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.

Laminins containing the beta2 chain modulate the precise organization of CNS synapses.

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate beta2-containing laminins, including perhaps laminins 13 (alpha3beta2gamma3) and 14 (alpha4beta2gamma3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

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.

Targeted deletion of the sciellin gene resulted in normal development and maturation.

Sciellin, together with other precursor proteins, was cross-linked by transglutaminase 1 to form the cornified envelope, an essential component of the physical barrier of the epidermis and stratified squamous epithelia. To more fully understand the function of sciellin in cornified envelope formation, we generated sciellin null mice. The mice appeared normal in their development and maturation and there were no structural features that distinguished them from littermate controls. Isolated cornified envelopes appeared normal in structure and were not more fragile to mechanical stress. There was no evidence of decreased barrier function or altered expression of other cornified envelope components. Transgenic mice expressing the repeat domain appeared to have a normal phenotype, like the null, and did not alter endogenous sciellin expression. We conclude that sciellin null mice had no structural anomalies and the transgenic mice did not act as a dominant-negative mutation.

Collagen XVII and BPAG1 expression in the retina: evidence for an anchoring complex in the central nervous system.

The ectoderm gives rise not only to the skin but also to the entire CNS. This common embryonic lineage suggests that some molecular isoforms might serve analogous functions in both tissues. Indeed, not only are laminins important components of dermal adhesion mechanisms, but they also regulate some aspects of synaptic development in both the CNS and the PNS. In the skin, laminins are part of a hemidesmosome complex essential for basal keratinocyte adhesion that includes collagen XVII (BP180) and BPAG1 (dystonin/BP230). Here, we show that CNS neurons also express collagen XVII and BPAG1 and that these molecules are expressed in the adult and developing retina. In the retina, isoforms of collagen XVII and BPAG1 are colocalized with laminins at photoreceptor synapses and around photoreceptor outer segments; both molecules are expressed by rods, whereas cones express collagen XVII but not BPAG1. Moreover, biochemical data demonstrate that collagen XVII complexes with retinal laminins. We propose that collagen XVII and BPAG1 isoforms may help to anchor elements of the rod photoreceptor cytomatrix to the extracellular matrix.

The expression of vitamin D-upregulated protein 1 in skin and its interaction with sciellin in cultured keratinocytes.

Sciellin, a precursor of the cornified envelope, contains a LIM domain that is known to function as a protein interaction module. In this study we used the yeast two-hybrid system to find proteins interacting with sciellin and identified vitamin D-upregulated protein 1 (VDUP1). This protein had not been reported in skin, but was shown in a number of cells to interact with reduced thioredoxin and regulate its function. Using an affinity VDUP1 column and an extract of cultured keratinocytes it was shown that VDUP1 and sciellin interacted. By immunohistochemistry VDUP1 was localized to the basal layer of normal human epidermis and the inner and outer root sheaths but not the matrix of the hair follicle. In the proliferative epidermis of psoriasis, VDUP1 was most highly expressed in the upper epidermal layers. In cultured keratinocytes, VDUP1 and sciellin were more highly expressed in cells undergoing differentiation. Colocalization of the proteins could be demonstrated by immunohistochemistry in parts of the follicle, psoriatic epidermis, and cultured keratinocytes. Our results suggested that VDUP1 could have a unique role in epidermis regulating the conversion of postmitotic cells to differentiating ones.