Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin ؉ entactin and collagen IV. Integrin-blocking antibodies demonstrated that ␣V␤5 integrins mediated adhesion to vitronectin, ␣5␤1 mediated adhesion to fibronectin, and ␣6␤1 mediated adhesion to laminin ؉ entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines. STEM

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Since the discovery of the ␣6␤4 integrin in the late 1980s, our understanding of its role in providing stable adhesion of epithelial cells to basement membranes (BM) has significantly increased. ␣6␤4 plays a key role in the formation and stabilization of junctional adhesion complexes called hemidesmosomes (HDs) that are connected to the intermediate filament (IF) system, as well as in the regulation of a variety of signaling processes. However, it is not clear as yet whether ␣6␤4 participates in cell signaling by serving as a substrate for tyrosine kinases and as an adaptor for their associated signaling proteins or whether its role in cellular processes is passive, involving regulation of the assembly and disassembly of HDs. In this review, we will discuss the roles attributed to ␣6␤4 and the controversies in the field. HISTORY OF THE ␣6␤4 INTEGRINThe integrin ␣6␤4 was discovered in the late 1980s by two different groups and was called either ␣E␤4 or Ic-Ic binding protein (Ic-IcBP) (36, 85). The Ic subunit had previously been shown to form a complex with glycoprotein IIa on platelets (86), which was subsequently identified as the common ␤1 subunit of the integrin family (63). Since the Ic subunit was immunologically and biochemically different from the five integrin ␣ subunits known at that time, it was named ␣6 and the complex of ␣6 with ␤1 was called VLA-6 (30). Subsequently IcBP, which for an integrin subunit had the unusual size of approximately 200 kDa, was found to be identical to the ␤4 subunit of the ␣E␤4 complex (29). The discovery of the integrin ␣6␤4 demonstrated that a particular ␣ subunit can dimerize with more than one ␤ subunit, a property that was then thought to be unique for ␤ subunits. In further studies, the tumor antigens TSP-180 and A9 were found to be identical to ␣6␤4 (39, 93). Increased expression of ␣6␤4 and changes in its distribution were then correlated with increased aggressiveness of tumors and poor prognosis (15,97). At the same time, ␣6␤4 was also found to be a component of HDs (34,84,87). Although ␣6␤4, like ␣6␤1, can interact with different laminin isoforms, its preferred ligand in the epidermal BM is laminin-5 (4, 57, 71).Sequencing of ␤4 revealed that its large size is due to an unusually long cytoplasmic domain of over 1,000 amino acids (31,89). This domain contains two pairs of type III fibronectin (FNIII) domains, separated by a connecting segment (CS) (Fig. 1). A Na-Ca exchanger (CalX) motif precedes the first FNIII domain, but its function is still not clear (77). Importantly, ␣6␤4 was found to be associated with keratin IFs instead of with actin like other integrins (26, 84). The association with IFs is mediated by the hemidesmosomal components plectin and BP230 (27,58,70). The importance of ␤4 for adhesion to the BM became evident in ␤4 knockout mice that developed severe blistering of the skin (14, 92). This was in line with findings, just prior to these studies, that a mutation in the ␤4 gene (ITGB4) is responsible for the pyloric atresia associated with junctional ...

We have previously shown that plectin is recruited into hemidesmosomes through association of its actin-binding domain (ABD) with the first pair of fibronectin type III (FNIII) repeats and a small part of the connecting segment (residues 1328 -1355) of the integrin ␤4 subunit. Here, we show that two proline residues (P1330 and P1333) in this region of the connecting segment are critical for supporting ␤4-mediated recruitment of plectin. Additional binding sites for the plakin domain of plectin on ␤4 were identified in biochemical and yeast two-hybrid assays. These sites are located at the end of the connecting segment (residues 1383-1436) and in the region containing the fourth FNIII repeat and the C-tail (residues 1570 -1752). However, in cells, these additional binding sites cannot induce the assembly of hemidesmosomes without the interaction of the plectin-ABD with ␤4. Because the additional plectin binding sites overlap with sequences that mediate an intramolecular association of the ␤4 cytoplasmic domain, we propose that they are not accessible for binding and need to become exposed as the result of the binding of the plectin-ABD to ␤4. Furthermore, these additional binding sites might be necessary to position the ␤4 cytoplasmic domain for an optimal interaction with other hemidesmosomal components, thereby increasing the efficiency of hemidesmosome assembly.