Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.
To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.
Barlow
S
S
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Gonzalez-Garay
M L
ML
West
R R
RR
Olmsted
J B
JB
Cabral
F
F
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002087:Butyrates / Q000633:toxicity; D020148:Butyric Acid; D016466:CHO Cells; D006224:Cricetinae; D004330:Drosophila; D004351:Drug Resistance; D005455:Fluorescent Antibody Technique; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure*; D012313:RNA / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D016875:tau Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism
D002087:Butyrates; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D016875:tau Proteins; D020148:Butyric Acid; D012313:RNA