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

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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Adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly

10.1101/389015 ◽

2018 ◽

Cited By ~ 1

Author(s):

Cameron MacQuarrie ◽

MariaSanta Mangione ◽

Robert Carroll ◽

Michael James ◽

Kathleen L. Gould ◽

...

Keyword(s):

Plasma Membrane ◽

Schizosaccharomyces Pombe ◽

Live Cell Imaging ◽

Cell Imaging ◽

Adaptor Protein ◽

Live Cell ◽

Endocytic Vesicle ◽

Actin Networks ◽

Binding Partner ◽

Actin Patch

ABSTRACTArp2/3 complex-nucleated branched actin networks provide the force necessary for endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including the Schizosaccharomyces pombe proteins WASp Wsp1 and myosin-1 Myo1. There are >40 known yeast endocytic proteins with distinct spatial and temporal localizations and functions; however, it is still unclear how these proteins work together to drive endocytosis. We used quantitative live cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with verprolin Vrp1 for Myo1 binding, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalize with the endocytic vesicle; however, in the absence of Bbc1, a portion of Vrp1-Wsp1 remains with Myo1 at the base of the invagination and endocytic invaginations are twice as long. We propose that Bbc1 disrupts a transient Myo1-Vrp1-Wsp1 interaction and limits Arp2/3 complex-nucleation of actin branches at the plasma membrane.

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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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EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

The Journal of Cell Biology ◽

10.1083/jcb.201711151 ◽

2018 ◽

Vol 217 (6) ◽

pp. 2047-2058 ◽

Cited By ~ 17

Author(s):

Chi-Lun Chang ◽

Yu-Ju Chen ◽

Carlo Giovanni Quintanilla ◽

Ting-Sung Hsieh ◽

Jen Liou

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Cellular Functions ◽

Binding Ability ◽

Trapping Mechanism ◽

Spatiotemporal Regulation

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER–plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1–EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER–PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER–PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1–EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

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Simultaneous visualization of callose deposition and plasma membrane for live-cell imaging in plants

Plant Cell Reports ◽

10.1007/s00299-020-02580-6 ◽

2020 ◽

Vol 39 (11) ◽

pp. 1517-1523

Author(s):

Masaki Kohari ◽

Naoto Shibuya ◽

Hanae Kaku

Keyword(s):

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Callose Deposition ◽

Simultaneous Visualization

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Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

Biochemical Society Transactions ◽

10.1042/bst0371056 ◽

2009 ◽

Vol 37 (5) ◽

pp. 1056-1060 ◽

Cited By ~ 9

Author(s):

Marek Cebecauer ◽

Dylan M. Owen ◽

Anna Markiewicz ◽

Anthony I. Magee

Keyword(s):

Plasma Membrane ◽

Physical Properties ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Eukaryotic Cells ◽

Dynamic Nature ◽

Membrane Components ◽

Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

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

10.1101/490011 ◽

2018 ◽

Cited By ~ 3

Author(s):

Hetty E Manenschijn ◽

Andrea Picco ◽

Markus Mund ◽

Jonas Ries ◽

Marko Kaksonen

Keyword(s):

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Turgor Pressure ◽

Type I ◽

Actin Network ◽

Actin Nucleation ◽

Genetic Perturbations ◽

Membrane Bending ◽

Dose Dependent

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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Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells

The Journal of Infectious Diseases ◽

10.1093/infdis/jiv083 ◽

2015 ◽

Vol 212 (suppl 2) ◽

pp. S160-S166 ◽

Cited By ~ 29

Author(s):

Gordian Schudt ◽

Olga Dolnik ◽

Larissa Kolesnikova ◽

Nadine Biedenkopf ◽

Astrid Herwig ◽

...

Keyword(s):

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Live Cell ◽

Imaging Analysis ◽

Infected Cells

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Live cell imaging of interleukin-6-induced targeting of “transcription factor” STAT3 to sequestering endosomes in the cytoplasm

AJP Cell Physiology ◽

10.1152/ajpcell.00220.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. C1374-C1382 ◽

Cited By ~ 37

Author(s):

Fang Xu ◽

Somshuvra Mukhopadhyay ◽

Pravin B. Sehgal

Keyword(s):

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Direct Evidence ◽

Live Cell ◽

Dominant Negative ◽

Adapter Protein ◽

Toll Like Receptor ◽

Caveolin 1 ◽

Induced Signal

Signal transducer and activator of transcription (STAT) family transcription factors are classically viewed as transducing cytokine- and growth factor-activated signals from the plasma membrane to the cell nucleus for the purpose of activating transcription. We report live cell imaging studies of fluorescently labeled STAT3 expressed in Hep3B hepatocytes that reveal interleukin (IL)-6-activated targeting of STAT3 and PY-STAT3 to relatively long-lived sequestering endosomes in the cytoplasm. This targeting was rapid but transient, required phosphorylation and integrity of Tyr 705 in STAT3, and was blocked by nocodazole, geldanamycin, and indirubin E804 and by overexpression of wild-type caveolin-1. Strikingly, overexpression of the dominant-negative (DN) mutant K44A of the GTPase dynamin II led to marked constitutive accumulation of STAT3 in the endocytic compartment with depletion of the STAT3 nuclear pool. Subsets of the native and K44A-generated STAT3- and PY-STAT3-sequestering endosomes colocalized with MyD88, an adapter protein that integrates pathways of Toll-like receptor and IL-1 transcriptional signaling and stabilization of mRNAs. These data provide direct evidence for the cytokine-induced “signal transduction” by STAT3 from the plasma membrane to a cytoplasmic membrane destination for yet to be elucidated function(s) in the cytoplasm including prolongation of signaling and/or cross talk.

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