scholarly journals A novel live cell imaging assay reveals regulation of endosome maturation

2021 ◽  
Author(s):  
Maria Podinovskaia ◽  
Cristina Prescianotto-Baschong ◽  
Dominik Buser ◽  
Anne Spang
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Communication ◽  
Cell Types ◽  
Cellular Systems ◽  
Live Cell ◽  
Endosomal Recycling ◽  
Endosomal Acidification ◽  
Turn Over ◽  
Endosome Maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1- and Rab11-dependent endosomal recycling occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

scholarly journals A novel live-cell imaging assay reveals regulation of endosome maturation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Maria Podinovskaia ◽  
Cristina Prescianotto-Baschong ◽  
Dominik P Buser ◽  
Anne Spang
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Communication ◽  
Cell Types ◽  
Cellular Systems ◽  
Live Cell ◽  
Recycling Endosome ◽  
Endosomal Acidification ◽  
Turn Over ◽  
Endosome Maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

scholarly journals Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kazuki Harada ◽  
Takami Chihara ◽  
Yuki Hayasaka ◽  
Marie Mita ◽  
Mai Takizawa ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Live Cell ◽  
Trace Detection ◽  
Lactate And Pyruvate ◽  
Lactate Levels ◽  
Green Fluorescent

Abstract Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them “Green Lindoblum” and “Green Pegassos,” respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.

Advances in the design of cell-permeable fluorescent probes for applications in live cell imaging

2018 ◽  
Vol 54 (97) ◽  
pp. 13641-13653 ◽  
Author(s):  
Samira Husen Alamudi ◽  
Young-Tae Chang
Keyword(s):  
Fluorescent Probes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Types ◽  
Design Strategy ◽  
Live Cell ◽  
Specific Cell ◽  
Cellular Environment

Advances in the design strategy of cell-permeable small fluorescent probes are discussed. Their applications in imaging specific cell types and intracellular bioanalytes, as well as the cellular environment in live conditions, are presented.

scholarly journals Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

2015 ◽  
Vol 112 (24) ◽  
pp. E3104-E3113 ◽  
Author(s):  
Paula Nunes ◽  
Isabelle Roth ◽  
Paolo Meda ◽  
Eric Féraille ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Cell Volume ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Chloride Concentration ◽  
Cell Types ◽  
Reaction Rates ◽  
Live Cell ◽  
Ionic Balance ◽  
Molecular Crowding ◽  
Ionic Imbalance

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking.

scholarly journals Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

2018 ◽  
Vol 217 (4) ◽  
pp. 1537-1552 ◽  
Author(s):  
Sascha Conic ◽  
Dominique Desplancq ◽  
Alexia Ferrand ◽  
Veronique Fischer ◽  
Vincent Heyer ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Live Cell ◽  
Biological Information ◽  
Live Cells

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

scholarly journals F-pili dynamics by live-cell imaging

2008 ◽  
Vol 105 (46) ◽  
pp. 17978-17981 ◽  
Author(s):  
Margaret Clarke ◽  
Lucinda Maddera ◽  
Robin L. Harris ◽  
Philip M. Silverman
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Recipient Cell ◽  
Cell Communication ◽  
Live Cell ◽  
Cell Contact ◽  
Gram Negative Bacteria ◽  
Donor Cells ◽  
Cell Cell

Bacteria have evolved numerous mechanisms for cell–cell communication, many of which have important consequences for human health. Among these is conjugation, the direct transfer of DNA from one cell to another. For Gram-negative bacteria, conjugation requires thin, flexible filaments (conjugative pili) that are elaborated by DNA donor cells. The structure, function, and especially the dynamics of conjugative pili are poorly understood. Here, we have applied live-cell imaging to characterize the dynamics of F-pili (conjugative pili encoded by the F plasmid of Escherichia coli). We establish that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event, such as contact with a recipient cell. When made, such contacts are able to survive the shear forces felt by bacteria in liquid media. Our data emphasize the role of F-pilus flexibility both in efficiently sampling a large volume surrounding donor cells in liquid culture and in establishing and maintaining cell–cell contact. Additionally and unexpectedly, we infer that extension and retraction are accompanied by rotation about the long axis of the filament.

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