In vivo coassembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function.
alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta-tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.
Joshi
H C
HC
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Yen
T J
TJ
Cleveland
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D004912:Erythrocytes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005796:Genes; D046911:Macromolecular Substances; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D014162:Transfection; D014404:Tubulin / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D046911:Macromolecular Substances; D014404:Tubulin