lens syncytium
| systems medicine | CELL COMMUNICATION PATHWAYS | other

Disrupted communication between fiber cells in the avascular lens tissue results in optical aberrations and cataract formation. We study proteins implicated in several parallel pathways of intercellular communication in the lens. In addition to “classical” connexin-based gap junctions and novel syncytial pathways characterized in our lab few years ago, we recently identified that the third, pannexin-mediated coupling, is also present in the lens. In the year 2004 our lab received a prestigious PECASE Award from the White House for the work on the lens syncytium.

Cell-cell communication in the lens.
The lens of the eye grows throughout life by the deposition of newly differentiated fiber cells at its surface. The mature lens is thus composed of a layer of young, metabolically-active, surface fiber cells overlying a core of relatively inactive, older fibers. Older fibers degrade their nuclei and other organelles, resulting in the formation of an organelle-free zone in the lens core. Communication between cells at the surface and those buried deep in the lens core is critical for normal tissue function.

Metabolites enter the surface cells and diffuse from cell to cell, via gap junctions, into the lens core. However, whereas small molecules are free to diffuse in this fashion, the proteinaceous components of fiber cells are not. Being too large to pass through gap junctions, proteins are trapped within the cells in which they were originally synthesized.
Recently, an alternative pathway has that support large molecule diffusion between lens cells been characterized in our lab. We used gene delivery technology and confocal microscopy to examine the properties of a novel protein-permeable pathway that forms between fiber cells in the core of the developing lens. This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions. Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core. Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers.
The discovery of this pathway is important because it suggests a new model of lens metabolism. Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse. Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells. This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.

The main focus of this project is to uncover protein players and elucidate molecular mechanism implicated in establishing of the syncytium. Although developmental cell fusion in myoblasts, osteoclasts and zygotes has been studied for years, the underlying molecular mechanism remains illusive. We will use immunohistochemical, molecular genetic and transgenic approaches to investigate the fiber cell fusion mechanism employed in the lens. Transgenic strains carrying dominant-negative and constitutively expressed ADAM12 protein have been constructed and are being bred with connexin KO strains as well as with diagnostic GFP-mosaic strain GFPU5Nagy. In vitro dye-coupling experiments using real-time imaging to probe fibers for communication properties will be utilized. We will also test whether other fusion-related proteins like CD9, INT1B, CD47/MFR, TRAIL, syncytin, are being expressed in lens fibers. Their potential role in the core syncytium formation will be evaluated.

Protein synthesis is thought to cease shortly after the nuclei and other organelles are degraded. This occurs as early as embryonic day 12 (E12), in the chicken lens (Bassnett and Beebe, 1992). As there is no cell turnover in the core of the lens, core fibers and their macromolecular contents must persist for the life of the individual. In the absence of new synthesis, lens proteins are exposed to a lifetime of potentially damaging environmental stress. Indeed, the accumulation of damage to aging proteins in the lens core is thought to result in cataract formation, the most common cause of blindness in the world (Young, 1991).

Membrane protein complexes are major constituents of the fiber cell plasma membrane that function to control the unique life long tissue physiology. Considerable proportion of these complexes are implemented in cell-cell communication, which is critical for lens homeostasis and transparency. Connexins, a classical type of communication proteins forming molecular channels, which interconnect cytoplasm of neighboring cells, has been shown to be a centerpiece of Gap Junctional Complexes. Our data, obtained in collaboration with Kumar Lab at UIC demonstrated that these complexes also include multifunctional adapter protein ZO1. Commonly found in epithelial tight junctions ZO1 exhibits a complex translocation pattern during fiber differentiation.

Aquaporin0 (MIP) form regular shaped square arrays, commonly found during EM imaging of fiber plasma membrane.
Adherence Junctional complexes provide structural integrity of lens fibers and lens in general. Until recently these abundant complexes formed by dosens of membrane proteins were largely overlooked by the research community. These complexes anchor cytoplasmic content to certain membrane microdomains supporting complex 3D envelope of the fiber’s plasma membrane.

A disintegrin/metalloprotease ADAM12 form microscopic plaque-like accumulations in fiber plasma membranes adjacent to gap junction complexes. This protein has multiple functions in mammalian tissues, but according to our data, in undifferentiated myoblasts and lens fibers ADAM12 is uniquely processed into 52 kDa isoform, implemented in membrane fusion. This may indicate that ADAM12 has a role in the core syncytium formation, that we recently characterized in the lens (in collaboration with Bassnett Lab at WashU). Few candidate membrane proteins are likely to be implemented in developmental cell-cell fusion. CD9, INT1B, CD47/MFR, TRAIL, Syncytin, ABC…, were reported to play role in mammalian developmental cell fusion but these data is still cell-type specific and controversial.

The role of pannexins in cell-cell communication and signaling

Panx-1 and -2 are the newly discovered members of pannexin family of gap junction-forming proteins functionally analogous to connexins related to invertebrate innexins. Pannexins has been demonstrated to form gap-junction-like molecular channels when expressed in frog oocytes. In addition, it has been shown to form a gated hemichannel capable of transporting ATP and Ca2+ ions through by-lipid layers of plasma membrane and endoplasmic reticulum. In collaboration with Dr. Gerhard Dahl, we demonstrated that Panx1 and Panx2 are abundantly expressed in the ER and plasma membranes in many cell types including lens fibers and epithelium and retinal ganglion cells. Based on our preliminary data and suggested molecular properties we hypothesize that pannexins in the lens form both full channels and hemichannels (half-channels) in fiber cell membranes. These abundant hemichannels may mediate ATP and Ca2+ release and, theoretically, contribute to Ca2+ signaling and/or cell volume regulation during accomodation. We developed Panx1-specific antibodies and will examine the role of Panx-1 in cell communication in the lens.