Organization of point contacts in neuronal growth cones

Abstract

Growth cones from rat dorsal root ganglia plated on laminin contain integrin clusters over the entire growth cone surface, and growth cones make transient adhesions at sites called point contacts. We examined, by immunocytochemistry and confocal microscopy, the composition and distribution of point contacts in neuronal growth cones. Vinculin was concentrated in the central domain of growth cones and at the tips of filopodia. Vinculin was specifically associated with integrin clusters at the membrane-substrate interface and thus marked point contacts. The cytoskeletal proteins paxillin and talin colocalized with beta1 integrin in a subpopulation of clusters restricted to the central domain of the growth cone and to the tips of filopodia. The neuron-specific kinase, FAK+ also distributed with the vinculin-positive clusters. The Rho family proteins RhoA, RhoB, and Cdc42 were present in growth cones, and a few Rho clusters were colocalized with vinculin. Examination of proteins resistant to detergent extraction in PC12 cells confirmed the retention of beta1 integrin, paxillin, talin, and vinculin with the cytoskeleton. Moreover, we detected FAK+ and RhoA in the detergent-resistant cytoskeleton, supporting their distribution to point contacts. Our observations indicate that two types of integrin clusters are present in growth cones: those associated with vinculin at the cell substratum interface, and those not associated with vinculin. Point contacts are mature adhesion sites defined by the presence of both beta1 integrin and vinculin, and they are associated with signaling proteins.

Eicosanoid Activation of Protein Kinase C ϵ

“…Unlike the fibroblast, which forms focal adhesions or adhesion plaques, the growth cone exhibits a broad belt of adherent spots along its distal rim, as seen by reflection interference microscopy (6). As shown here and elsewhere, this distribution is mirrored by a punctate pattern of adhesion site proteins, including paxillin, dispersed throughout the plasmalemma adherent substrate contact area (6,50). It is evident in our images that MARCKS and PKC⑀ puncta do not overlap with those of paxillin but that the three proteins form coextensive patterns in the same optical section (adhesive plane).…”

“…For reference, growth cones also were labeled with phalloidin to reveal F-actin and with an antibody to paxillin, a known adhesion site protein. In growth cones, plasmalemmal adhesive area is defined by a punctate pattern (rather than plaques) of proteins, such as ␤ 1 -integrin, paxillin, talin, and focal adhesion kinase, at least on a laminin substratum (6,50). The optical sections shown in Fig.…”

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

“…To confirm the distinctive subcellular distribution of each Dp71 isoform, we carried out immunoblotting analyses of nuclear and cytosolic protein extracts. To verify the fidelity of our fractionation procedure, we employed vinculin, a focal adhesion protein [26], and calnexin, a membrane protein from the endoplasmic reticulum, as cytoplasmic markers. On Cells, cultured on glass coverslips for confocal microscopy analysis, were immunostained with the primary monoclonal antibodies described above, and the specific signal was developed using a fluorescein-conjugated secondary antibody.…”

Conventional myosins – unconventional functions

“…Key to adhesion are proteins such as integrin, vinculin or paxillin, which are found in adherent cells (Zamir and Geiger 2001) including those of neuronal origin, where they mediate adhesive interactions of the growth cone (Gomez et al 1996;de Curtis and Malanchini 1997;Renaudin et al 1999). These adhesive foci are also sites for signal transmission between the extracellular matrix and the cell interior, using mechanisms that require myosin 2-generated contractility (Burridge and ChrzanowskaWodnicka 1996).…”

“…Each adhesion focus acts as a mechanosensor, its size increasing in direct proportion to the forces sensed (Bershadsky et al 2003); typically, mammalian cells exert stress forces of the order 2-10 nN/μm 2 at these sites (Bershadsky et al 2003). As a cell migrates, adhesion sites mature, beginning as small point contacts near the leading edge and developing to larger focal adhesions distal to the lamellipodia (Renaudin et al 1999;Rottner et al 1999). While the formation of nascent adhesions within the lamellipodium appears to be independent of myosin 2 activity (Choi et al 2008), maturation of these nascent sites, behind the lamellipodium, requires myosin 2 action.…”