The long-term goal of this project is to elucidate the functional roles of lipid raft domains in lens fiber-cell membranes, and their associated proteins, which are crucial in maintaining the transparency of the lens. In many types of cells, lipid rafts are functionally associated with various membrane processes such as protrusions, microvilli, filopodia and lamellipodia. The lipid rafts function as platforms to recruit proteins for signal transduction, calcium transport and cholesterol transport. In lens fiber cells of all species, large numbers of interlocking domains (in the forms of ball-and-socket, protrusion and microvillus processes) protrude into neighboring cells and increase cell membrane surface area. Since the lens is a vascular organ, this unique structural configuration of interlocking domains (IDs) would be ideal for facilitating molecular exchange between cells. In this project, we focus on identification of key lipid and protein components of lipid rafts in IDs of embryonic and adult chicken lenses. Because IDs share several unique features with lipid rafts of other cells, we hypothesize that the lens-specific IDs are the lipid rafts in lens fiber cells.
Aim 1 tests the prediction from this hypothesis that cholesterol is enriched in various IDs, by using filipin cytochemistry with freeze-fracture TEM.
Aim 2 tests the prediction that caveolin-1, PMCA, Src kinase and other key raft-associated proteins are enriched in IDs, by using immunogold EM with high-pressure freezing procedures.
Aim 3 tests the corollary of our hypothesis that cav-1-connexin interaction in ball-and- socket domains and flat cell membranes is critical for gap junction assembly and maturation in lens fibers, by using freeze-fracture immunogold replica labeling (FRIL) and biochemistry. Specifically, the roles of cav-1 in trafficking of cholesterol will be examined for the transformation of cholesterol-rich to cholesterol-free gap junctions during junction assembly, maturation, and aging using our innovative methodology combining filipin cytochemistry with FRIL at EM level. Quantitative analysis will be applied for assessing the association of cav-1 with cholesterol- containing gap junctions for their putative role as lipid-raft domains in the lens. Because the functional roles of lipid rafts critically depend on their cytoskeletal organization, Aim 4 will determine the actin branching network configuration and identify the actin-associated proteins (e.g., Arp2/3, ezrin and paralemmin) in developing raft-like domains, by using high pressure freezing procedures.
|Hu, Zhengping; Shi, Wen; Riquelme, Manuel A et al. (2017) Connexin 50 Functions as an Adhesive Molecule and Promotes Lens Cell Differentiation. Sci Rep 7:5298|
|Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K et al. (2016) Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells. Invest Ophthalmol Vis Sci 57:4084-99|
|Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan et al. (2015) Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells. Am J Physiol Cell Physiol 308:C835-47|
|Biswas, Sondip K; Brako, Lawrence; Gu, Sumin et al. (2014) Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse. Exp Eye Res 127:132-42|
|Biswas, Sondip K; Brako, Lawrence; Lo, Woo-Kuen (2014) Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice. Exp Eye Res 125:9-19|
|Lo, Woo-Kuen; Biswas, Sondip K; Brako, Lawrence et al. (2014) Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens. Invest Ophthalmol Vis Sci 55:1202-12|
|Zhang, Cheng; Asnaghi, Laura; Gongora, Celine et al. (2011) A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye. Eur J Cell Biol 90:440-8|
|Biswas, Sondip K; Lee, Jai Eun; Brako, Lawrence et al. (2010) Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens. Mol Vis 16:2328-41|
|Biswas, Sondip K; Jiang, Jean X; Lo, Woo-Kuen (2009) Gap junction remodeling associated with cholesterol redistribution during fiber cell maturation in the adult chicken lens. Mol Vis 15:1492-508|
|Steele Jr, E C; Wang, J H; Lo, W K et al. (2000) Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant. Mol Vis 6:85-94|
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