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PubMed1985

Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.

We have developed a new method based on total internal reflection fluorescence to map the shape of the region between glass and the lower surface of a living cell spread upon it. Fluorescently labeled nonadsorbing volume marker molecules that cannot penetrate into the cell are locally stimulated so that they fluoresce only very near the glass/medium interface. The total fluorescence intensity at any point beneath the cell depends on the cell-to-glass separation. Focal contacts appear as dark areas owing to dye exclusion, whereas when the gap exceeds approximately 150 nm, fluorescence asymptotes to the bright background level. Our technique provides greater contrast than does interference reflection microscopy and is free from errors due to cytoplasmic thickness and refractive index inhomogeneities arising from cytoplasmic inclusions. We have shown that sufficiently large molecules suffer steric exclusion from regions accessible to small molecules, which gives new information about lateral penetrability in the apposition region.

Animals↗
PubMed1986

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Actinin↗
PubMed1987

The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading.

Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.

Amino Acid Sequence↗
PubMed1987

Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact.

The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F-actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F-actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F-actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear.

Actins↗
PubMed1988

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

Actins↗
PubMed1988

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata.

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.

Actin Cytoskeleton↗
PubMed1988

Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix.

Transforming growth factor beta (TGF-beta) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-beta. We have correlated this inhibitory effect with an increase in cellular fibronectin synthesis and deposition in a two-dimensional culture system using specific matrix coatings. The inhibitory effect was mimicked by addition of soluble fibronectin to cultures. In contrast, TGF-beta was found to elicit the formation of tube-like structures (mimicking angiogenesis) when microvascular endothelial cells were grown in three-dimensional collagen gels. In this culture system TGF-beta elicited rapid extensive formation of complex, branching, tube-like structures, while cell proliferation was not inhibited. These data confirm and support the hypothesis that TGF-beta is angiogenic and may exert some of its effects through modulation of matrix synthesis and are consistent with the hypothesis that the organization of the extracellular environment influences cellular responses to this "panregulin."

Angiogenesis Inducing Agents↗
PubMed1988

Fluorescent microtubules break up under illumination.

We have synthesized three new fluorescent analogues of tubulin, using fluorescein or rhodamine groups attached to N-hydroxy-succinimidyl esters, and have partially characterized the properties of these analogues. We have also further characterized the tubulin derivatized with dichlorotriazinyl-aminofluorescein that has previously been used in this and other laboratories. Our results show that all four analogues assemble into microtubules which break up when exposed to light of the wavelengths that excite fluorescence. This sensitivity places severe constraints on the use of these analogues in studies of microtubule dynamics.

Animals↗
PubMed1989

Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

Guanosine Diphosphate↗
PubMed1990

Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement.

Both cellular motility and intracellular particle movement are compared between normal Dictyostelium amebae of strain AX4 and amebae of a myosin II heavy chain null mutant, HS2215, using the computer assisted "Dynamic Morphology System." In AX4 cells rapidly translocating in buffer, cytoplasmic expansion is apical and the majority of intracellular particles move anteriorly, towards the site of expansion. When these cells are pulsed with 10(-6) M cAMP, the peak concentration of the natural cAMP wave, cells stop translocating and average particle velocity decreases threefold within 2-4 s after cAMP addition. After 8 s, there is a partial rebound both in cytoplasmic expansion and particle velocity, but in both cases, original apical polarity is lost. In HS2215 cells in buffer, both cellular translocation and average particle velocity are already at the depressed levels observed in normal cells immediately after cAMP addition, and no anterior bias is observed in either the direction of cytoplasmic expansion or the direction of particle movement. The addition of cAMP to myosin-minus cells results in no additional effect. The results demonstrate that myosin II is necessary for (a) the rapid rate of intracellular particle movement, (b) the biased anterior directionality of particle movement, and (c) the rapid inhibition of particle movement by cAMP.

Biological Transport↗
PubMed1990

Mechanics of fibroblast locomotion: quantitative analysis of forces and motions at the leading lamellas of fibroblasts.

Shapes, motions, and forces developed in lamellipodia and ruffles at the leading edges of primary chick embryo heart fibroblasts were characterized by differential interference contrast microscopy and digital video enhancement techniques. The initial extension of the cell edge to form a thin, planar lamellipodium parallel to the substrate surface was analyzed in two dimensions with temporal and spatial resolution of 3 s and 0.2 micron, respectively. An extension begins and ends with brief, rapid acceleration and deceleration separated by a long period of nearly constant velocity in the range of 4-7 microns/min. Extensions and retractions were initiated randomly over time. As demonstrated by optical sectioning microscopy, the extended lamellipodia formed ruffles by sharply bending upward at hinge points 2-4 microns behind their tips. Surprisingly, ruffles continued to grow in length at the same average rate after bending upward. They maintained a straight shape in vertical cross section, suggesting the ruffles were mechanically stiff. The forces required to bend ruffles of these cells and of BC3H1 cells were measured by pushing a thin quartz fishpole probe against the tip of a ruffle 7-10 microns from its base either toward or away from the center of the cell. Force was determined by measuring the bending of the probe monitored by video microscopy. Typically the probe forced the ruffle to swing rigidly in an arc about an apparent hinge at is base, and ruffles rapidly, and almost completely, recovered their shape when the probe was removed. Hence, ruffles appeared to be relatively stiff and to resist bending with forces more elastic than viscous, unlike the cell body. Ruffles on both types of cells resisted bending with forces of 15-30 mudyn/microns of displacement at their tips when pushed toward or away from the cell center. The significance of the observations for mechanisms of cell locomotion is discussed.

Animals↗
PubMed1990

Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin alpha 3 beta 1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins alpha 6 beta 4 and alpha 3 beta 1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that alpha 6 beta 4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with alpha 3 beta 1-FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs. (b) Inhibition of HFK adhesion with combined anti-alpha 3 beta 1 (P1B5) and anti-alpha 6 beta 4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while alpha 3 beta 1 played a dominant role in spreading. (c) alpha 6 beta 4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both alpha 6 beta 4-SACs and alpha 3 beta 1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to alpha 3 beta 1-FAs, alpha 6 beta 4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for alpha 3 beta 1-FAs. (e) alpha 6 beta 4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with alpha 6 beta 4. (f) We suggest that alpha 6 beta 4/BPA-SACs in culture restrict migration of HFKs on ECM while alpha 3 beta 1-FAs form dynamic adhesions in spreading and migrating cells. alpha 6 beta 4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.

Autoantigens↗
PubMed1991

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes.

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.

Basement Membrane↗
PubMed1991

Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts.

The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.

Actins↗
PubMed1991

Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

Guanosine Triphosphate↗
PubMed1992

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

Actin Cytoskeleton↗
PubMed1992

Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue.

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.

Animals↗
PubMed1992

Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.

Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell movement, time-lapse, low-light video microscopy was used to image integrins on living cells tagged with a fluorescent anti-beta 1 integrin antibody. Integrin macroaggregates remain fixed with respect to the substratum, despite the fact that they fluctuate in size, density, and shape over a period of minutes. Upon detachment of the cell rear, as much as 85% of the beta 1 integrin density of a macroaggregate remains behind on the substrate, along with both alpha 5 integrin and fibronectin. Release of the cell rear does not involve cleavage of the beta 1 integrin cytoplasmic domain from the remainder of the protein. These results indicate that cell motility does not require regulated detachment of integrin receptors from the substrate. On the other hand, cytoskeletal components and a variable fraction of the integrins are carried forward with the cell during detachment, suggesting that some type of cortical disassembly process does occur. Integrin macroaggregate structures are not recycled intact after detachment of the cell rear from the substrate. They do not persist on the cell surface, nor can they be seen to be engulfed by vesicles; yet, some of the individual integrins that make up these macroaggregates are eventually transported forward by both vesicular and cell-surface routes. Antibody-tagged integrins accumulate in dense patches at the lateral edges and dorsal surface of the cell, and move forward on the cell surface. The tagged integrins also enter cytoplasmic vesicles, which move forward within the cytoplasm. Macroaggregates generally form and grow at the cell front; however, application of fluorescent antibody causes integrins to disappear from the leading edge. Therefore, it has not been possible to directly visualize the recycling of the forward moving tagged integrins into new macroaggregates at the cell front. Surprisingly, under these conditions cells move normally despite the absence of any delivery of tagged integrin to the leading edge, indicating that recycling of integrins to the lamella is not required for apparently normal motility.

Animals↗