EXPRESSION OF KINESIN HEAVY-CHAIN ISOFORMS IN RETINAL-PIGMENT EPITHELIAL-CELLS

To examine the possible role of kinesin in pigment granule migration i n the retinal pigment epithelium (RPE) of teleosts, we investigated th e expression and distribution of kinesin heavy chain (KHC) in RPE. Blo ts of fish RPE lysates probed with two well-characterized antibodies t o KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC an tibody (SUK4) recognized a band at 118 kD. The 118 kD band was also oc casionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase cha in reaction (RT-PCR) of mRNA from RPE using primers homologous to cons erved regions of the KHC motor domain resulted in the identification o f two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microt ubules in pigment granule aggregation in RPE. In addition, the reporte d microtubule polarity orientation in RPE apical projections is consis tent with a role for kinesin in pigment granule aggregation. Immunoflu orescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable se lective association with pigment granules, even in cells fixed while a ggregating pigment granules. Microinjected KHC antibodies had no effec t on pigment granule aggregation or dispersion, although each of the t hree antibodies has been shown to block kinesin function in other syst ems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other m icrotubule-dependent processes in RPE. (C) 1995 Wiley-Liss, Inc.