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The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-alpha, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-delta. GFAP-delta has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-alpha, assembly was restored providing GFAP-delta levels were kept low (approximately 10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-delta is naturally part of the endogenous intermediate filaments, although levels were low (approximately 10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease-causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-delta and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of alphaB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-delta splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein- and filament-filament associations as well as Jnk phosphorylation.

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.

The R120G mutation in alphaB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of alphaB-crystallin for desmin filaments. The apparent dissociation constant of R120G alphaB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G alphaB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type alphaB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G alphaB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in alphaB-crystallin.

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2alpha, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2alpha was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2alpha, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2alpha into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2alpha is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2alpha. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2alpha-lamin A/C complexes.