Impaired cytoskeletal organization and membrane integrity in lens fibers of a Rho GTPase functional knockout transgenic mouse.
To investigate the effects of Rho GTPase inactivation on lens fiber cell cytoskeletal and morphological integrity, a transgenic mouse model expressing C3-exoenzyme (a bacterial toxin) in a lens-specific manner was utilized. Cryosections of whole eyes from C3 transgenic mice and littermate controls were stained for F-actin with rhodamine-phalloidin or immunostained for beta-catenin, aquaporin-0 or connexin-50, and confocal images were recorded. Lens fiber cell morphology was examined at both light and electron microscopic levels. To investigate the influence of Rho GTPase inactivation on the profiles of gene expression, cDNA libraries generated from transgenic and littermate control mouse lenses were screened by cDNA microarray analysis. In contrast to the wild-type lens, fiber cells of the transgenic lens were grossly swollen and disorganized, with abnormal membrane architecture. Staining of F-actin, beta-catenin, aquaporin-0 and connexin-50 was reduced dramatically in the C3 transgenic lens as compared to controls. Western blot analysis and cDNA microarray analysis did not reveal any noticeable decreases in actin, beta-catenin and aquaporin-0 protein levels or expression in C3 transgenic lenses, indicating that altered cytoskeletal organization in response to Rho GTPase inactivation might underlie the noted changes in staining for these proteins. Additionally, cDNA microarray analysis of C3 lens revealed altered expression (at least two-fold, compared to littermate controls) of 44 genes. These include genes encoding extracellular matrix and basement membrane proteins, cell survival and apoptotic pathways, and ion and protein transport. These data indicate that disruption of Rho GTPase function in the developing mouse lens results in abnormal cytoskeletal organization, fiber cell interactions, impaired lens fiber cell morphology and altered gene expression of cellular proteins involved in diverse functions. This work reveals that the morphological and cytoskeletal abnormalities triggered upon Rho GTPase inactivation in lens could be one of the important insults associated with cataract formation in C3 transgenic mouse lens.
Maddala
Rupalatha
R
Department of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA.
Deng
Pei-Feng
PF
Costello
Joseph M
JM
Wawrousek
Eric F
EF
Zigler
Jacob S
JS
Rao
Vasantha P
VP
D000199:Actins / Q000378:metabolism; D000818:Animals; D020346:Aquaporins / Q000378:metabolism; D001483:Base Sequence; D017630:Connexins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology*; D004247:DNA / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D007908:Lens, Crystalline / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020741:rho GTP-Binding Proteins / Q000172:deficiency* / Q000235:genetics
D000199:Actins; D020346:Aquaporins; D017630:Connexins; D003598:Cytoskeletal Proteins; D004247:DNA; D020741:rho GTP-Binding Proteins