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

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

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

2018 ◽  
Vol 217 (6) ◽  
pp. 2047-2058 ◽  
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.

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

2009 ◽  
Vol 37 (5) ◽  
pp. 1056-1060 ◽  
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.

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

2008 ◽  
Vol 180 (6) ◽  
pp. 1219-1232 ◽  
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.

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

2007 ◽  
Vol 293 (4) ◽  
pp. C1374-C1382 ◽  
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.

scholarly journals Live‐cell imaging of endogenous Ras‐GTP illustrates predominant Ras activation at the plasma membrane

EMBO Reports ◽  
2006 ◽  
Vol 7 (1) ◽  
pp. 46-51 ◽  
Author(s):  
Martin Augsten ◽  
Rico Pusch ◽  
Christoph Biskup ◽  
Knut Rennert ◽  
Ute Wittig ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Ras Activation

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

2018 ◽  
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.

scholarly journals A small-molecule fluorescent probe for live-cell imaging of endocytosis

2021 ◽  
Author(s):  
Ryo Seino ◽  
Hidefumi Iwashita ◽  
Masataka Takahashi ◽  
Takatoshi Ezoe ◽  
Munetaka Ishiyama ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Probe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Endocytic Pathway ◽  
Fluorescence Signal ◽  
Scientific Interest ◽  
Acidic Conditions ◽  
Acidic Vesicles

Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.

scholarly journals Investigating the morphological dynamics of the plasma membrane by high-speed atomic force microscopy

2021 ◽  
Vol 134 (17) ◽  
Author(s):  
Yiming Yu ◽  
Shige H. Yoshimura
Keyword(s):  
Atomic Force Microscopy ◽  
Plasma Membrane ◽  
Living Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Live Cell ◽  
Force Microscopy ◽  
Atomic Force ◽  
Recent Developments

ABSTRACT Despite numerous recent developments in bioimaging techniques, nanoscale and live-cell imaging of the plasma membrane has been challenging because of the insufficient z-resolution of optical microscopes, as well as the lack of fluorescent probes to specifically label small membrane structures. High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualising the dynamics of a specimen surface and is therefore suitable for observing plasma membrane dynamics. Recent developments in HS-AFM for live-cell imaging have enabled the visualisation of the plasma membrane and the network of cortical actin underneath the membrane in a living cell. Furthermore, correlative imaging with fluorescence microscopy allows for the direct visualisation of morphological changes of the plasma membrane together with the dynamic assembly or disassembly of proteins during the entire course of endocytosis in a living cell. Here, we review these recent advances in HS-AFM in order to analyse various cellular events occurring at the cell surface.

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