scholarly journals ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

2014 ◽  
Vol 25 (7) ◽  
pp. 1111-1126 ◽  
Author(s):  
Merja Joensuu ◽  
Ilya Belevich ◽  
Olli Rämö ◽  
Ilya Nevzorov ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Network Architecture ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Actin Depolymerization ◽  
Specific Subset ◽  
3D Network ◽  
3D Electron Microscopy

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

2021 ◽  
Author(s):  
Alice Sandmeyer ◽  
Lili Wang ◽  
Wolfgang Hübner ◽  
Marcel Müller ◽  
Benjamin Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Cost Effective ◽  
Live Cell ◽  
Virological Synapse ◽  
Hiv 1

scholarly journals CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

scholarly journals High Rac1 activity is functionally translated into cytosolic structures with unique nanoscale cytoskeletal architecture

2019 ◽  
Vol 116 (4) ◽  
pp. 1267-1272 ◽  
Author(s):  
Daniel J. Marston ◽  
Karen L. Anderson ◽  
Mark F. Swift ◽  
Marie Rougie ◽  
Christopher Page ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Network Architecture ◽  
Structural Changes ◽  
Epithelial Mesenchymal Transition ◽  
Spatial Cues ◽  
Mesenchymal Transition ◽  
Rac1 Activity ◽  
Structural Regime ◽  
Electron Cryotomography

Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures—such as those mandated by epithelial−mesenchymal transition, for example—or resetting the region by completely dissipating.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

2009 ◽  
Vol 48 (9) ◽  
pp. 097003 ◽  
Author(s):  
Hieu M. Dang ◽  
Takehito Kawasumi ◽  
Gen Omura ◽  
Toshiyuki Umano ◽  
Shin'ichiro Kajiyama ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Live Cell ◽  
Emission Microscopy

scholarly journals Mechanisms of actin disassembly

2013 ◽  
Vol 24 (15) ◽  
pp. 2299-2302 ◽  
Author(s):  
William Brieher
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Depolymerization ◽  
Filament Dynamics ◽  
Physiological Demands ◽  
In Cells

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility and organize cytoplasm. Although much is known about how cells control actin polymerization, we do not understand how actin filaments depolymerize inside cells. I briefly describe how the combination of imaging actin filament dynamics in cells and using in vitro biochemistry progressively altered our views of actin depolymerization. I describe why I do not think that the prevailing model of actin filament turnover—cofilin-mediated actin filament severing—can account for actin filament disassembly detected in cells. Finally, I speculate that cells might be able to tune the mechanism of actin depolymerization to meet physiological demands and selectively control the stabilities of different actin arrays.

scholarly journals Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

2021 ◽  
Author(s):  
Sam Walcott ◽  
David M Warshaw
Keyword(s):  
Phase Transition ◽  
Actin Filament ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Fold Increase ◽  
Coarse Grained ◽  
Actin Network ◽  
Cargo Delivery ◽  
Myosin Va ◽  
Filament Networks

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc, is a geometric property of the actin network that depends only on the position and polarity of the actin filaments and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

scholarly journals Interchromosomal interactions: A genomic love story of kissing chromosomes

2018 ◽  
Vol 218 (1) ◽  
pp. 27-38 ◽  
Author(s):  
Philipp G. Maass ◽  
A. Rasim Barutcu ◽  
John L. Rinn
Keyword(s):  
Gene Expression ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Nuclear Architecture ◽  
Specific Gene ◽  
Love Story ◽  
Fundamental Properties ◽  
Disease States ◽  
Gene Positioning

Nuclei require a precise three- and four-dimensional organization of DNA to establish cell-specific gene-expression programs. Underscoring the importance of DNA topology, alterations to the nuclear architecture can perturb gene expression and result in disease states. More recently, it has become clear that not only intrachromosomal interactions, but also interchromosomal interactions, a less studied feature of chromosomes, are required for proper physiological gene-expression programs. Here, we review recent studies with emerging insights into where and why cross-chromosomal communication is relevant. Specifically, we discuss how long noncoding RNAs (lncRNAs) and three-dimensional gene positioning are involved in genome organization and how low-throughput (live-cell imaging) and high-throughput (Hi-C and SPRITE) techniques contribute to understand the fundamental properties of interchromosomal interactions.

scholarly journals The repressive genome compartment is established early in the cell cycle before forming the lamina associated domains

2018 ◽  
Author(s):  
TR Luperchio ◽  
MEG Sauria ◽  
VE Hoskins ◽  
X Wong ◽  
E DeBoy ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulation Of Gene Expression ◽  
Three Dimensional ◽  
Nuclear Lamina ◽  
Early Event ◽  
Chromosome Conformation ◽  
3D Genome ◽  
Dynamic Relationship ◽  
Organizational Principles

AbstractThree-dimensional (3D) genome organization is thought to be important for regulation of gene expression. Chromosome conformation capture-based studies have uncovered ensemble organizational principles such as active (A) and inactive (B) compartmentalization. In addition, large inactive regions of the genome associate with the nuclear lamina, the Lamina Associated Domains (LADs). Here we investigate the dynamic relationship between A/B-compartment organization and the 3D organization of LADs. Using refined algorithms to identify active (A) and inactive (B) compartments from Hi-C data and to define LADs from DamID, we confirm that the LADs correspond to the B-compartment. Using specialized chromosome conformation paints, we show that LAD and A/B-compartment organization are dependent upon chromatin state and A-type lamins. By integrating single-cell Hi-C data with live cell imaging and chromosome conformation paints, we demonstrate that self-organization of the B-compartment within a chromosome is an early event post-mitosis and occurs prior to organization of these domains to the nuclear lamina.

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