No abstract

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β4 subunit of the basement membrane laminin receptor integrin α6β4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β4 subunits and cytokeratin filaments.

Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.

Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin's function in a living organism, we have disrupted its gene in mice. Plectin (−/−) mice died 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (−/−) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (−/−) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.

Summary A specific mutation (ΔE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3α co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3α. This association with human nesprin-3 appeared to be stronger for torsinAΔE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3 was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.