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First published online 23 April 2003
doi: 10.1242/jcs.00445

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Differential regulation of cell adhesive functions by integrin subunit cytoplasmic tails in vivo

Department of Cell Biology, University of Virginia School of Medicine, PO Box 800732, Charlottesville, VA 22908, USA

View larger version (48K): [in a new window]   Fig. 1. Expression of 4 constructs. (A) A4EX recognizes the extracellular domain of Xenopus integrin 4. Embryos injected with full-length Xenopus 4 (4wt) or partial cytoplasmic tail deletion 4 mutant (4RR) were lysed and subjected to western blot with A4EX (raised against the 4 extracellular domain) and D2AP (raised against the 4 cytoplasmic tail). Protein equivalent to one embryo was loaded per lane. Arrows indicated bands of 140 kDa, 80 kDa and 60 kDa. A4EX can detect both 4wt and 4RR, whereas D2AP only recognizes 4wt. (B) 4 constructs are expressed on embryonic cell surface. Cell-surface biotinylated 4 tail truncation and chimera proteins (as indicated) are immunoprecipitated by A4EX. Each lane contains protein precipitated from seven embryos. Arrows indicate bands of 80 kDa and 60 kDa. Note that the 4 cytoplasmic tail deletion mutants and chimeras are present at the cell surface predominantly in the cleaved form. 42 is expressed on the surface less well than other constructs. Quantification of the pixel densities of the 4 bands indicates a less-than-twofold variation in surface expression from sample to sample, with the exception of 42. (C) 4 constructs form heterodimers with the ß1 subunit. Embryos co-injected with RNA transcripts of 4 constructs and ß1 were lysed at stage 15 and immunoprecipitated with mAb 8c8. The immunoprecipitated proteins were then separated on an 8% polyacrylamide gel and western blotted with A4EX. Precipitate equivalent to ten embryos was loaded per lane. Both full-length and cleaved forms of 4 can associate with ß1 subunit.

View larger version (182K): [in a new window]   Fig. 2. 4 Constructs are able to mediate the attachment of dissociated animal cap cells to the V-region of FN. The adhesive substrate and the 4 construct injected are as indicated on each panel. Scale bar for all panels is shown in A.

View larger version (166K): [in a new window]   Fig. 3. Animal cap cell spreading in response to activin induction. The 4 construct injected and the substrate are indicated on each panel. Scale bar for all panels is shown in A.

View larger version (15K): [in a new window]   Fig. 4. `Spider' graphs of individual DIMZ cell migration tracks. The 4 constructs expressed are as indicated. The `control' is GFP transcript-injected DIMZ cells and the substrate used is 9.11-GST (A). For all the 4 constructs, the substrate is V-GST (B-H). Each `spider' graph contains 15 representative cell migration tracks with the start point all set to `0,0'. The scale is as indicated in (G). Note that 4KV and 4 RR have reduced paths compared with 4 chimeras with full-length cytoplasmic tails. Among all the 4 chimeras, 45 has slightly shorter and more coiled migration tracks.

View larger version (27K): [in a new window]   Fig. 5. DIMZ cell migration rate mediated by 4 constructs. The travel rate (A) and the radial displacement rate (B) were measured using the Nanotrack function within the ISEE software package, then plotted as bar graphs. The 4 constructs expressed and substrates used are as indicated. For each construct, 40 to 65 cells from three different batches of embryos were measured. Error bars indicate standard deviations. Normal DIMZ cells cannot adhere to V-GST, thus their travel rate and radial displacement are not available. 4KV and 4RR cells migrate significantly slower than 4 chimeras, whereas 45 cells migrate the slowest compared with other 4 chimeras. The statistical significance among different 4 constructs is summarized in Table 2.

View larger version (28K): [in a new window]   Fig. 6. DMZ explant migration assay. (A) A DMZ explant. The bottom layer is the mesoderm tissue sheet (red), which encounters the substrate, and the top layer is ectoderm tissue (blue). The dorsal lip edge is positioned up and the arrow indicates the direction of mesoderm tissue sheet migration. (B) The morphology of DMZ explants expressing GFP or 45, shown as a representative example. The construct injected and the adhesive substrate are as indicated. Arrows point to the migrating sheet of mesodermal tissue. (C) Migration rate of DMZ explants. The distance of leading edge cell advancement in 1 hour is measured and plotted as a bar graph. The substrate and construct injected are as indicated.

View larger version (136K): [in a new window]   Fig. 7. Only some cytoplasmic domains are able to support FN fibrillogenesis. (A) FN fibril assembly rescue assay. 4 Constructs were injected into the animal pole regions of embryos at the two-cell stage. At stage 9, mAb P8D4 were injected into the blastocoel of these embryos. The embryos were cultured until the end of gastrulation. Then animal caps were dissected and subjected to immunostaining for FN. (B-J) Immunofluorescence of FN on the blastocoel roof. The 4 construct expressed is indicated on each panel. Endogenous FN fibril network is formed on the blastocoel roof at the end of gastrulation (B). P8D4 blocked fibril formation (C). In the presence of P8D4, 4 chimeras with the 5 and 6 cytoplasmic domains were able to mediate the assembly of significant amount of FN fibrils (H,I). A few fibrils were also observed on the BCR injected with 43 (G). The mAb 4H2 was used to immunostain FN.

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