scholarly journals cNap1 bridges centriole contact sites to maintain centrosome cohesion

2021 ◽  
Author(s):  
Robert Mahen
Keyword(s):  
Cell Migration ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spatial Proximity ◽  
Contact Site ◽  
Physical Force ◽  
Cellular Processes ◽  
Contact Sites ◽  
Reduced Viscosity ◽  
Membrane Bound

Centrioles are non-membrane bound organelles that participate in fundamental cellular processes through their ability to form physical contacts with other structures. During interphase, two mature centrioles can associate to form a single centrosome - a phenomenon known as centrosome cohesion. Centrosome cohesion is important for processes such as cell migration, and yet how it is maintained is unclear. Current models indicate that pericentriolar fibres termed rootlets, also known as the centrosome linker, entangle to maintain centriole proximity. Here, I uncover a new centriole-centriole contact site and mechanism of centrosome cohesion, based on coalescence of the proximal centriole component cNap1. Using live-cell imaging of endogenously tagged cNap1, I show that proximal centrioles form dynamic contacts in response to physical force from the cytoskeleton. Expansion microscopy reveals that cNap1 bridges between these contact sites, physically linking proximal centrioles on the nanoscale. When ectopically tethered to organelles such as lysosomes, cNap1 forms viscous and cohesive condensates that promote organelle spatial proximity. Conversely, cNap1 mutants with reduced viscosity are unable to maintain centrosome cohesion. These results define a previously unrecognised mechanism of centrosome cohesion by cNap1 assemblies at the proximal centriole and illustrate how a non-membrane bound organelle forms dynamic organelle contact sites.

scholarly journals A general fluorescent light-up probe for staining and quantifying protein

2019 ◽  
Vol 6 (8) ◽  
pp. 190580 ◽  
Author(s):  
Jiawei Zou ◽  
Gangyi Chen ◽  
Feng Du ◽  
Yi Yuan ◽  
Xin Huang ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Visual Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescent Light ◽  
Cell Penetration ◽  
Cellular Processes ◽  
Low Toxicity ◽  
Protein Rich

Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. The visual analysis of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be realized in 5 min by using PyMDI-Zn as a light-up dye. Based on its cell penetration and low toxicity, PyMDI-Zn could also be applied to locate protein-rich regions and organelles in live cell imaging. Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds.

scholarly journals Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration

2011 ◽  
Vol 193 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Zhizhan Gu ◽  
Erika H. Noss ◽  
Victor W. Hsu ◽  
Michael B. Brenner
Keyword(s):  
Functional Analysis ◽  
Cell Migration ◽  
Growth Factor ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Focal Adhesions ◽  
Initial Step ◽  
Ventral Surface ◽  
Platelet Derived Growth Factor ◽  
Trailing Edges

During cell migration, integrins are redistributed from focal adhesions undergoing disassembly at the cell’s trailing edges to new focal adhesions assembling at leading edges. The initial step of integrin redistribution is thought to require clathrin-mediated endocytosis. However, whether clathrin-mediated endocytosis functions in different contexts, such as basal versus stimulated migration, has not been determined. In this paper, we examine the spatial and temporal redistribution of integrins from focal adhesions upon stimulation by growth factors. Four-dimensional confocal live-cell imaging along with functional analysis reveals that surface integrins do not undergo significant endocytosis at ventral focal adhesions upon cell stimulation with the platelet-derived growth factor. Rather, they abruptly redistribute to dorsal circular ruffles, where they are internalized through macropinocytosis. The internalized integrins then transit through recycling endosomal compartments to repopulate newly formed focal adhesions on the ventral surface. These findings explain why integrins have long been observed to redistribute through both surface-based and internal routes and identify a new function for macropinocytosis during growth factor–induced cell migration.

scholarly journals Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Michelle Seiko Lu ◽  
David G. Drubin
Keyword(s):  
Cell Migration ◽  
Genetic Analysis ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Cytoskeletal Remodeling ◽  
Cell Divisions ◽  
Rho Family

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.

scholarly journals Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

2018 ◽  
Author(s):  
Jan Huebinger ◽  
Jessica Spindler ◽  
Kristin J. Holl ◽  
Björn Koos
Keyword(s):  
Technological Progress ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cultured Cells ◽  
Chemical Fixation ◽  
Cellular Processes ◽  
Cytoplasmic Proteins ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Key Parameter

AbstractTo understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes along with long acquisition times and high phototoxicity. Physiological live cell imaging is therefore often unfeasible and chemical fixation is employed. However, fixation methods have not been rigorously reviewed to preserve patterns at the resolution at which they can be nowadays imaged. A key parameter for this is the time span until fixation is completed. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from the cells. Other small aldehydes like glyoxal or acrolein showed inferior results. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely antagonized by supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

scholarly journals Rear-polarized Wnt5a-receptor-actin-myosin-polarity (WRAMP) structures promote the speed and persistence of directional cell migration

2017 ◽  
Vol 28 (14) ◽  
pp. 1924-1936 ◽  
Author(s):  
Mary Katherine Connacher ◽  
Jian Wei Tay ◽  
Natalie G. Ahn
Keyword(s):  
Cell Migration ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Movement ◽  
Leading Edge ◽  
Cellular Mechanism ◽  
Directional Movement ◽  
New Directions ◽  
And Function ◽  
Directional Cell Migration

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration.

scholarly journals The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

2020 ◽  
Author(s):  
Xavier Pichon ◽  
Konstadinos Moissoglu ◽  
Emeline Coleno ◽  
Tianhong Wang ◽  
Arthur Imbert ◽  
...  
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Feedback Loop ◽  
Cell Imaging ◽  
Rna Localization ◽  
Dependent Manner ◽  
Rna Transport ◽  
Cellular Functions ◽  
Transport Pathway

AbstractRNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly showed single mRNAs transported by single KIF1C motors, with the 3’UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal an RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role both in transporting RNAs and in promoting their clustering within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

scholarly journals Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

2020 ◽  
Author(s):  
Andre Arashiro Pulschen ◽  
Delyan R. Mutavchiev ◽  
Kim Nadine Sebastian ◽  
Jacques Roubinet ◽  
Marc Roubinet ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Halophilic Archaea ◽  
Live Cell ◽  
Tight Coupling ◽  
Dna Compaction ◽  
Cellular Processes ◽  
Daughter Cells

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

scholarly journals Automated live cell imaging of cell migration across a microfluidic-controlled chemoattractant gradient

2015 ◽  
Vol 12 (11) ◽  
pp. vii-viii ◽  
Author(s):  
Philip Lee ◽  
Cindy S Y Chen ◽  
Terry Gaige ◽  
Paul J Hung
Keyword(s):  
Cell Migration ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell
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