scholarly journals Myosin-X is dispensable for spindle morphogenesis and positioning in the mouse oocyte

Development ◽  
2021 ◽  
Vol 148 (7) ◽  
Author(s):  
Flora Crozet ◽  
Christelle Da Silva ◽  
Marie-Hélène Verlhac ◽  
Marie-Emilie Terret
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Actin Assembly ◽  
Force Transmission ◽  
Developmental Potential ◽  
Spindle Positioning ◽  
Chromosome Alignment ◽  
Asymmetric Divisions ◽  
Myosin X ◽  
Meiosis I

ABSTRACT Off-center spindle positioning in mammalian oocytes enables asymmetric divisions in size, which are important for subsequent embryogenesis. The migration of the meiosis I spindle from the oocyte center to its cortex is mediated by F-actin. Specifically, an F-actin cage surrounds the microtubule spindle and applies forces to it. To better understand how F-actin transmits forces to the spindle, we studied a potential direct link between F-actin and microtubules. For this, we tested the implication of myosin-X, a known F-actin and microtubule binder involved in spindle morphogenesis and/or positioning in somatic cells, amphibian oocytes and embryos. Using a mouse strain conditionally invalidated for myosin-X in oocytes and by live-cell imaging, we show that myosin-X is not localized on the spindle, and is dispensable for spindle and F-actin assembly. It is not required for force transmission as spindle migration and chromosome alignment occur normally. More broadly, myosin-X is dispensable for oocyte developmental potential and female fertility. We therefore exclude a role for myosin-X in transmitting F-actin-mediated forces to the spindle, opening new perspectives regarding this mechanism in mouse oocytes, which differ from most mitotic cells.

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 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 The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover

2019 ◽  
Vol 218 (10) ◽  
pp. 3415-3435 ◽  
Author(s):  
M. Angeles Juanes ◽  
Daniel Isnardon ◽  
Ali Badache ◽  
Sophie Brasselet ◽  
Manos Mavrakis ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Adenomatous Polyposis ◽  
Actin Assembly ◽  
Polyposis Coli ◽  
Capture Event ◽  
Focal Adhesion Turnover ◽  
Microtubule Capture

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin’s roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state “primed” for microtubule-induced turnover.

scholarly journals Actin dynamics drive microvillar motility and clustering during brush border assembly

2018 ◽  
Author(s):  
Leslie M Meenderink ◽  
Matthew J. Tyska
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Driving Force ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Actin Dynamics ◽  
Apical Surface ◽  
Actin Assembly ◽  
Brush Borders ◽  
Surface Remodeling

SUMMARYDuring differentiation, transporting epithelial cells generate large arrays of microvilli known as a brush borders to enhance functional capacity. To develop our understanding of brush border formation, we used live cell imaging to visualize apical surface remodeling during early stages of this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ~0.2 μm/min. Perturbation studies with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed-ends of core bundles, which in turn is linked to robust treadmilling of these structures. Because the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately leads to their clustering and consolidation into higher order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.

scholarly journals Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis

2014 ◽  
Vol 205 (5) ◽  
pp. 721-735 ◽  
Author(s):  
Alexandre Grassart ◽  
Aaron T. Cheng ◽  
Sun Hae Hong ◽  
Fan Zhang ◽  
Nathan Zenzer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Cell Imaging ◽  
Actin Assembly ◽  
Specific Proteins ◽  
Quantitative Analyses ◽  
And Function

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2–4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

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