| systems medicine | cell communication pathways | other

Role of the syncytium in the lens core.

We have previously characterized formation of a true syncytium in the core region of the ocular lens during development. The syncytium is a unique cellular structure which function is required to support life-long cellular homeostasis and transparency of the lens. Disruption of the syncytium leads to the disruption of the optical quality of the lens and cataract formation in the mouse model. Within the syncytium all cells become interconnected by macromolecule-permeable pathway allowing large molecules to freely diffuse between neighboring cells. We are currently testing potential contribution of ADAM12, connexin 46, CD9 and MP19/Lim2 proteins to the lens syncytium formation using molecular and genetic (transgenic) approaches. This project has won the prestigious US government Presidential (PECASE) award in 2004 and is funded by NIH/NEI R01 grant.

We developed a method to analyze tightly spaced developmental events using microarrays and applied it to study the regulatory pathways implicated in the syncytium formation. The lens samples microdissected using the precision of the Laser Capture Microscopy were used for RNA extraction, amplification and microarray probe preparation. The comparison of transcriptional profiles of young elongating fibers with the such in the mature, syncytial tissue revealed the group of differentially expressed genes, that has been further analyzed in MetaCore. We utilyzed the MetaCore™ Analytical suite (in collaboration with GeneGo Inc.) for data mining the results of microarray gene expression data obtained from the affected cells in the lens and the optic nerve tissues. Such “network” analysis allows us to utilize various types of data, including high throughput microarray and proteomic data, to complement for gaps in imperfections in these datasets, and reveal conserved network modules that are most relevant to pathophysiology. Our analysis identified a small number of highly interconnected "hub" proteins that included p53, NFkB, PKAcat (cAMP dependent), C/EBPbeta, CREB-binding protein and SP1 to be major hubs (circled at the network picture) and less connected JNK/MAPK(8-10), p70S6 kinase1, Ubiquitin, Syntaxin, STAT3, Glutaredoxin, Calcineurin and ADAM12 (polygons) to represent minor ones. These hub proteins interact to control expression of multiple genes within the network (framed at the picture) and may be implicated in the fiber cell maturation. Several representative networks are shown below.



Relevant publications.
1. D. Ivanov, G. Dvoriantchikova, A. Pestova, V.I. Shestopalov (2004) Microarray analysis of the fiber cell maturation in the lens. FEBS Letts. 579:1213-1219
2. Shestopalov V.I, Bassnett S. Development of a macromolecular diffusion pathway in the lens. (2003) J Cell Sci. v.15, p.4191-9
3. Shestopalov V.I., Missey H., Bassnett S. Gene delivery to lens cells using particle bombardment. (2002) Experim. Eye Res, v.74, p. 639-649.
4. Shestopalov V.I, Bassnett S. (2000) Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core. J. Cell Science. v 113, 1913-1921.