PROGRAM | Biological Sciences

By: David Scheiblin Chair: Melinda Duncan

ABSTRACT

For vision to occur, there needs to be coordination between tissues that detect light such as the retina, and those that control image quality, such as the iris, cornea, and lens. The lens contains two main cell types; epithelial cells, a monolayer of cells found on the anterior lens surface, and fiber cells, which make up the majority of the lens. For the lens to refract light, it must remain transparent which is regulated by a combination of inter-molecular interactions and its highly specialized cellular structure. Changes in the morphology of lens cells can result in improper light refraction and even cataract, a clouding of the ocular lens. The investigations in this dissertation contribute to the growing knowledge of the molecular control of lens fiber cell lateral membrane structure.

β1 integrin, a heterodimeric transmembrane cell adhesion molecule, is the most abundant β integrin in the lens and has multifaceted roles in lens biology. Conditional deletion of β1 integrin from all lens cells during embryonic development results in profound lens defects; however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional deletion approach was used to delete β1 integrin solely from the lens fiber cells, while leaving lens epithelial expression intact. This deletion resulted in two distinct phenotypes, with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of “clear” conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with loss of the F-actin network. Physiological measurements found that the lens fiber cells had a two-fold increase in gap junctional coupling, perhaps due to the differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In conclusion, I show that β1-integrin has a large role in maintaining/specifying the structure of the lens fiber cell membrane during differentiation of the LFCs, presumably due to its function in maintaining the F-actin cytoskeleton underlying the lateral membrane, as opposed to functions mediated via its interactions with ECM.

The lateral membranes contain a microstructure known as a membrane protrusion, which are the most commonly seen microstructure on lens fiber cells. However, little has been published on what the function of membrane protrusions are, although it could be speculated that they are needed to increase surface area and communication between cells in an avascular lens. Notably, elaborate membrane curvature is a key characteristic of these protrusions although little is known about the molecules that regulate these structures. However, in the past decade, a superfamily of proteins known as BAR domain proteins was discovered that are known to sense and induce membrane curvature. Bridging integrator protein 3 (Bin3), a ubiquitously expressed and evolutionarily conserved BAR domain protein, was first implicated in lens transparency since mice lacking this gene develop cataracts. However, at the beginning of my dissertation work, little was known about either the pathophysiology of the cataract or the possible underlying mechanisms by which Bin3 regulated lens transparency. The lack of cellular organization in the Bin3 null lenses implied that Bin3 may play a role in lens fiber cell differentiation as cells develop increased surface area along the lateral membranes. Bin3 null lenses displayed minor defects in lens packing and vacuole formation early in development (one to two weeks) followed by a complete loss of organization of fiber cell morphology by eight months, resulting in a loss of transparency and possibly disrupted ion transport. Further analysis of Bin3 null lenses revealed hypertrophy of interdigitations, a loss of spatial regulation in where these protrusions form, and disruption of the F-actin network. Bin3 null lenses were analyzed for Cdc42 since Bin3 is known to recruit Rho GTPases and Rho GEFs to allow for cytoskeletal remodeling. Cdc42, one of the most common RhoGTPases in the lens, was mislocalized to the broad sides of the membrane. The Rho GEF, Tuba, was downregulated in the Bin3 null lenses. Thus, the mislocalization of Cdc42, the down regulation of Tuba, and the disrupted F-actin network, may all lead to the disruption in cellular morphology and hypertrophied interdigitations. In conclusion, we proposed a mechanistic model for the formation of interdigitations during fiber differentiation. Bin3 may be needed to recruit Tuba (a Rho GEF) to the fiber cell membranes. Tuba in turn could then interact with Cdc42 (a Rho GTPase), and possibly WAVE2, activating an ARP2/3 complex to initiate nucleation and branching of cortical F-actin along the membrane pulling the membrane inward forming an invagination. (Abstract shortened by UMI.)

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