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Carlo Iomini, Andrea Blitzer, Luca Gusella, John Danias, Marek Mlordzik; The Intraflagellar Transport Component Ift88 Orchestrates Cell Polarization During Development And Repair Of The Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6664.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial cells (CEC) migrate or stretch with a precise directionality during development and repair of the corneal endothelium (CE). Directed cell migration generally requires polarization of cellular organelles such as the basal body (bb) and the Golgi apparatus. Very little is known, however, about how extracellular cues instruct intracellular polarity. Our recent studies have shown that primary cilia are required for CE patterning and assemble during development and repair of CE but disassemble during normal adult CE steady state. Here we test the hypothesis that cilia are required for CEC polarization during directed cell migration in development and repair of the CE.
Identification and localization of cilia, bb, cell-cell contacts and IFT20 was achieved by immuno-confocal microscopy using antibodies directed to acetylated tubulin, γ-tubulin, ZO1 and IFT20, respectively. Morphometric analysis was conducted with ImageJ and graphs were generated with Prism (GraphPad) and ROSE software. The use of wild-type and IFT88orpk mice was in adherence to the ARVO statement for the use of animals in ophthalmic and vision research.
To assess cell polarization during postnatal development we have determined the position of the bb and IFT20 within CEC located at the center and periphery of the cornea. IFT20 is a component of the intraflagellar transport machinery required for assembly and maintenance of cilia that not only localizes at the bb and cilia like all the IFTs but also at the Golgi. In central CEC of wild-type mice the majority of the bb and the IFT20 were localized near the cell center. In contrast, the bb and IFT20 were shifted toward the periphery in CEC located at the CE periphery. Strikingly, the position of the bb and distribution of IFT20 in peripheral CEC was random in orpk mice that carry a mutation in the IFT88 gene and show short cilia and defective CE patterning. A similar cell polarization was also found in CEC involved in in vivo wound healing of a mechanical injury. After 30h healing, we found that cytoplasmic IFT20 also polarized toward the leading edge of cells close to the wound whereas in intact CE, IFT20 localized perinuclearly and at the bb. We are using a conditional mutant for IFT88 to determine whether cellular polarization during repair also requires intact cilium.
We have found that the cilium instructs cell polarization of CEC involved in directed cell migration during development and repair.
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