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Whitehead JL
,
Wang SY
,
Bost-Usinger L
,
Hoang E
,
Frazer KA
,
Burnside B
.
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The heterotrimeric microtubule motor kinesin II has been shown to be required for morphogenesis and maintenance of both motile flagella and immotile sensory cilia. Recently, we showed that the KIF3A subunit of kinesin II is concentrated in the inner segment and connecting cilium of fish photoreceptors. Here we report the gene structure of human KIF3A (HsKIF3A) and describe its localization in human and monkey retina. We also describe the localization of both KIF3A and KIF3Bkinesin II subunits in Xenopus retina. Using a portion of HsKIF3A we had amplified from adult human retinal cDNA, we found by a GenBank database search that an identical sequence had already been obtained by the Human Genome Center at Lawrence Berkeley National Laboratories in a direct sequencing analysis of 680 kb of human chromosome 5q31. By comparing the genomic sequence of HsKIF3A to the open reading frame (ORF) of the highly homologous mouse Kif3A, we determined that the HsKIF3A gene has 17 exons and an ORF of approximately 2.1 kb, predicting a protein of 80.3 kDa. Antibodies against sea urchin KRP85, a KIF3A homologue, bound to a single band of approximately 85 kDa in immunoblots of total retina protein from human, monkey and Xenopus. In these same samples, a single band of approximately 95 kDa is recognized by antibodies against Xklp3, a Xenopus KIF3B homologue. In sections of Xenopus retina, both antibodies strongly labelled photoreceptor inner segments and the outer limiting membrane. Both antibodies also labelled photoreceptor axonemes. The axonemal localization of kinesin II subunits suggests that kinesin II may play a role in transport of materials from the photoreceptor cell body to the outer segment.
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10548469
???displayArticle.link???Exp Eye Res ???displayArticle.grants???[+]
Fig. 1. Comparison of the human KIF3A amino acid sequence with other kinesin family members. PCR primers used to amplify
KIF3A from human retina are marked by the arrows. Primers sequences are as follows: PCR4 is 5«TT(T}C)GC-
(A}T}G}C)CA(T}C)AT(A}T}C)TT(T}C)GG (A}T}G}C)CA (T}C)-3«, PCR5 is 5«-CAT(A}G)TC(A}G)TC(A}T}G}C)G C(A}G)-
TT(A}G)TT-3« and PCR6 is 5«(A}T}G}C)A(A}G)(A}T}G}C)GTCAT(A}T}G)AT (A}T}G}C)C(G}T)(A}G)TC-3«. Abbreviations
used, and accession numbers for the KIF3 family members are given in parentheses: human KIF3A (HsKIF3A; AF041853),
sea urchin KRP85 (SpKRP85; L16993), human KIF3B (HsKIF3B; AB002357), Xenopus laevis Xklp3 (Xklp3; AJ009839),
human KIF3C (HsKIF3C; AF035621). Shading indicates amino acids conserved in three or more of the KIFs in the pileup.
Fig. 2. Genomic organization of the KIF3A gene. (A) The organization of KIF3A was determined by analysis of clones H23-P1
and H11-P1. Exons and introns are drawn to scale, except for introns represented by a hatched line which have been scaled
down. (B) Schematic diagram of KIF3A showing exons only. The nucleotides and corresponding amino acids are numbered as
shown.
Fig. 3. Immunoblot analysis of total retina protein from human, monkey and Xenopus with antibodies against kinesin II
subunits. (A) K2±4, a monoclonal antibody which recognizes a KIF3A homologue (KRP85) in sea urchin KRP85, cross reacts
with a band of C85 kDa in retina in all three species. (B) Antibodies raised against the Xenopus KIF3B homologue, bind to a
band of C95 kDa in retina from human, monkey and Xenopus. (C) Antibodies against the human KAP protein (SMAP)
recognize a major band of C105 kDa in human and monkey retina and a fainter band of C95 kDa. These antibodies do not
cross react with Xenopus protein.
Fig. 4. Immunolocalization of KIF3A and opsin in rhesus monkey retina. Control retinas were incubated with (A) 5% fetal
bovine serum or (B) monoclonal antibodies to bovine opsin, followed by secondary antibodies conjugated to horseradish
peroxidase. Retinas incubated with monoclonal antibodies raised against sea urchin KRP85 (KIF3A) are shown in (C) and (D).
Stain is predominantly localized to photoreceptor inner segments (IS), the outer limiting membrane (OLM) and glial Muller
cells. OS, outer segments; ONL, outer nuclear layer; IPL, inner plexiform layer; INL, inner nuclear layer; IPL, inner plexiform
layer. All images represent ¬430 magni®cation.
Fig. 5. Immunolocalization of KIF3A in human retina
using antibodies raised against sea urchin KRP85, as described
in Fig. 4. Stain is predominantly localized to photoreceptor
inner segments (IS), outer limiting membrane (OLM) and
ganglion cells. Arrows in B and C indicate distal staining of
photoreceptor inner segments. (A) ¬430; (B, C) ¬1080.
ONL, outer nuclear layer; OPL, outer plexiform layer; INL,
inner nuclear layer; IPL, inner plexiform layer.
Fig. 6. Confocal scanning microscopic images of KIF3A and KIF3B localization patterns in Xenopus retina. In (A±C), methanol-
®xed retinas were incubated with antibodies against (A) sea urchin KRP85 (KIF3A), (B) Xenopus Xklp3 (KIF3B), or (C) no
primary antibody, followed by Cy3-labelled secondary antibodies. In (D±E), paraformaldehyde-®xed retinas were incubated
with (D) anti-KRP85 antibodies or (E) no primary antibodies, followed by Cy3-labelled secondary antibodies. Analysis was
carried out by confocal scanning microscopy. OS, outer segments; IS, inner segments; OLM, outer limiting membrane; ONL,
outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
(¬650).
Fig. 7. KIF3A and KIF3B are present in photoreceptor axonemes. Five micron sections of Xenopus retina were incubated with
antibodies against (A, B, G) sea urchin KRP85 (KIF3A) and (D, E) Xenopus Xklp3 (KIF3B), followed by ¯uorescent secondary
antibodies. Photoreceptor axonemes are marked by arrows. Phase contrast images (C and F) accompany ¯uorescent images B
and E respectively. (A±F) ¬1000; (G) ¬1800.
