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Romell Gletten, Zhen Wang, Kerry Walker, Angus C Grey, Paul J Donaldson, Kevin L Schey; Proteomic and Immunohistochemical Characterization of Lens Fiber Cell Vesicles. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3075.
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© ARVO (1962-2015); The Authors (2016-present)
To study the mechanisms regulating the trafficking of aquaporins to the plasma membrane of lens fiber cells by characterizing the proteins present in bovine lens cortical vesicles and their associated posttranslational modifications.
nanoESI LC-MS/MS Decapsulated frozen, bovine lens cortical fiber cells were isolated via dissection and homogenized. Vesicle fractions were isolated via differential and sucrose density gradient centrifugation. Proteins in vesicle fractions were trypsinized, their peptides separated via nanoHPLC, and the column eluate was directly infused into a Velos LTQ ion trap mass spectrometer with a nanoelectrospray ionization source. Tandem mass spectra were searched against a bovine protein database as well as interpreted manually. Immunohistochemistry To verify the association of the identified proteins with the vesicular fraction lenses were fixed, cryosectioned, immunolabelled and imaged using confocal microscopy.
LC-MS/MS analyses revealed the concomitant presence of AQP0 and vesicular trafficking marker proteins in vesicle fractions including Arf proteins, Rab proteins, clathrin, VPS 35, etc. Sucrose density centrifugation revealed the presence of vesicular markers including clathrin, dynactin, synaptobrevin homolog YKT6, etc. Preliminary immunohistochemical analyses focused on the Rab GTPases family that regulates vesicle trafficking. Rab 4,5,7,9, and 11 were differentially expressed through the outer cortex of the lens in punctate staining patterns typical of vesicle immunostaining, but no signal for of the Rab proteins was found in the lens core.
LC-MS/MS and IHC were used to characterize the proteomics of the bovine lens cortical vesicles. Our results suggest the presence of vesicular aquaporins and common vesicle associated proteins in the lens outer cortex. Future work will involve optimization of our sucrose density centrifugation protocol to improve vesicle isolation efficiency and subsequent EM confirmation of the presence of vesicles. Additional immunohistochemistry will also be used to further validate our proteomics data set.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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