Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin.

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

BPAG1 isoform-b: complex distribution pattern in striated and heart muscle and association with plectin and alpha-actinin.

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Compositional differences between infant and adult human corneal basement membranes.

PURPOSE: Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS: Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS: The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.

Retinoschisin forms a multi-molecular complex with extracellular matrix and cytoplasmic proteins: interactions with beta2 laminin and alphaB-crystallin.

PURPOSE: X-linked juvenile retinoschisis is a rare early-onset retinal degeneration characterized by the formation of cysts and loss of the electroretinogram "b" wave. The affected gene normally codes for retinoschisin (Rs1), a secreted protein containing a large discoidin homology domain. Rs1 seems to be principally synthesized in the photoreceptors, but its structure and spectrum of effects when mutated indicates association with other proteins. The present study searched for retinal proteins capable of interacting with Rs1. METHODS: Western blotting and RT-PCR of isolated outer nuclear (photoreceptors), inner nuclear and ganglion cell layers, and cell culture compartments were performed to verify sites of Rs1 synthesis and distribution. Potential Rs1 binding partners were searched for with affinity columns generated using specific Rs1- and beta2 laminin-antisera. Following loading with total protein extracts from porcine retina, bound proteins were acid eluted and visualized by Coomassie blue staining of SDS-polyacrylamide gels, and selected bands were excised for tryptic peptide digestion and sequencing. Using single and double labeled immunohistochemistry, candidate binding partner distributions with that of Rs1. RESULTS: Whereas Rs1 mRNA was confined to the outer nuclear layer, Rs1 protein was found throughout the retina, including within the ganglion cell layer. One protein that was retained on Rs1 affinity columns was identified as alphaB crystallin, which showed partially overlapping distribution with Rs1 in the retina, mainly in the interphotoreceptor matrix and outer plexiform layer. Also, beta2 laminin columns retained Rs1, and again shared partial distribution patterns. Finally, unidentified peanut agglutinin-binding proteins from the retina also bound to Rs1, alphaB crystallin and beta2 laminin. CONCLUSIONS: Taken together, these data demonstrate that Rs1 associates with different proteins during its synthesis and secretion, forming a multimolecular complex which presumably forms a stabilizing scaffold for retinal synapses, and possibly overall tissue integrity.