Proteomic characterization of isolated retinal pigment epithelium microvilli.
Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and function of each epithelium. The molecular basis of this heterogeneity is poorly understood. However, differences in function will undoubtedly be reflected in the specific molecular composition of the apical surface in each epithelial subtype. We have exploited a method for isolating microvilli from the mouse eye using wheat germ agglutinin (WGA)-agarose beads to begin to understand the specific molecular composition of apical microvilli of the retinal pigment epithelium (RPE) and expand our knowledge of the potential function of this interface. Initially, apical RPE plasma membranes bound to WGA beads were processed for morphological analysis using known apical and basolateral surface markers. The protein composition of the apical microvilli was then established using proteomic analysis. Over 200 proteins were identified, including a number of proteins previously known to be localized to RPE microvilli, as well as others not known to be present at this surface. Localization of novel proteins identified with proteomics was confirmed by immunohistochemistry in both mouse and rat eye tissue. The data generated provides new information on the protein composition of the RPE apical microvilli. The isolation technique used should be amenable for isolating microvilli in other epithelia as well, allowing new insights into additional functions of this important epithelial compartment.
Bonilha
Vera L
VL
The Cole Eye Institute, Department of Ophthalmic Research, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA. bonilhav@ccf.org
Bhattacharya
Sanjoy K
SK
West
Karen A
KA
Sun
Jian
J
Crabb
John W
JW
Rayborn
Mary E
ME
Hollyfield
Joe G
JG
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D016764:Cell Polarity / Q000502:physiology; D005136:Eye Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000648:ultrastructure; D040901:Proteomics; D051381:Rats; D014909:Wheat Germ Agglutinins / Q000737:chemistry
D005136:Eye Proteins; D014909:Wheat Germ Agglutinins