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 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 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 Live Cell Imaging in Fission Yeast

2017 ◽  
Vol 2017 (10) ◽  
pp. pdb.top090621 ◽  
Author(s):  
Daniel P. Mulvihill
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

scholarly journals Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

2005 ◽  
Vol 170 (4) ◽  
pp. 637-648 ◽  
Author(s):  
Vladimir Sirotkin ◽  
Christopher C. Beltzner ◽  
Jean-Baptiste Marchand ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Biochemical Analysis ◽  
Cell Imaging ◽  
Activator Proteins ◽  
Myosin I ◽  
Class 1 ◽  
Dynamic Structures ◽  
Actin Patch

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6–9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.

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 Light-dependent inhibition of clathrin-mediated endocytosis in yeast

2021 ◽  
Author(s):  
Davia Prischich ◽  
Javier Encinar del Dedo ◽  
Maria Cambra ◽  
Judit Prat ◽  
Nuria Camarero ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Genetic Model ◽  
Peptide Inhibitor ◽  
Pharmacological Agents ◽  
Evolutionarily Conserved ◽  
Endocytic Protein ◽  
Dependent Inhibition ◽  
Endocytic Machinery

Clathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.

scholarly journals Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

2018 ◽  
Author(s):  
Brian Cook ◽  
Fred Chang ◽  
Ignacio Flor-Parra ◽  
Jawdat Al-Bassam
Keyword(s):  
Fission Yeast ◽  
Functional Role ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Self Organization ◽  
Structural Studies ◽  
Overexpressed Gene

AbstractThe XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four αβ-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two αβ-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that αβ-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.

scholarly journals Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

2005 ◽  
Vol 16 (4) ◽  
pp. 1756-1768 ◽  
Author(s):  
Isabelle Loïodice ◽  
Jayme Staub ◽  
Thanuja Gangi Setty ◽  
Nam-Phuong T. Nguyen ◽  
Anne Paoletti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Microtubule Organization ◽  
Cellular Processes ◽  
Spindle Midzone ◽  
Fluorescence Live Cell Imaging

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

scholarly journals Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

2021 ◽  
Author(s):  
Keiichiro Sakai ◽  
Yohei Kondo ◽  
Hiroyoshi Fujioka ◽  
Mako Kamiya ◽  
Kazuhiro Aoki ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Live Cell Imaging ◽  
Near Infrared ◽  
Cell Imaging ◽  
Infrared Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Yeast Cells ◽  
Color Palette

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with near-infrared excitation and emission maxima. Unlike the other conventional fluorescent proteins, iRFP requires biliverdin (BV) as a chromophore. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP-PCB was higher than that of iRFP-BV in vitro and in fission yeast. We introduced SynPCB, a PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids carrying the iRFP-fused marker proteins together with SynPCB. These tools not only enable the easy use of the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

scholarly journals Transient Breakage of the Nucleocytoplasmic Barrier Controls Spore Maturation via Mobilizing the Proteasome Subunit Rpn11 in the Fission Yeast Schizosaccharomyces pombe

2020 ◽  
Vol 6 (4) ◽  
pp. 242
Author(s):  
Hui-Ju Yang ◽  
Haruhiko Asakawa ◽  
Chizuru Ohtsuki ◽  
Tokuko Haraguchi ◽  
Yasushi Hiraoka
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nuclear Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Leading Edge ◽  
Imaging Analysis ◽  
Transmembrane Region ◽  
Nuclear Envelope Breakdown ◽  
Yeast Meiosis

Forespore membrane (FSM) closure is a process of specialized cytokinesis in yeast meiosis. FSM closure begins with the contraction of the FSM opening and finishes with the disassembly of the leading-edge proteins (LEPs) from the FSM opening. Here, we show that the FSM opening starts to contract when the event of virtual nuclear envelope breakdown (vNEBD) occurs in anaphase II of the fission yeast Schizosaccharomyces pombe. The occurrence of vNEBD controls the redistribution of the proteasomal subunit Rpn11 from the nucleus to the cytosol. To investigate the importance of Rpn11 re-localization during vNEBD, Rpn11 was sequestered at the inner nuclear membrane by fusion with the transmembrane region of Bqt4 (Rpn11-GFP-INM). Remarkably, in the absence of endogenous rpn11+, the cells carrying Rpn11-GFP-INM had abnormal or no spore formation. Live-cell imaging analysis further reveals that the FSM opening failed to contract when vNEBD occurred, and the LEP Meu14 was persistently present at the FSM in the rpn11-gfp-INM cells. The results suggest that the dynamic localization of Rpn11 during vNEBD is essential for spore development.

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