Effect of colcemid on the distribution of pseudopodial activity in fibroblasts. Microtubule-independent stabilization of cell surface

The effect of microtubule-destroying drugs on the pseudopodial activity of cultured fibroblasts was examined. As previously described, colcemid and similar drugs caused almost complete disappearance of inactive zones of the edge of normal mouse fibro-blasts. However, similarly treated, poorly spread, transformed TLSF cells often had inactive zones at the edges; these zones in colcemid-treated cells were shorter than similar zones in control TLSF cells. It is suggested that in addition to previously described microtubule-dependent stabilization of cell surface there exists another, more local and less effective microtubule-independent stabilization process.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts

Microtubules are involved in actin-based protrusion at the leading-edge lamellipodia of migrating fibroblasts. Here we show that the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole activates Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. We suggest that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration.     

Locomotion of epithelial cells : Factors involved in extension of the leading edge

Epithelial cells, like fibroblasts, spread by the active extension of their flattened leading edge. The role of microtubules, microfilaments, and internal hydrostatic pressure in this spreading was studied. While colchicine and colcemid have no effect, cytochalasin B, which affects the microfilament meshwork in the region of the leading edge without affecting general protein synthesis, and sorbitol, which presumably reduces the cell's internal hydrostatic pressure, reversibly prevent the extension of the flattened leading edge. These results are discussed in relation to the mechanism of this extension.     

Directional motility induced by epidermal growth factor requires Cdc42

Cell motility is actuated by a host of intracellular signaling cascades that result in movement of the cell in one direction, even without an external gradient. Phospholipase C-gamma (PLCgamma) has been shown to be important for growth factor-induced lamellipodial protrusion at the front of the cell while Cdc42 has been implicated in both filopodium formation at the leading edge and control of polarity of migrating cells. We asked whether these asymmetries in effector molecules may be linked. When we overexpressed either constitutively active, dominant negative, or GFP-tagged Cdc42, wild-type NR6 fibroblasts lost directionality, as expected. On epidermal growth factor (EGF) exposure these cells produced multiple, transient protrusions in every direction; these extensions failed to result in productive motility. GFP-tagged Cdc42 appeared transiently at edges of newly formed protrusions in EGF-stimulated cells while they moved haphazardly. While PLCgamma is distributed throughout the cell, the ratio of active, tyrosyl-phosphorylated PLCgamma was increased at the leading edge, where phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis is concentrated. This co-localization of activities may be due to Cdc42 directing PLCgamma to the cell front, as PLCgamma associated with Cdc42 in an EGF-dependent manner. We conclude that Cdc42 controls cell polarity, likely in part, through its binding to active PLCgamma.     

Morphology of the microtubule system in mouse kidney epithelial cells]

The cultured mouse kidney cells forming epithelial sheets were studied using an indirect immunofluorescence microscopy with antibodies against tubulin. These cells, as well as fibroblasts, were found to contain a well developed microtubular system sensitive to colcemid. The assembly of microtubules after washing out of colcemid began from one or two perinuclear centers, associated with the cilium-like structure. There were certain differences between the microtubular systems in epithelial cells and fibroblasts: 1) Microtubules in the fibroblasts penetrated the whole cytoplasm including the peripheral lamella whereas in the epithelial cells the lamellar cytoplasm was often free from microtubules. 2) The orientation of microtubules in the epithelial cells, unlike in the fibroblasts, was not correlated with the stable or active state of the cell margin. A possible role of microtubular system in the epithelial cells and fibroblasts is compared and discussed.     

Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.     

The arrangement of microfilaments and microtubules in the periphery of spreading fibroblasts and glial cells

Studies of spreading fibroblasts and glial cells showed that the initial phase of the spreading process on a solid substratum proceeds by sequential development of different kinds of protrusions. Initially there is a high blebbing activity which is followed by development of small lamellipodia and somewhat later microspikes are formed. In the periphery of the spreading cells several types of microfilament organizations are displayed, these seem to be related to different stages in the cycles of extensions and retractions performed by the lamellipodia. The presence of microtubules and their relation to the different microfilament organizations are also shown.     

Maturation of Filopodia Shaft Adhesions Is Upregulated by Local Cycles of Lamellipodia Advancements and Retractions

While cell-substrate adhesions that form between the protruding edge of a spreading cell and flat surfaces have been studied extensively, processes that regulate the maturation of filopodia adhesions are far less characterized. Since little is known about how the kinetics of formation or disassembly of filopodia adhesions is regulated upon integration into the lamellum, a kinetic analysis of the formation and disassembly of filopodia adhesions was conducted at the leading edge of β3-integrin-EGFP-expressing rat embryonic fibroblasts spreading on fibronectin-coated glass or on soft polyacrylamide gels. Filopodia β3-integrin adhesions matured only if the lamellipodium in their immediate vicinity showed cyclic protrusions and retractions. Filopodia β3-integrin shaft adhesions elongated rapidly when they were overrun by the advancing lamellipodium. Subsequently and once the lamellipodium stopped its advancement at the distal end of the filopodia β3-integrin adhesion, these β3-integrin shaft adhesions started to grow sidewise and colocalize with the newly assembled circumferential actin stress fibers. In contrast, the suppression of the cyclic protrusions and retractions of the lamellipodium by blocking myosin light chain kinase suppressed the growth of filopodia adhesion and resulted in the premature disassembly of filopodia adhesions. The same failure to stabilize those adhesions was found for the advancing lamellipodium that rapidly overran filopodia shaft adhesions without pausing as seen often during fast cell spreading. In turn, plating cells on soft polyacrylamide gels resulted in a reduction of lamellipodia activity, which was partially restored locally by the presence of filopodia adhesions. Thus filopodia adhesions could also mature and be integrated into the lamellum for fibroblasts on soft polyacrylamide substrates.     

Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts

Myosin I is present in Swiss 3T3 fibroblasts and its localization reflects a possible involvement in the extension and/or retraction of protrusions at the leading edge of locomoting cells and the transport of vesicles, but not in the contraction of stress fibers or transverse fibers. An affinity-purified polyclonal antibody to brush border myosin I colocalizes with a polypeptide of 120 kD in fibroblast extracts. Within initial protrusions of polarized, migrating fibroblasts, myosin I exhibits a punctate distribution, whereas actin is diffuse and myosin II is absent. Myosin I also exists in linear arrays parallel to the direction of migration in filopodia and microspikes, established protrusions, and within the leading lamellae of migrating cells. Myosin II and actin colocalize along transverse fibers in the lamellae of migrating cells, while myosin I displays no definitive organization along these fibers. During contractions of actin-based fibers, myosin II is concentrated in the center of the cell, while the distribution of myosin I does not change. Thus, myosin I is found at the correct location and time to be involved in the extension and/or retraction of protrusions and the transport of vesicles. Myosin II-based contractions in more posterior cellular regions could generate forces to separate cells, maintain a polarized cell shape, maintain the direction of locomotion, maximize the rate of locomotion, and/or aid in the delivery of cytoskeletal/contractile subunits to the leading edge.     

Intensification and redistribution of protrusive activity is a feature of tumor transformation and is associated with an increase of the invasive potential of cells

The abilities of tumor cells to invade and metastasize are frequent causes of death of cancer patients. Studying the mechanisms of cell motility alterations and acquisition of enhanced metastatic potential as the result of transformation is an important aspect in current cell biology. The initial and determinant step of cell motility is the formation of active cell edge with protrusions based on the Arp2/3-dependent actin polymerization. We used three different cell systems as examples of different models of tumor transformation to study the alteration and redistribution of protrusive activity caused by transformation in fibroblasts. We analyzed relationships between detected alterations and the acquisition of increased invasive potential by cells. Active edge of untransformed fibroblasts occupies about 50% of the cell perimeter and is concentrated at the cell front. There are well pronounced stable regions at the lateral cell edges. Tumor transformation causes redistribution of protrusive activity of fibroblasts irrespective of their origin and the nature of transforming agents. The length of active edges significantly increases, up to 92% of the total perimeter in fibrosarcoma cells of tumor origin. These cells have practically no stable edges. The intensity of protrusive activity of transformed cells is also increased. Single transformed cells show a decrease in the directionality and rate of migration on 2D substrate without special stimulation. Instead, they gain the capacity to migrate in 3D and to invade matrigel. These abilities increase in parallel with the intensification of edge activity. We showed that invasive abilities are not associated with the activation of matrix metalloproteinases in the studied cell systems. Our data demonstrate that the increase of length of active edge could be considered as an additional feature of cell transformation together with the reduction of stress fiber and focal adhesions and that the excessive protrusive activity results in the development of explorative migration of tumor cells.     

Local and global measures of shape dynamics

The shape and motion of cells can yield significant insights into the internal operation of a cell. We present a simple, yet versatile, framework that provides multiple metrics of cell shape and cell shape dynamics. Analysis of migrating Dictyostelium discoideum cells shows that global and local metrics highlight distinct cellular processes. For example, a global measure of shape shows rhythmic oscillations suggestive of contractions, whereas a local measure of shape shows wave-like dynamics indicative of protrusions. From a local measure of dynamic shape, or boundary motion, we extract the times and locations of protrusions and retractions. We find that protrusions zigzag, while retractions remain roughly stationary along the boundary. We do not observe any temporal relationship between protrusions and retractions. Our analysis framework also provides metrics of the boundary as whole. For example, as the cell speed increases, we find that the cell shape becomes more elongated. We also observe that while extensions and retractions have similar areas, their shapes differ.     

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.     

Analysis of changes induced by oncogene <Emphasis Type="Italic">N-RAS</Emphasis> expression in pattern and distribution of pseudopodial activity of fibroblasts

It is not known which morphological properties of fibroblasts induced by malignant transformation modulate their migration pattern. We studied the changes in the distribution and dynamics of the leading edge of 10(3) mouse fibroblasts after their transformation by oncogene N-RAS ^asp13 and analyzed the changes in the pattern of cell migration. Transformation proved to increase the leading edge proportion and to considerably redistribute pseudopodial activity along the cell edge. As the result of transformation, small pseudopodia are formed in the stable lateral regions of the cell edge typical of normal fibroblasts, i.e., the lateral edge is no more truly stable. In addition, pseudopodial activity of the leading edge in transformed fibroblasts proved higher compared to normal ones. It is necessary to notice, the leading edge activity is equally high immediately after induction in both normal and transformed fibroblasts; although, it is suppressed with time in normal cells but not in transformed ones where it remains steadily high. These properties promote the random component of malignant cell motility and modify the cell migration pattern after transformation     

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules. Cancer-related truncation mutations lack direct and indirect binding sites for microtubules in APC, suggesting that loss of this function contributes to defects in APC-mutant cells. In this study, we show that loss of APC results in disappearance of cellular protrusions and decreased cell migration. These changes are accompanied by a decrease in overall microtubule stability and also by a decrease in posttranslationally modified microtubules in the cell periphery particularly the migrating edge. Consistent with the ability of APC to affect cell shape, the overexpression of APC in cells can induce cellular protrusions. These data demonstrate that cell migration and microtubule stability are linked to APC status, thereby revealing a weakness in APC-deficient cells with potential therapeutic implications.     

Microtubule targeting of substrate contacts promotes their relaxation and dissociation.

We recently showed that substrate contact sites in living fibroblasts are specifically targeted by microtubules (Kaverina, I., K. Rottner, and J.V. Small. 1998. J. Cell Biol. 142:181-190). Evidence is now provided that microtubule contact targeting plays a role in the modulation of substrate contact dynamics. The results are derived from spreading and polarized goldfish fibroblasts in which microtubules and contact sites were simultaneously visualized using proteins conjugated with Cy-3, rhodamine, or green fluorescent protein.For cells allowed to spread in the presence of nocodazole the turnover of contacts was retarded, as compared with controls and adhesions that were retained under the cell body were dissociated after microtubule reassembly. In polarized cells, small focal complexes were found at the protruding cell front and larger adhesions, corresponding to focal adhesions, at the retracting flanks and rear. At retracting edges, multiple microtubule contact targeting preceded contact release and cell edge retraction. The same effect could be observed in spread cells, in which microtubules were allowed to reassemble after local disassembly by the application of nocodazole to one cell edge. At the protruding front of polarized cells, focal complexes were also targeted and as a result remained either unchanged in size or, more rarely, were disassembled. Conversely, when contact targeting at the cell front was prevented by freezing microtubule growth with 20 nM taxol and protrusion stimulated by the injection of constitutively active Rac, peripheral focal complexes became abnormally enlarged. We further found that the local application of inhibitors of myosin contractility to cell edges bearing focal adhesions induced the same contact dissociation and edge retraction as observed after microtubule targeting.Our data are consistent with a mechanism whereby microtubules deliver localized doses of relaxing signals to contact sites to retard or reverse their development. We propose that it is via this route that microtubules exert their well-established control on cell polarity.     

KINESIN-ASSOCIATED TRANSPORT IS INVOLVED IN THE REGULATION OF CELL ADHESION

It has been recently shown that depolymerization of microtubules induces the elongation of focal contacts at the leading edge. On the other hand, cell shape and pseudopodial activity were found to depend on the microtubule-based motor kinesin. In this paper, we examine whether kinesin is involved in controlling the dynamics of adhesive structures at the cell surface. Microinjection of an antiblocking kinesin activityin vitrocauses focal contact elongation similar to the effect of microtubule-depolymerizing drugs. Thus, the role of microtubules in cell adhesion lies in the supporting kinesin-based transport to the adhesion sites.     

Organization of the migrating chick epiblast edge: attachment sites, cytoskeleton, and early developmental changes

The radial expansion of the chick extraembryonic epiblast on the inner side of the vitelline membrane in yolk sac formation provides a useful system for study of adhesion and migration of an epithelial cell sheet. A band of specialized cells at the epiblast edge adheres by its dorsal side to the overlying vitelline membrane. The attached edge was examined by scanning electron microscopy. The attachment region (av 0.06 mm wide) extends from the advancing edge to a transitional ridge. The ridge appears to be an area of adhesion and de-adhesion. The attached surface is smooth with small surface projections and filopodia. These become more numerous and prominent with cold treatment. Epiblast cells display a filopodial/lamellipodial mode of migration in vivo and in vitro. The distribution of 4- to 7-nm microfilaments in edge cells is examined using transmission electron microscopy of whole cells. Decoration with heavy meromyosin shows that these components of the cytoskeleton contain actin. Treatment of intact blastoderms and dissociated edge cells with cytochalasin B and cold suggests that microfilaments rather than microtubules are primarily responsible for edge cell morphology. Early blastoderm cells which have not initiated migration respond to cytochalasin B, cold, and colcemid in the same way as migrating edge cells. This suggests that the differentiative change that produces the rapidly migrating edge cells does not involve a shift in the relative contribution of microtubules and microfilaments to the cytoskeleton.     

Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating Swiss 3T3 fibroblasts

The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235-247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613-1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449-478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrusions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.     

"Gap guidance" of fibroblasts and epithelial cells by discontinuous edged surfaces

Cell adhesion, shape, and directed migration are some of the fundamental processes underlying tissue development and organization. The setting of geometric limits on cellular behavior has led to the hypothesis that a continuous edge is required to elongate a cell and guide its direction of movement. The aim of this study was to examine the validity of this hypothesis by examining the response of human gingival fibroblasts and periodontal ligament epithelial cells, to microfabricated surfaces that incorporate discontinuous edges. Cell response was assessed through spreading, morphology, cytoskeletal organization, and time-lapse microscopy, on substrata with a pattern of repeated open boxes with gaps at the corners. Fibroblasts attached and spread within 6 h, adopting either a square, triangular, or diagonally elongated morphology. Epithelial cells took longer to adhere, but were observed to adopt morphologies similar to those of the fibroblasts. Addition of colcemid or cytochalasin-D attenuated the orientation and alignment of both fibroblasts and epithelial cells. Fibroblasts and epithelial cell migration was guided diagonally in their movement through gaps in the square pattern, demonstrating that a continuous edge is not a prerequisite for guided cell migration.