HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

Morphogenetic machines revealed: Microscopy of living cells in the embryo of the frog, Xenopus laevis

1993 ◽  
Vol 51 ◽  
pp. 172-173
Author(s):  
Ray Keller
Keyword(s):  
Image Processing ◽  
Fluorescent Dyes ◽  
Cell Movement ◽  
Living Cells ◽  
Ease Of Use ◽  
Low Light ◽  
Amphibian Embryo ◽  
Large Populations ◽  
Light Fluorescence ◽  
Video Image Processing

The amphibian embryo offers advantages of size, availability, and ease of use with both microsurgical and molecular methods in the analysis of fundamental developmental and cell biological problems. However, conventional wisdom holds that the opacity of this embryo limits the use of methods in optical microscopy to resolve the cell motility underlying the major shape-generating processes in early development.These difficulties have been circumvented by refining and adapting several methods. First, methods of explanting and culturing tissues were developed that expose the deep, nonepithelial cells, as well as the superficial epithelial cells, to the view of the microscope. Second, low angle epi-illumination with video image processing and recording was used to follow patterns of cell movement in large populations of cells. Lastly, cells were labeled with vital, fluorescent dyes, and their behavior recorded, using low-light, fluorescence microscopy and image processing. Using these methods, the details of the cellular protrusive activity that drives the powerful convergence (narrowing)

scholarly journals Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

2006 ◽  
Vol 176 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Erik Sahai ◽  
Raquel Garcia-Medina ◽  
Jacques Pouysségur ◽  
Emmanuel Vial
Keyword(s):  
Cell Migration ◽  
Tumor Cell ◽  
Rho Gtpases ◽  
Rho Kinase ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Periphery ◽  
Tumor Cell Migration ◽  
Tumor Cell Motility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.

Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells

1999 ◽  
Vol 112 (17) ◽  
pp. 2833-2842 ◽  
Author(s):  
M. Mishima ◽  
E. Nishida
Keyword(s):  
Amino Acids ◽  
Cell Spreading ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Cellular Slime Mold ◽  
Cell Periphery ◽  
Terminal Amino ◽  
Wd Repeat ◽  
Leading Edges

Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64–299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64–299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.

scholarly journals Visualization of mRNA translation in living cells

2006 ◽  
Vol 175 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Alexis J. Rodriguez ◽  
Shailesh M. Shenoy ◽  
Robert H. Singer ◽  
John Condeelis
Keyword(s):  
Mrna Translation ◽  
Leading Edge ◽  
Intercellular Junctions ◽  
Mrna Localization ◽  
Living Cells ◽  
C2c12 Cells ◽  
Cell Periphery ◽  
Actin Mrna ◽  
Cell Asymmetry ◽  
Cellular Compartments

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of β-actin mRNA. Constructs coding for β-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with β-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. β-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of β-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.

scholarly journals Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hamouche ◽  
Cyrille Billaudeau ◽  
Anna Rocca ◽  
Arnaud Chastanet ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Model Organism ◽  
Living Cells ◽  
Cell Periphery ◽  
Rnase E ◽  
Content Type ◽  
Endonucleolytic Cleavage ◽  
Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

scholarly journals Specificity of Plant Rhabdovirus Cell-to-Cell Movement

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Xin Zhou ◽  
Wenye Lin ◽  
Kai Sun ◽  
Shuo Wang ◽  
Xueping Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Core Protein ◽  
Rna Viruses ◽  
Rna Virus ◽  
Cell Movement ◽  
Tomato Mosaic Virus ◽  
Cell Periphery ◽  
Nucleic Acid Binding ◽  
Core Proteins

ABSTRACTPositive-stranded RNA virus movement proteins (MPs) generally lack sequence-specific nucleic acid-binding activities and display cross-family movement complementarity with related and unrelated viruses. Negative-stranded RNA plant rhabdoviruses encode MPs with limited structural and functional relatedness with other plant virus counterparts, but the precise mechanisms of intercellular transport are obscure. In this study, we first analyzed the abilities of MPs encoded by five distinct rhabdoviruses to support cell-to-cell movement of two positive-stranded RNA viruses by usingtrans-complementation assays. Each of the five rhabdovirus MPs complemented the movement of MP-defective mutants of tomato mosaic virus and potato X virus. In contrast, movement of recombinant MP deletion mutants of sonchus yellow net nucleorhabdovirus (SYNV) and tomato yellow mottle-associated cytorhabdovirus (TYMaV) was rescued only by their corresponding MPs, i.e., SYNV sc4 and TYMaV P3. Subcellular fractionation analyses revealed that SYNV sc4 and TYMaV P3 were peripherally associated with cell membranes. A split-ubiquitin membrane yeast two-hybrid assay demonstrated specific interactions of the membrane-associated rhabdovirus MPs only with their cognate nucleoproteins (N) and phosphoproteins (P). More importantly, SYNV sc4-N and sc4-P interactions directed a proportion of the N-P complexes from nuclear sites of replication to punctate loci at the cell periphery that partially colocalized with the plasmodesmata. Our data show that cell-to-cell movement of plant rhabdoviruses is highly specific and suggest that cognate MP-nucleocapsid core protein interactions are required for intra- and intercellular trafficking.IMPORTANCELocal transport of plant rhabdoviruses likely involves the passage of viral nucleocapsids through MP-gated plasmodesmata, but the molecular mechanisms are not fully understood. We have conducted complementation assays with MPs encoded by five distinct rhabdoviruses to assess their movement specificity. Each of the rhabdovirus MPs complemented the movement of MP-defective mutants of two positive-stranded RNA viruses that have different movement strategies. In marked contrast, cell-to-cell movement of two recombinant plant rhabdoviruses was highly specific in requiring their cognate MPs. We have shown that these rhabdovirus MPs are localized to the cell periphery and associate with cellular membranes, and that they interact only with their cognate nucleocapsid core proteins. These interactions are able to redirect viral nucleocapsid core proteins from their sites of replication to the cell periphery. Our study provides a model for the specific inter- and intracellular trafficking of plant rhabdoviruses that may be applicable to other negative-stranded RNA viruses.

scholarly journals Actin Cytoskeleton Is Involved in Targeting of a Viral Hsp70 Homolog to the Cell Periphery

2005 ◽  
Vol 79 (22) ◽  
pp. 14421-14428 ◽  
Author(s):  
Alexey I. Prokhnevsky ◽  
Valera V. Peremyslov ◽  
Valerian V. Dolja
Keyword(s):  
Actin Cytoskeleton ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Fluorescent Proteins ◽  
Cell Movement ◽  
Plant Viruses ◽  
Cell Periphery ◽  
Virus Particles ◽  
Shock Proteins

ABSTRACT The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular ∼70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.

scholarly journals Myosin rings and spreading in mouse blastomeres.

1984 ◽  
Vol 99 (3) ◽  
pp. 1145-1150 ◽  
Author(s):  
J S Sobel
Keyword(s):  
Contact Region ◽  
Cell Spreading ◽  
Cell Contact ◽  
Cell Periphery ◽  
Cell Substrate ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Filament Bundles ◽  
Surface Polysaccharides ◽  
Induced Changes

The relationship between myosin organization and cell spreading in the preimplantation mouse embryo was studied by indirect immunofluorescence in embryos cultured on lectin-coated substrates. Binding of cell surface polysaccharides to substrate-bound concanavalin A and wheat germ agglutinin induced changes in myosin distribution that resembled those which occur during cell-cell contact interaction. This involved an initial loss of myosin from the contact region that was associated with the development of stable cell-substrate attachments. In addition, a ring of myosin was formed along the edge of the cells' contact to the substrate. The presence of such a ring may be related to the potential for subsequent cell spreading. A myosin ring was also identified in the apical junctional region of the outer morula cells where it similarly separated the cell periphery into contacted and free peripheral domains. Following these changes in myosin organization the embryos spread on the substrate by extension of lamellipodia. These movements were coupled to the dissolution of the myosin ring and the reorganization of myosin into filament bundles. The sequence of changes in the pattern of myosin distribution suggests that contact regulation of myosin organization plays an important role in controlling the spreading behavior of blastomeres and perhaps more generally in the organization of cells into epithelia.

scholarly journals Nanometer targeting of microtubules to focal adhesions

2003 ◽  
Vol 161 (5) ◽  
pp. 853-859 ◽  
Author(s):  
Olga Krylyshkina ◽  
Kurt I. Anderson ◽  
Irina Kaverina ◽  
Irene Upmann ◽  
Dietmar J. Manstein ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Dorsal Surface ◽  
Cell Periphery ◽  
Specific Targeting ◽  
Substrate Adhesion ◽  
Site Targeting ◽  
Molecular Dimensions ◽  
Adhesion Site

Although cell movement is driven by actin, polarization and directional locomotion require an intact microtubule cytoskeleton that influences polarization by modulating substrate adhesion via specific targeting interactions with adhesion complexes. The fidelity of adhesion site targeting is precise; using total internal reflection fluorescence microscopy (TIRFM), we now show microtubule ends (visualized by incorporation of GFP tubulin) are within 50 nm of the substrate when polymerizing toward the cell periphery, but not when shrinking from it. Multiple microtubules sometimes followed similar tracks, suggesting guidance along a common cytoskeletal element. Use of TIRFM with GFP- or DsRed-zyxin in combination with either GFP-tubulin or GFP–CLIP-170 further revealed that the polymerizing microtubule plus ends that tracked close to the dorsal surface consistently targeted substrate adhesion complexes. This supports a central role for the microtubule tip complex in the guidance of microtubules into adhesion foci, and provides evidence for an intimate cross-talk between microtubule tips and substrate adhesions in the range of molecular dimensions.

scholarly journals The lamellipodium is a myosin independent mechanosensor

2017 ◽  
Author(s):  
Patrick W. Oakes ◽  
Tamara C. Bidone ◽  
Yvonne Beckham ◽  
Austin V. Skeeters ◽  
Guillermina R. Ramirez-San Juan ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Substrate Stiffness ◽  
Cell Physiology ◽  
Cell Periphery ◽  
Initial Adhesion ◽  
Kinetics Of

AbstractThe ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young’s modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.Significance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

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