Determination of microtubule polarity in vitro by the use of video-enhanced differential-interference contrast light microscopy and Chlamydomonas flagellar axonemal pieces.
Microtubules nucleated by sea urchin sperm-tail axonemes have polar ends that differ both functionally and structurally but cannot be distinguished from one another when viewed by light microscopy. Ambiguity and circularity surround any classification of microtubule polarity by conventional methods. Chlamydomonas flagellar axonemal pieces have distinct morphological differences at their plus- and minus-ends, and microtubules nucleated from these pieces can be distinguished as plus- or minus-ended based on the morphological differences present in the Chlamydomonas flagellar axonemal pieces. Plus- and minus-ended microtubules were polymerized in this fashion and analyzed for differences in growth rates, shortening rates, and frequencies of transitions. The results were in good agreement with similar data generated by the more time-consuming and difficult use of kinesin-coated beads (R. J. Kowalski, and R. C. Williams, Jr. (1993) Cell Motil. Cytoskeleton 26, 282-290) to determine microtubule polarity. This is a relatively simple and effective method for determining the polarity of microtubules in vitro by video-enhanced differential-interference contrast light microscopy.
Gamblin
T C
TC
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Williams
R C
RC
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000648:ultrastructure; D016547:Kinesins; D008297:Male; D008857:Microscopy, Interference / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D008863:Microspheres; D008870:Microtubules / Q000737:chemistry / Q000502:physiology*; D013082:Sperm Tail / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D016547:Kinesins