RESUMO
Changes in cell shape, anchorage and motility are all associated with the dynamic reorganisation of the architectural arrays of actin filaments that make up the actin cytoskeleton. The relative expression of these functionally different actin filament arrays is intimately linked to the pattern of contacts that a cell develops with its extracellular substrate. Cell polarity is acquired by the development of an asymmetric pattern of substrate contacts, effected in a specific, site-directed manner by the delivery of adhesion-site modulators along microtubules.
Assuntos
Actinas/química , Citoesqueleto/química , Animais , Transporte Biológico , Adesão Celular , Linhagem Celular , Movimento Celular , Polaridade Celular , Tamanho Celular , Dineínas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Proteínas rac de Ligação ao GTP , Proteína rhoB de Ligação ao GTPRESUMO
The dynamic turnover of actin filaments plays a central role in the locomotion of metazoan cells. Based on results obtained with actin labelled with a caged fluorescent probe, Theriot and Mitchison proposed a 'nucleation-release' model for the fast-moving fish keratocyte, which predicts the existence of short non-oriented filaments in the motile lamellipodium. More recent structural data on keratocyte cytoskeletons do not support this model, but are consistent with the treadmilling of long actin filaments of graded length. Taken together with Theriot and Mitchison's demonstration that the cytoskeleton remains stationary relative to the substrate in the moving keratocyte, the structural data raise the possibility that a lateral flow of filaments plays a role in lamellipodia motility.
RESUMO
The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.
Assuntos
Actinas/metabolismo , Células Cultivadas/ultraestrutura , Citoesqueleto/ultraestrutura , Animais , Adesão Celular , Movimento Celular , Embrião de Galinha , Citoesqueleto/efeitos dos fármacos , Fixadores/farmacologia , Glutaral/farmacologia , Tetróxido de Ósmio/farmacologia , Polietilenoglicóis , ÁguaRESUMO
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.
Assuntos
Membrana Celular/fisiologia , Movimento Celular , Citoesqueleto/fisiologia , Microtúbulos/fisiologia , Actinas/análise , Animais , Membrana Celular/ultraestrutura , Citoesqueleto/ultraestrutura , Fibroblastos , Imunofluorescência , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Filmes Cinematográficos , Proteínas Musculares/análise , Tubulina (Proteína)/análise , VinculinaRESUMO
Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells. The characteristics of pseudopodia differ according to the infected cell type. In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell. In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm. We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them. It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail. This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium. The tapering of the tail is attributed to a grading in length of the long, axial filaments. The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin. We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane. A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.
Assuntos
Actinas/fisiologia , Listeria monocytogenes/química , Listeria monocytogenes/ultraestrutura , Listeriose , Pseudópodes/química , Animais , Proteínas de Bactérias/fisiologia , Imunofluorescência , Células HeLa/microbiologia , Humanos , Listeria monocytogenes/fisiologia , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Microscopia Eletrônica , Microscopia de Vídeo , Pseudópodes/ultraestruturaRESUMO
From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.
Assuntos
Actinas/fisiologia , Peixes/fisiologia , Actinas/ultraestrutura , Animais , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Locomoção , Microscopia Eletrônica , Microscopia de VídeoRESUMO
By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a "cross talk" between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.
Assuntos
Microtúbulos/fisiologia , Células 3T3 , Animais , Linhagem Celular , Filamentos Intermediários/fisiologia , Camundongos , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , RatosRESUMO
We investigated the mode of association of vinculin with areas of contact between the termini of microfilament bundles and the cell membrane in sites of focal contact with the substrate by selective removal of actin from these areas. Opened-up substrate-attached membranes of chick fibroblasts as well as detergent-permeabilized cells were treated with fragmin from Physarum in the presence of Ca+2. This treatment removed actin filaments from the cytoplasmic faces of the membranes, along with several actin-associated proteins (alpha-actinin, tropomyosin, myosin, and filamin). Vinculin distribution was not affected by treatment. Moreover, rhodamine- or fluorescein-conjugated vinculin, when added to these preparations, became specifically associated with the focal contacts regardless of whether the latter were pretreated with fragmin or not. We conclude that the association of vinculin with focal contacts is largely actin-independent. We discuss the implications of these findings in the molecular mechanisms of microfilament membrane association in areas of cell contact.
Assuntos
Actinas/farmacologia , Membrana Celular/metabolismo , Proteínas Musculares/metabolismo , Animais , Cálcio/farmacologia , Comunicação Celular , Embrião de Galinha , Citoplasma/metabolismo , Fluorescência , Proteínas Musculares/farmacologia , Rodaminas/metabolismo , VinculinaRESUMO
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.
Assuntos
Adesão Celular , Polaridade Celular , Fibroblastos/citologia , Microtúbulos/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Carpa Dourada , Peptídeos e Proteínas de Sinalização Intracelular , Microtúbulos/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Nocodazol/farmacologia , Paclitaxel/farmacologia , Polímeros , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Pseudópodes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transfecção , Tubulina (Proteína)/metabolismo , Vinculina/metabolismo , Proteínas rac de Ligação ao GTP , Quinases Associadas a rhoRESUMO
Isolated cell preparations from chicken gizzard smooth muscle typically contain a mixture of cell fragments and whole cells. Both species are spontaneously permeable and may be preloaded with externally applied phalloidin and antibodies and then induced to contract with Mg ATP. Labeling with antibodies revealed that the cell fragments specifically lacked certain cytoskeletal proteins (vinculin, filamin) and were depleted to various degrees in others (desmin, alpha-actinin). The cell fragments showed a unique mode of supercontraction that involved the protrusion of actin filaments through the cell surface during the terminal phase of shortening. In the presence of dextran, to minimize protein loss, the supercontracted products were star-like in form, comprising long actin bundles radiating in all directions from a central core containing myosin, desmin, and alpha-actinin. It is concluded that supercontraction is facilitated by an effective uncoupling of the contractile apparatus from the cytoskeleton, due to partial degradation of the latter, which allows unhindered sliding of actin over myosin. Homogenization of the cell fragments before or after supercontraction produced linear bipolar dimer structures composed of two oppositely polarized bundles of actin flanking a central bundle of myosin filaments. Actin filaments were shown to extend the whole length of the bundles and their length averaged integral to 4.5 microns. Myosin filaments in the supercontracted dimers averaged 1.6 microns in length. The results, showing for the first time the high actin to myosin filament length ratio in smooth muscle are readily consistent with the slow speed of shortening of this tissue. Other implications of the results are also discussed.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Músculo Liso/ultraestrutura , Actinas/análise , Animais , Galinhas , Proteínas do Citoesqueleto/análise , Moela das Aves/citologia , Moela das Aves/ultraestrutura , Immunoblotting , Microscopia Eletrônica , Microscopia Imunoeletrônica , Músculo Liso/citologiaRESUMO
Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin-containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.
Assuntos
Citoesqueleto/ultraestrutura , Contração Muscular , Proteínas Musculares/análise , Músculo Liso/ultraestrutura , Actinina/análise , Actinas/análise , Animais , Galinhas , Citoesqueleto/fisiologia , Imunofluorescência , Moela das Aves/citologia , Moela das Aves/fisiologia , Moela das Aves/ultraestrutura , Imuno-Histoquímica , Modelos Estruturais , Músculo Liso/citologia , Músculo Liso/fisiologiaRESUMO
The distribution of contractile and cytoskeletal proteins in smooth muscle has been mapped by immunocytochemical methods, with special reference to the localization of the actin-binding protein, filamin. Immunolabeling of ultrathin sections of polyvinylalcohol-embedded smooth muscle distinguished two domains in the smooth muscle cell: (a) actomyosin domains, made up of continuous longitudinal arrays of actin and myosin filaments, and (b) longitudinal, fibrillar, intermediate filament domains, free of myosin but containing actin and alpha-actinin-rich dense bodies. Filamin was found to be localized specifically in the latter intermediate filament-actin domains, but was excluded from the core of the dense bodies. Filamin was also localized close to the cell border at the inner surface of the plasmalemma-associated plaques. In isolated cells the surface filamin label showed a rib-like distribution similar to that displayed by vinculin. It is speculated that the two domains distinguished in these studies may reflect the existence of two functionally distinct systems: an actomyosin system required for contraction and an intermediate filament-actin system, with associated gelation proteins, that is responsible, at least in part, for the slow relaxation and tone peculiar to smooth muscle.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso/ultraestrutura , Animais , Galinhas , Citoesqueleto/ultraestrutura , Filaminas , Imunofluorescência , Moela das Aves/ultraestrutura , Cobaias , Técnicas Imunológicas , Filamentos Intermediários/metabolismo , Mitocôndrias Musculares/ultraestrutura , Músculo Liso/metabolismo , SuínosRESUMO
Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.
Assuntos
Citoplasma/fisiologia , Citoplasma/ultraestrutura , Células Epiteliais/fisiologia , Receptores de Hialuronatos/fisiologia , Ácido Hialurônico/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Citoplasma/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Feminino , Receptores de Hialuronatos/efeitos dos fármacos , Ácido Hialurônico/fisiologia , Glândulas Mamárias Animais , Camundongos , Microscopia de Vídeo , Proteínas Recombinantes/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.
Assuntos
Movimento Celular/fisiologia , Córnea/citologia , Animais , PeixesRESUMO
The association and interaction of plectin (Mr 300,000) with intermediate filaments and filament subunit proteins were studied. Immunoelectron microscopy of whole mount cytoskeletons from various cultured cell lines (rat glioma C6, mouse BALB/c 3T3, and Chinese hamster ovary) and quick-frozen, deep-etched replicas of Triton X-100-extracted rat embryo fibroblast cells revealed that plectin was primarily located at junction sites and branching points of intermediate filaments. These results were corroborated by in vitro recombination studies using vimentin and plectin purified from C6 cells. Filaments assembled from mixtures of both proteins were extensively crosslinked by oligomeric plectin structures, as demonstrated by electron microscopy of negatively stained and rotary-shadowed specimens as well as by immunoelectron microscopy; the binding of plectin structures on the surface of filaments and cross-link formation occurred without apparent periodicity. Plectin's cross-linking of reconstituted filaments was also shown by ultracentrifugation experiments. As revealed by the rotary-shadowing technique, filament-bound plectin structures were oligomeric and predominantly consisted of a central globular core region of 30-50 nm with extending filaments or filamentous loops. Solid-phase binding to proteolytically degraded vimentin fragments suggested that plectin interacts with the helical rod domain of vimentin, a highly conserved structural element of all intermediate filament proteins. Accordingly, plectin was found to bind to the glial fibrillar acidic protein, the three neurofilament polypeptides, and skin keratins. These results suggest that plectin is a cross-linker of vimentin filaments and possibly also of other intermediate filament types.
Assuntos
Citoesqueleto/análise , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/análise , Proteínas/análise , Animais , Linhagem Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Citoesqueleto/ultraestrutura , Fibroblastos , Técnica de Congelamento e Réplica , Glioma , Imuno-Histoquímica , Junções Intercelulares/análise , Junções Intercelulares/ultraestrutura , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica , Plectina , Proteínas/metabolismo , Ratos , Células Tumorais Cultivadas , Ultracentrifugação , Vimentina/análise , Vimentina/metabolismoRESUMO
The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.
Assuntos
Caveolinas , Distrofina/metabolismo , Músculo Liso/ultraestrutura , Sarcolema/ultraestrutura , Vinculina/metabolismo , Animais , Caveolina 1 , Compartimento Celular , Galinhas , Proteínas da Matriz Extracelular/metabolismo , Cobaias , Imuno-Histoquímica , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Espectrina/metabolismoRESUMO
Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.
Assuntos
Movimento Celular/fisiologia , Adesões Focais/fisiologia , Actomiosina/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Simulação por Computador , Fibroblastos/citologia , Fibroblastos/fisiologia , Carpa Dourada , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Método de Monte Carlo , Pseudópodes/fisiologia , Estresse Mecânico , TransfecçãoRESUMO
Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.
Assuntos
Actinina/análise , Proteínas de Transporte/análise , Proteínas Contráteis/análise , Pulmão/ultraestrutura , Proteínas dos Microfilamentos/análise , Músculo Liso/ultraestrutura , Miocárdio/ultraestrutura , Animais , Anticorpos , Embrião de Galinha , Epitélio/ultraestrutura , Filaminas , Imunofluorescência , Moela das Aves/ultraestrutura , Ouro , Microscopia Eletrônica , Subfragmentos de Miosina/análise , Fragmentos de Peptídeos/análise , Coloração e RotulagemRESUMO
Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo-electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub-class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo-electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo-electron tomography of snap-frozen intact cells will be required to finally clarify the three-dimensional arrangement of actin filaments in lamellipodia in vivo.
Assuntos
Pseudópodes/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Microscopia Crioeletrônica , Microscopia Eletrônica de Transmissão , Coloração NegativaRESUMO
Filopodia are rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3DeltaDAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3DeltaDAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3DeltaDAD at their tips. These data suggest Drf3-induced filopodia form and extend by de novo nucleation of actin filaments instead of convergent elongation. Finally, Drf3DeltaDAD also induced the formation of unusual, lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines, reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.