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.

The outer nuclear membrane proteins nesprin-1 and nesprin-2 are retained at the nuclear envelope through an interaction of their klarsicht/ANC-1/syne homology (KASH) domain with Sun proteins present at the inner nuclear membrane. We investigated the requirements for the localization of nesprin-3α at the outer nuclear membrane and show that the mechanism by which its localization is mediated is similar to that reported for the localization of nesprin-1 and nesprin-2: the last four amino acids of the nesprin-3α KASH domain are essential for its interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results in a redistribution of nesprin-3α away from the nuclear envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker protein plectin. Both nesprin-3α and plectin can form dimers, and dimerization of plectin is required for its interaction with nesprin-3α at the nuclear envelope, which is mediated by its N-terminal actin-binding domain. Additionally, overexpression of the plectin actin-binding domain stabilizes the actin cytoskeleton and prevents the recruitment of endogenous plectin to the nuclear envelope. Our studies support a model in which the actin cytoskeleton influences the binding of plectin dimers to dimers of nesprin-3α, which in turn are retained at the nuclear envelope through an interaction with Sun proteins.

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 ...

The nucleus in eukaryotic cells can move within the cytoplasm, and its position is crucial for many cellular events, including migration and differentiation. Nuclear anchorage and movement can be achieved through association of outer nuclear membrane (ONM) proteins with the three cytoskeletal systems. Two decades ago studies described C. elegans mutants with defects in such events, but only recently has it been shown that the strategies for nuclear positioning are indeed conserved in C. elegans, Drosophila, mammals and potentially all eukaryotes. The integral ONM proteins implicated in these processes thus far all contain a conserved Klarsicht/ANC-1/Syne homology (KASH) domain at their C-terminus that can associate with Sad1p/UNC-84 (SUN)-domain proteins of the inner nuclear membrane within the periplasmic space of the nuclear envelope (NE). The complex thus formed is responsible not only for association with cytoplasmic elements but also for the integrity of the NE itself. Journal of Cell Science 5022 region and residues that lie within the PS, and a cytoplasmic N-terminus that does not show sequence similarity to any known protein (McGee et al., 2006). UNC-84 contains several potential transmembrane domains and a C-terminal region that is homologous to the yeast protein Sad1p and, accordingly, is called the Sad1p/UNC-84 (SUN) domain (Malone et al., 1999;McGee et al., 2006). The localization of UNC-84 to the INM is not dependent upon its SUN domain but rather on the presence of nuclear lamins, which probably interact with its Nterminus (Lee et al., 2002). Deletion of UNC-84, mutations in the UNC-84 SUN domain or mutations within the UNC-83 KASH domain can prevent the localization of UNC-83 at the ONM (McGee et al., 2006;Starr et al., 2001), presumably because of a loss of direct interaction between the UNC-83 KASH and UNC-84 SUN domains within the PS (McGee et al., 2006) (Fig. 2). This explains why the nuclear migration defects in unc-83 and unc-84 mutant worms are very similar (Malone et al., 1999). Curiously, UNC-83 is present at the NE in a limited number of cell types (including P, hyp7, intestinal, pharyngeal and uterine cells), unlike UNC-84, which is localized at the NE in nearly all cells (Starr et al., 2001).UNC-83 and UNC-84 were originally proposed to tether the nucleus to centrosomes, which was hypothesized to drive nuclear migration (Malone et al., 1999;Reinsch and Gonczy, 1998); however, later studies showed a normal association between the centrosomes and nuclei in unc-83 and unc-84 mutant cells whose nuclei fail to migrate (Lee et al., 2002;Starr et al., 2001). The proteins that interact with the N-terminus of UNC-83 to facilitate nuclear migration have not been identified. Recently, another ONM protein, was shown to mediate an association between the centrosomes and Journal of Cell Science 119 (24) the nucleus in C. elegans. In zygote defective (zyg)-12 mutant worms, the centrosome detachment defect in the developing embryo results in death as a consequence of chromosome segregation defects (...

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