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1.
Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances. 相似文献
2.
Neha Gadhari Mirren Charnley Mattia Marelli Jürgen Brugger Matthias Chiquet 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2013,1833(12):3415-3425
Randomly spread fibroblasts on fibronectin-coated elastomeric membranes respond to cyclic strain by a varying degree of focal adhesion assembly and actin reorganization. We speculated that the individual shape of the cells, which is linked to cytoskeletal structure and pre-stress, might tune these integrin-dependent mechanotransduction events. To this aim, fibronectin circles, squares and rectangles of identical surface area (2000 μm2) were micro-contact printed onto elastomeric substrates. Fibroblasts plated on these patterns occupied the corresponding shapes. Cyclic 10% equibiaxial strain was applied to patterned cells for 30 min, and changes in cytoskeleton and cell-matrix adhesions were quantified after fluorescence staining. After strain, megakaryocytic leukemia-1 protein translocated to the nucleus in most cells, indicating efficient RhoA activation independently of cell shape. However, circular and square cells (with radial symmetry) showed a significantly greater increase in the number of actin stress fibers and vinculin-positive focal adhesions after cyclic strain than rectangular (bipolar) cells of identical size. Conversely, cyclic strain induced larger changes in pY397-FAK positive focal complexes and zyxin relocation from focal adhesions to stress fibers in bipolar compared to symmetric cells. Thus, radially symmetric cells responded to cyclic strain with a larger increase in assembly, whereas bipolar cells reacted with more pronounced reorganization of actin stress fibers and matrix contacts. We conclude that integrin-mediated responses to external mechanical strain are differentially modulated in cells that have the same spreading area but different geometries, and do not only depend on mere cell size. 相似文献
3.
In the present work the behavior of mitochondria and lysosomes during cell spreading has been investigated in normal conditions and under ATP-synthesis inhibitors: sodium aside and N,N-dicyclohexylcarbodiimide (DCCD). In the control culture, microtubules run along the stable edge and perpendicular to the leading edge in most of spreading cells. As a whole, microtubules form a dense network in these cells. However, the radial cells contain bundles of microtubules, radiating from the perinuclear area or form circular arrays around the nucleus. The microtubule network is more dense under inhibitory treatment, than in control conditions. In the control culture the spherical cells display numerous small mitochondria (staining with Rhodamine 123). In the process of cell spreading some elongated mitochondria appear, most of them being localized in the perinuclear area. The mitochondria of cells with radial microtubule organization are directed towards the cell periphery, while in cells with circular bundles of microtubules the mitochondria are localized chaotically. Under DCCD treatment the mitochondria retain the staining for 2-3 h. In the spreading cells, round mitochondria may be distributed all over the cytoplasm. In the presence of sodium aside the mitochondria are not stained. However, by means of phase contrast microscopy some disoriented thread-shaped structures are observed, obviously corresponding to mitochondria. In the control conditions, lysosomes (stained with Acridine orange) in spreading cells are dispersed chaotically, all over the cytoplasm, or are localized in the perinuclear area. In the presence of sodium aside lysosomes are observed only in the perinuclear area. Under DCCD treatment lysosomes do not accumulate the dye. Thus, the cytoskeleton modification and changes in the properties of membrane organelles, induced by ATP-synthesis inhibitors, do not prevent attachment, spreading or cell polarization. 相似文献
4.
Stress fibers are actin bundles encompassing actin filaments, actin-crosslinking, and actin-associated proteins that represent the major contractile system in the cell. Different types of stress fibers assemble in adherent cells, and they are central to diverse cellular processes including establishment of the cell shape, morphogenesis, cell polarization, and migration. Stress fibers display specific cellular organization and localization, with ventral fibers present at the basal side, and dorsal fibers and transverse actin arcs rising at the cell front from the ventral to the dorsal side and toward the nucleus. Perinuclear actin cap fibers are a specific subtype of stress fibers that rise from the leading edge above the nucleus and terminate at the cell rear forming a dome-like structure. Perinuclear actin cap fibers are fixed at three points: both ends are anchored in focal adhesions, while the central part is physically attached to the nucleus and nuclear lamina through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we discuss recent work that provides new insights into the mechanism of assembly and the function of these actin stress fibers that directly link extracellular matrix and focal adhesions with the nuclear envelope. 相似文献
5.
We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles. Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF-2 granules moving to the cell periphery and FM4-64-labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF-2 movements toward the cell periphery. The FGF-2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased. FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus. FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms. 相似文献
6.
Moving and stationary actin filaments are involved in spreading of postmitotic PtK2 cells 总被引:6,自引:3,他引:3
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《The Journal of cell biology》1993,122(4):833-843
We have investigated spreading of postmitotic PtK2 cells and the behavior of actin filaments in this system by time-lapse microscopy and photoactivation of fluorescence. During mitosis PtK2 cells round up and at cytokinesis the daughter cells spread back to regain their interphase morphology. Normal spreading edges are quite homogenous and are not comprised of two distinct areas (lamellae and lamellipodia) as found in moving edges of interphase motile cells. Spreading edges are connected to a network of long, thin, actin-rich fibers called retraction fibers. A role for retraction fibers in spreading was tested by mechanical disruption of fibers ahead of a spreading edge. Spreading is inhibited over the region of disruption, but not over neighboring intact fibers. Using photoactivation of fluorescence to mark actin filaments, we have determined that the majority of actin filaments move forward in spreading edges at the same rate as the edge. As far as we are aware, this is the first time that forward movement of a cell edge has been correlated with forward movement of actin filaments. In contrast, actin filaments in retraction fibers remain stationary with respect to the substrate. Thus there are at least two dynamic populations of actin polymer in spreading postmitotic cells. This is supported by the observation that actin filaments in some spreading edges not only move forward, but also separate into two fractions or broaden with time. A small fraction of postmitotic cells have a spreading edge with a distinct lamellipodium. In these edges, marked actin polymer fluxes backward with respect to substrate. We suggest that forward movement of actin filaments may participate in generating force for spreading in postmitotic cells and perhaps more generally for cell locomotion. 相似文献
7.
The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h incubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell survival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells attached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth. 相似文献
8.
Beardsley A Fang K Mertz H Castranova V Friend S Liu J 《The Journal of biological chemistry》2005,280(5):3541-3547
The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration. 相似文献
9.
Hazuki Yamada Koji Tsutsumi Yuki Nakazawa Yoshio Shibagaki Seisuke Hattori Yasutaka Ohta 《PloS one》2016,11(1)
FilGAP is a Rac-specific GTPase-activating protein (GAP) that suppresses lamellae formation. In this study, we have identified RBM10 (RNA Binding Motif domain protein 10) as a FilGAP-interacting protein. Although RBM10 is mostly localized in the nuclei in human melanoma A7 cells, forced expression of Src family tyrosine kinase Fyn induced translocation of RBM10 from nucleus into cell peripheries where RBM10 and FilGAP are co-localized. The translocation of RBM10 from nucleus appears to require catalytic activity of Fyn since kinase-negative Fyn mutant failed to induce translocation of RBM10 in A7 cells. When human breast carcinoma MDA-MB-231 cells are spreading on collagen-coated coverslips, endogenous FilGAP and RBM10 were localized at the cell periphery with tyrosine-phosphorylated proteins. RBM10 appears to be responsible for targeting FilGAP at the cell periphery because depletion of RBM10 by siRNA abrogated peripheral localization of FilGAP during cell spreading. Association of RBM10 with FilGAP may stimulate RacGAP activity of FilGAP. First, forced expression of RBM10 suppressed FilGAP-mediated cell spreading on collagen. Conversely, depletion of endogenous RBM10 by siRNA abolished FilGAP-mediated suppression of cell spreading on collagen. Second, FilGAP suppressed formation of membrane ruffles induced by Fyn and instead produced spiky cell protrusions at the cell periphery. This protrusive structure was also induced by depletion of Rac, suggesting that the formation of protrusions may be due to suppression of Rac by FilGAP. We found that depletion of RBM10 markedly reduced the formation of protrusions in cells transfected with Fyn and FilGAP. Finally, depletion of RBM10 blocked FilGAP-mediated suppression of ruffle formation induced by EGF. Taken together, these results suggest that Src family tyrosine kinase signaling may regulate FilGAP through association with RBM10. 相似文献
10.
Renu Vishavkarma Swetavalli Raghavan Chandrashekar Kuyyamudi Abhijit Majumder Jyotsna Dhawan Pramod A. Pullarkat 《PloS one》2014,9(9)
It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus. 相似文献
11.
SYNOPSIS. Mechanism of locomotion of deep cells of Fundulusheteroclitus was studied in vivo during gastrulation with theaid of time lapse cinemicrography (Nomarski differential interferencecontrast optics), scanning electron microscopy of cells knownto be moving at the time of fixation, and cell culture. Theseare our findings. 1) Deep cells usually move rapidly, at about1015 µ/min, regardless of whether they move byblebbing or spreading. Evidence suggests that this high speedis associated with weak adhesion of the trailing edge: it remainsrounded, without large retraction fibers, and it advances continuouslywith advance of the leading edge, not sporadically, as it wouldif it adhered strongly. 2) In contrast, when stationary cellsin close contact separate, they remain connected by retractionfibers, suggesting strong punctate adhesions. 3) Locomotionby shortening of a long lobopodium is really a form of spreadingmovement; the tip of a lobopodium always spreads. Also, sincespeed of shortening decreases with continuance, it may dependprimarily on elastic recoil rather than active contraction.4) Fundulus deep cells appear to move in two ways: a) protrusionof blebs, followed by much cytoplasmic flow; b) protrusion oflamellipodia, accompanied by filopodia and frequent cell shortening.5) Filopodia were not found except at the leading edge of aspreading lamellipodium and often spread themselves; perhapsfilopodia and lamellipodia are interconvertible. 6) A lamellipodialmargin may form undulations in vivo that move backward likeruffles in vitro. 7) At all times, whether stationary or moving,the surface of deep cells is smooth, raising unanswered questionsconcerning the source of surface for their rapid protrusiveactivity. 相似文献
12.
The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor. 相似文献
13.
T J Mitchison 《Cell motility and the cytoskeleton》1992,22(2):135-151
When PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase-dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 microns/minute. As they move in they increase progressively in phase-density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed. 相似文献
14.
T M Svitkina 《Tsitologiia》1988,30(7):861-866
Spread fibroblasts contain a dense microfilament sheath under the dorsal cell surface in the endoplasmic region. The formation of the sheath during spreading of mouse embryo fibroblasts was studied using electron microscopy of platinum replicas. At the first stages of spreading the actin meshwork comprising the pseudopodial cytoskeleton arises at the cell edges. The actin of unattached pseudopodia moves centripetally and forms a circular microfilament bundle at the endoplasm periphery. Simultaneously, the microfilament cortex in the endoplasm appears to disassemble. Due to a continuous supply of polymerized actin from the periphery to the circular bundle the latter becomes wider to cover gradually the endoplasm and to form the microfilament sheath. Anchoring of centripetally moving microfilaments at the sites of cellular contacts with the substratum leads to the formation of radial actin bundles. 相似文献
15.
《Cell biology international reports》1981,5(10):975-980
Curved microfilamentous structures are frequently found in the leading lamellae of cultured fibroblasts. These structures, termed arcs, form parallel to, and about 8 μm from, the lamellar margin. Arcs move centripetally through the lamella beneath the dorsal surface at speeds of 1.5 to 3.0 μm min−1 and they disappear in front of the nucleus. The movement of arcs is related to the movement of particles transported on the cell surface. Arcs show some structural and biochemical similarities to the actomyosin stress fibres, but arcs are not obviously functional in applying force against the substratum during cell locomotion. Arcs provide clues to the organisation and regulation of the microfilament system for movement. 相似文献
16.
Cell shape provides global control of focal adhesion assembly 总被引:1,自引:0,他引:1
Chen CS Alonso JL Ostuni E Whitesides GM Ingber DE 《Biochemical and biophysical research communications》2003,307(2):355-361
Cell spreading was controlled independently of the amount and density of immobilized integrin ligand by culturing cells on single adhesive islands of different sizes (100-2500 microm(2)) and shapes (squares, circles, and lines) or on many smaller (3-5 microm diameter) circular islands that were coated with a saturating density of fibronectin and separated by non-adhesive regions. The amount of focal adhesions (FAs) containing vinculin and phosphotyrosine increased in direct proportion to cell spreading under all conditions. FAs localized asymmetrically along the periphery of the small islands that experienced highest tensional stress, and FA staining increased when cytoskeletal tension was stimulated with thrombin, whereas inhibitors of contractility promoted FA disassembly. Thus, these findings demonstrate the existence of an "inside-out" mechanism whereby global cell distortion produces increases in cytoskeletal tension that feed back to drive local changes in FA assembly. This complex interplay between cell morphology, mechanics, and adhesion may be critical to how cells integrate from and function in living tissues. 相似文献
17.
Yuan Li David Lovett Qiao Zhang Srujana Neelam Ram?Anirudh Kuchibhotla Ruijun Zhu Gregg?G. Gundersen Tanmay?P. Lele Richard?B. Dickinson 《Biophysical journal》2015,109(4):670-686
The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes. 相似文献
18.
A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific. 相似文献
19.
Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 microM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. 'Cytoskeletons' were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of alpha-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized. 相似文献
20.
We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment, cells start blebbing, which continues for different times and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic way through stochastic formation of lamellipodia. A second mode that occurs in 15% of cells was rapid, isotropic spreading via formation of circular lamella. The rate of spreading was maximal at the beginning and decreased during the first hour according to a logarithmic law. After 60 min, many cells formed stable edges and started to migrate on the substrate. However, the cell area slowly continued to increase. Actin bundles were formed 20 min after cell attachment. They first run along the cell boundary. This system disassembled within 20–40 min and was substituted with stress fibers crossing the cell. In isotropically spread cells, no actin bundles were seen. Microtubules in the spreading cells enter into large blebs and all nascent lamellas; later, they form a radial array. When MTs have been depolymerized or stabilized blebbing started, before cells attach to the substrate and continue much longer than in control cells. After both treatments, the initial rate of spreading decreases several-fold and remains constant for many hours. After 24 h, the mean area occupied by cells with an altered MT system was the same as in control. Alteration of the MT system had a moderate effect on the actin system: formation of actin cables occurred at the same time as in control (within 20 min upon cell attachment); however, they started growing even in cells undergoing prolonged blebbing. Actin cables running along the cell margin were similar to those in control cells, but they did not disappear for up to 1 h. When stabilized, MTs form a chaotic array: they do not enter blebs and, in spread cells, run parallel to the cell margin at a distance of 3–5 μm. We conclude that dynamic MTs speed up completion of blebbing and promote early stages of fibroblast spreading. 相似文献