Abstract
Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in β4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in β4 integrin-deficient cells by inducing expression of a truncated form of β4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated β4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and α6β4 integrin containing the truncated β4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated β4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-α6β4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to α6β4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.
Highlights
Re-epithelialization, the process by which epidermal cells undergo directed migration and move over an exposed wound bed, is a critical aspect in the closure of epithelial wounds
Input lysates (In) addition to rearrangement of the actin cytoskeleton, lamellipodium stability is dependent upon adhesion to the underlying substratum, with cell to matrix adhesion being a prerequisite for generation of sufficient traction forces to drive forward cell motility [1, 7]
By knocking down Col XVII in keratinocytes expressing either full-length or truncated 4 integrin, we demonstrate that Col XVII is required for Rac activity, lamellipodial dynamics, and processive migration
Summary
Col XVII, collagen XVII; BPAG1e, bullous pemphigoid antigen 1e; BPAG2, bullous pemphigoid antigen 2; CA, constitutively active; GABEB, generalized atrophic benign epidermolysis bullosa; JEB, junctional epidermolysis bullosa; HEK, human epidermal keratinocyte; FRAP, fluorescence recovery after photobleaching; IP, immunoprecipitation; GFP, green fluorescent protein. Following epidermal wounding, ␣64 integrin relocates from within hemidesmosomal structures to the leading front of the migrating epithelial cells where it regulates cell polarity and directed migration through activation of the small GTPase Rac and the actin-remodeling protein cofilin [16, 17, 20]. Our recent study presented evidence indicating that BPAG1e, one of the two bullous pemphigoid antigens that interact with ␣64 integrin, is required for signaling to Rac and, plays a role in directed skin cell migration [17]. Our findings provide novel insight into the mechanisms that determine keratinocyte motile behavior
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