May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Microtubule Disassembly Induces Activation of RhoA in Corneal Endothelial Cells
Author Affiliations & Notes
  • S.S. Jalimarada
    Sch of Optometry, Indiana University, Bloomington, IN
  • S. Srinivas
    Sch of Optometry, Indiana University, Bloomington, IN
  • Footnotes
    Commercial Relationships  S.S. Jalimarada, None; S. Srinivas, None.
  • Footnotes
    Support  EY14415(SPS) EY14415 (SPS)
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1616. doi:
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      S.S. Jalimarada, S. Srinivas; Microtubule Disassembly Induces Activation of RhoA in Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1616.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Microtublule (MT) disassembly induces MLC phosphorylation leading to disruption of actin cytoskeleton and barrier dysfunction in epithelial monolayers. Since certain RhoA specific Guanine–nucleotide Exchange Factors, GEFs (e.g, p190RhoGEF) co–localize with MT (van Horck, J. Biol. Chem, 276, 4948–56), we have examined activation of RhoA secondary to pharmacological disruption of MT by Nocodazole (ND).

Methods: : Experiments were performed with cultured Bovine Corneal Endothelial cells (BCEC). The RhoA activity was assessed by the RhoA activation assay (affinity precipitation and immunoblot analysis). The assay involves the binding of activated RhoA (RhoA GTP) to Rho Binding Domain of Rhotekin. The activated RhoA bound protein complex was determined by SDS– PAGE followed by Western blot analysis. MLC phosphorylation was assayed by urea glycerol gel electrophoresis followed by Western blotting. The barrier integrity was assessed as permeability to horseradish peroxidase (HRP).

Results: : Exposure to ND (2 µM) for 30 min led to significant RhoA activation compared to control and adenosine (200 µM) treated cells. ND also induced increase in MLC phosphorylation (∼ 120% of control) as well as several fold increase in the permeability to HRP. The increase in MLC phosphorylation by ND was suppressed by co–treatment with forskolin (10 µM), adenosine (200 µM), or ATP (100 µM), agents which are known to elevate cAMP. ND led to disruption of a dense assembly of cortical actin characteristically found in BCEC.

Conclusions: : This study establishes the activation of RhoA and mobilization of Rho kinase in response to MT disassembly. Therefore, microtubule disassembly induces MLC phosphorylation and barrier dysfunction in CE by Rho kinase induced inhibition of MLC phosphatase. Involvement of specific RhoGEFs in this phenomenon needs to be investigated.

Keywords: cornea: endothelium • cytoskeleton • signal transduction 

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