Type-I myosins promote actin polymerization to drive membrane bending in endocytosis

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization.

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Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane

The Journal of Cell Biology ◽

10.1083/jcb.200708060 ◽

2008 ◽

Vol 180 (6) ◽

pp. 1219-1232 ◽

Cited By ~ 113

Author(s):

Fatima-Zahra Idrissi ◽

Helga Grötsch ◽

Isabel M. Fernández-Golbano ◽

Cristina Presciatto-Baschong ◽

Howard Riezman ◽

...

Keyword(s):

Plasma Membrane ◽

Immunoelectron Microscopy ◽

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Live Cell ◽

Endocytic Vesicle ◽

Myosin I ◽

Initial Membrane ◽

Membrane Bending

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475–487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.

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Discovery of functional interactions among actin regulators by analysis of image fluctuations in an unperturbed motile cell system

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0110 ◽

2018 ◽

Vol 373 (1747) ◽

pp. 20170110 ◽

Cited By ~ 7

Author(s):

Tadamoto Isogai ◽

Gaudenz Danuser

Keyword(s):

Cell Biology ◽

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Cell System ◽

Feedback Loops ◽

Actin Dynamics ◽

Actin Network ◽

Theme Issue ◽

Functional Interactions

Cell migration is driven by propulsive forces derived from polymerizing actin that pushes and extends the plasma membrane. The underlying actin network is constantly undergoing adaptation to new mechano-chemical environments and intracellular conditions. As such, mechanisms that regulate actin dynamics inherently contain multiple feedback loops and redundant pathways. Given the highly adaptable nature of such a system, studies that use only perturbation experiments (e.g. knockdowns, overexpression, pharmacological activation/inhibition, etc.) are challenged by the nonlinearity and redundancy of the pathway. In these pathway configurations, perturbation experiments at best describe the function(s) of a molecular component in an adapting (e.g. acutely drug-treated) or fully adapted (e.g. permanent gene silenced) cell system, where the targeted component now resides in a non-native equilibrium. Here, we propose how quantitative live-cell imaging and analysis of constitutive fluctuations of molecular activities can overcome these limitations. We highlight emerging actin filament barbed-end biology as a prime example of a complex, nonlinear molecular process that requires a fluctuation analytic approach, especially in an unperturbed cellular system, to decipher functional interactions of barbed-end regulators, actin polymerization and membrane protrusion. This article is part of the theme issue ‘Self-organization in cell biology’.

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Faculty Opinions recommendation of Type-I myosins promote actin polymerization to drive membrane bending in endocytosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736356373.793589289 ◽

2021 ◽

Author(s):

Britta Qualmann

Keyword(s):

Actin Polymerization ◽

Type I ◽

Membrane Bending

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Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Journal of Experimental Medicine ◽

10.1084/jem.20062324 ◽

2007 ◽

Vol 204 (9) ◽

pp. 2213-2224 ◽

Cited By ~ 123

Author(s):

Dale A. Moulding ◽

Michael P. Blundell ◽

David G. Spiller ◽

Michael R.H. White ◽

Giles O. Cory ◽

...

Keyword(s):

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Human Gene ◽

Filamentous Actin ◽

Gene Encoding ◽

Activating Mutations ◽

Spindle Midzone ◽

Underlying Mechanisms ◽

Syndrome Protein

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

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A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

Virology Journal ◽

10.1186/s12985-019-1267-9 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Yuki Takamatsu ◽

Olga Dolnik ◽

Takeshi Noda ◽

Stephan Becker

Keyword(s):

Live Cell Imaging ◽

Intracellular Transport ◽

Actin Polymerization ◽

Ebola Virus ◽

Cell Imaging ◽

Temporal Dynamics ◽

Imaging System ◽

Marburg Virus ◽

Live Cell ◽

Infected Cells

Abstract Background Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. Methods To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Results Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. Conclusions We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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Intracellular mechanisms of fungal space searching in microenvironments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816423116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13543-13552 ◽

Cited By ~ 2

Author(s):

Marie Held ◽

Ondřej Kašpar ◽

Clive Edwards ◽

Dan V. Nicolau

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Turgor Pressure ◽

Fungal Growth ◽

Time Lapse ◽

Growth Direction ◽

Medical Applications ◽

Initial Growth ◽

Space Partitioning ◽

Constrained Environments

Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

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High-throughput, image-based screening of genetic variant libraries

10.1101/143966 ◽

2017 ◽

Author(s):

George Emanuel ◽

Jeffrey R. Moffitt ◽

Xiaowei Zhuang

Keyword(s):

High Throughput ◽

Genetic Variants ◽

High Throughput Screening ◽

Live Cell Imaging ◽

Cell Imaging ◽

Genetic Variant ◽

Fluorescent Protein ◽

Screening Method ◽

Wild Type ◽

Genetic Perturbations

AbstractImage-based, high-throughput, high-content screening of pooled libraries of genetic perturbations will greatly advance our understanding biological systems and facilitate many biotechnology applications. Here we introduce a high-throughput screening method that allows highly diverse genotypes and the corresponding phenotypes to be imaged in numerous individual cells. To facilitate genotyping by imaging, barcoded genetic variants are introduced into the cells, each cell carrying a single genetic variant connected to a unique, nucleic-acid barcode. To identify the genotype-phenotype correspondence, we perform live-cell imaging to determine the phenotype of each cell, and massively multiplexed FISH imaging to measure the barcode expressed in the same cell. We demonstrated the utility of this approach by screening for brighter and more photostable variants of the fluorescent protein YFAST. We imaged 20 million cells expressing ~60,000 YFAST mutants and identified novel YFAST variants that are substantially brighter and/or more photostable than the wild-type protein.

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