽  
Fluorescent Proteins ◽  
Adaptor Proteins ◽  
Actin Assembly ◽  
Characteristic Peak ◽  
Capping Proteins ◽  
Membrane Invagination ◽  
Actin Patch

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2905–2915), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

scholarly journals Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast.

1995 ◽  
Vol 128 (4) ◽  
pp. 599-615 ◽  
Author(s):  
R Li ◽  
Y Zheng ◽  
D G Drubin
Keyword(s):  
Cell Growth ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Budding Yeast ◽  
Yeast Cells ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Polarized Cell Growth ◽  
Nucleation Activity

We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.

scholarly journals A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis

2015 ◽  
Vol 26 (21) ◽  
pp. 3841-3856 ◽  
Author(s):  
Yidi Sun ◽  
Nicole T. Leong ◽  
Tiffany Wong ◽  
David G. Drubin
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Actin Assembly ◽  
Dynamic Interactions ◽  
Membrane Invagination ◽  
C Terminus ◽  
Cortical Localization ◽  
Necessary And Sufficient ◽  
Related Proteins

More than 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3 and the CIN85-related protein Sla1 are known to interact with each other in vitro, and they all appear after endocytic-site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here we used live-cell imaging in parallel with genetics and biochemistry to explore comprehensively the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites before Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, whereas the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated, using the auxin-inducible degron system. The results lead us to propose that endocytic-site initiation and actin assembly are separable processes linked by a Pan1/End3/Sla1 complex. Finally, our study provides mechanistic insights into how Pan1 and End3 function with Sla1 to coordinate cargo capture with actin assembly.

scholarly journals Fission Yeast Myosin-I, Myo1p, Stimulates Actin Assembly by Arp2/3 Complex and Shares Functions with Wasp

2000 ◽  
Vol 151 (4) ◽  
pp. 789-800 ◽  
Author(s):  
Wei-Lih Lee ◽  
Magdalena Bezanilla ◽  
Thomas D. Pollard
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
Double Mutant ◽  
Actin Assembly ◽  
Myosin I ◽  
Polarized Cell Growth ◽  
Cortical Localization ◽  
Nucleation Activity ◽  
Synthetically Lethal ◽  
Acidic Region

Fission yeast myo1+ encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1+ is not lethal, Δmyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1+ is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Δmyo1 defects. However, it fails to rescue the Δmyo1 Δwsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

1989 ◽  
Vol 35 (12) ◽  
pp. 1081-1086 ◽  
Author(s):  
Byron F. Johnson ◽  
L. C. Sowden ◽  
Teena Walker ◽  
Bong Y. Yoo ◽  
Gode B. Calleja
Keyword(s):  
Electron Microscopy ◽  
Fission Yeast ◽  
Budding Yeast ◽  
The Other ◽  
Yeast Cells ◽  
Scanning Electron Micrographs ◽  
Soft Or ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Scanning Electron

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.Key words: flocculation, budding yeast, fission yeast, scanning, transmission.

scholarly journals Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

PLoS Genetics ◽  
2016 ◽  
Vol 12 (2) ◽  
pp. e1005839 ◽  
Author(s):  
Gautam Chatterjee ◽  
Sundar Ram Sankaranarayanan ◽  
Krishnendu Guin ◽  
Yogitha Thattikota ◽  
Sreedevi Padmanabhan ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Candida Tropicalis ◽  
Budding Yeast

scholarly journals Cleavage of Model Replication Forks by Fission Yeast Mus81-Eme1 and Budding Yeast Mus81-Mms4

2002 ◽  
Vol 278 (9) ◽  
pp. 6928-6935 ◽  
Author(s):  
Matthew C. Whitby ◽  
Fekret Osman ◽  
Julie Dixon
Keyword(s):  
Fission Yeast ◽  
Budding Yeast ◽  
Replication Forks

scholarly journals Functional Comparison of the Tup11 and Tup12 Transcriptional Corepressors in Fission Yeast

2005 ◽  
Vol 25 (2) ◽  
pp. 716-727 ◽  
Author(s):  
Fredrik Fagerström-Billai ◽  
Anthony P. H. Wright
Keyword(s):  
Gene Expression ◽  
Gene Duplication ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Functional Difference ◽  
Evolutionary Mechanism ◽  
Genome Wide ◽  
Number Of Genes ◽  
Transcriptional Corepressors ◽  
Genome Wide Gene Expression

ABSTRACT Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11 + and tup12 + , that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11 − and tup12 − mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11 − and tup12 − mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11 + and tup12 + genes. Many of these genes are differentially derepressed in tup11 − mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12 − mutants require the Ssn6 protein for their repression. As for Tup12, Ssn6 is also required for efficient adaptation to KCl- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.

Sign in / Sign up

Export Citation Format

Share Document