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Integrin clustering induces kinectin accumulation

¹ Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4370, USA
² Department of Tissue Biology, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855, USA

View larger version (37K): [in a new window]   Fig. 7. N-terminal and C-terminal fragments of kinectin associating with FN-coated beads. Schematic diagram depicting full-length and various portions of kinectin tagged with VSV (A). VSV-immunoblots of the kinectin (KNT) fragments found to cluster with FN-coated beads (B) and expression levels of each kinectin fragment by NIH3T3 cells (C); indicated on the right are the molecular masses (kDa) of marker proteins. (D) Immunofluorescence staining of HFF transfected with VSV empty vector (a), VSV-kinectin fragments I, II, III and IV (b,c,d,e, respectively) and full-length kinectin (f). Integrins in cells expressing the different constructs were clustered with FN-coated beads. After 20 minutes incubation with the beads, the cells were fixed and stained with a monoclonal antibody to VSV. Cy3-conjugated goat anti-mouse IgG was used for detection of the bound antibodies. Each inset shows a higher magnification focusing on the equator of the bead marked by arrowhead. For full-length VSV-kinectin, 52% of the beads were scored as positive (see Materials and Methods for the semi-quantitative criteria); fragment I was 39% positive; fragments II and III were 9% and 8% positive, respectively; fragment IV was 53% positive; and fragment V (containing three-quarters of kinectin) was 82% positive. The mean values for induction of kinectin localization by fragments II and III were confirmed by ANOVA to be significantly lower than full-length kinectin (P<0.01), whereas the values for fragments I, IV and V were not significantly different statistically (P>0.05). Bar, 20 µm.

View larger version (27K): [in a new window]   Fig. 1. Immunoblot identification of accumulation of a 160 kDa protein with FN-coated beads. Unbound fractions (lane 1), bead-bound protein complexes (lane 2) from either ConA- or FN-coated beads, and rat brain extract (10 µg) were electrophoresed on an SDS-containing 4-12% polyacrylamide gel under reducing conditions and transferred to nitrocellulose. The membrane was probed with a putative PKC monoclonal antibody. Indicated on the right are the molecular masses (kDa) of marker proteins.

View larger version (24K): [in a new window]   Fig. 2. Partial amino acid sequence alignment of 160 kDa polypeptides and human kinectin. The sequence of Lys-C-digested peptides of 160 kDa protein were aligned with human kinectin (GenBank accession no. Z22551). The numbers represent the amino acid positions of kinectin.

View larger version (23K): [in a new window]   Fig. 3. Kinectin is specifically associated with adhesion complexes induced by FN-coated beads and 5ß1 integrin. (A) Bead-associated protein complexes induced by FN, fibronectin fragment III5-10, or mutant fragment III5-10KGE. (B) Bead-associated protein complexes were induced by mAb13 against the ß1 integrin or control antibody ES66. (C) Concanavalin A-coated (ConA) or fibronectin-coated (FN) beads were either mixed directly with HFF homogenates (Lysate) or incubated with intact, living HFF (Cells). Bead-bound protein complexes were then isolated, separated on 4-12% SDS-containing polyacrylamide gels, transferred to nitrocellulose, and probed with kinectin KR160.9 monoclonal antibody. Note that kinectin is present only in the integrin-based adhesion complexes.

View larger version (56K): [in a new window]   Fig. 4. Kinectin is markedly enriched in adhesion complexes induced by FN and FN III5-10 fragment-coated beads. Beads coated with fibronectin (FN), a fibronectin fragment containing type III domains 5-10 (III5-10RGD), or the same fragment with an inactivated RGD site (III5-10KGE) were used to obtain bead-associated protein complexes (Beads) or unbound fractions (Sup). The samples were separated on a 4-12% SDS-containing polyacrylamide gel, transferred to nitrocellulose, and probed with the indicated antibodies. Note that in the beads fraction, kinectin is remarkably enriched, while kinesin and tubulin are virtually absent. In this particular experiment, 91% of total cellular kinectin was found in the beads fraction, while the corresponding percentages of total -actinin and actin in the same fraction were 22% and 23%, respectively. Also note that no proteins are observed in the unbound fractions using the functionally inactivated fibronectin fragment (III5-10KGE), because the absence of integrin binding and adhesion to this control polypeptide resulted in no cells being isolated at the first step of the procedure where beads and bound cells are isolated using a magnetic concentrator.

View larger version (107K): [in a new window]   Fig. 5. Cellular localization of kinectin. Human fibroblasts were allowed to spread for 30 minutes on fibronectin-coated coverslips, fixed and double-stained for (A) kinectin (a,a') and -actinin (b,b') or (B) kinectin (a,a') and F-actin (b,b'). The green and the red signals are merged in panels c and c', and cell morphology is shown in the phase-contrast images in panels d and d'. Arrows point to areas that are enlarged on panels a' to d'. Note that kinectin is observed not only within the perinuclear part of the cell body, but also in fibril-like patterns that follow the actin cytoskeleton. Bar, 5 µm.

View larger version (99K): [in a new window]   Fig. 6. Fibronectin-induced clustering of kinectin and cytoskeletal proteins. Immunofluorescence staining of HFF clustered with either FN- (A,C,E,G,I,K) or polylysine-coated beads (B,D,F,H,J,L). Each inset shows a higher magnification that focuses on the equator of the bead marked by the arrowhead. After 20 minutes incubation with beads, cells were fixed and stained with antibodies against kinectin (A,B), calreticulin (C,D), RAP (E,F), paxillin (G,H), ß1 integrin (I,J), or tubulin (K,L) as described in Materials and Methods. Bar, 20 µm.

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