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

被引:12
作者
KINGSMITH, C [1 ]
BOSTUSINGER, L [1 ]
BURNSIDE, B [1 ]
机构
[1] UNIV CALIF BERKELEY,DEPT MOLEC & CELL BIOL,BERKELEY,CA 94720
来源
CELL MOTILITY AND THE CYTOSKELETON | 1995年 / 31卷 / 01期
关键词
MICROTUBULE MOTOR PROTEINS; IMMUNOLOCALIZATION; RT-PCR; NORTHERN SOUTHERN BLOTS; MICROINJECTION;
D O I
10.1002/cm.970310108
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE. (C) 1995 Wiley-Liss, Inc.
引用
收藏
页码:66 / 81
页数:16
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