3-panel darkfield micrograph

© 1994 Springer-Verlag. Reproduced with permission.
(from Durso & Cyr 1994, Protoplasma 180: 99-105)

An EF-1a homolog from carrot bundles plant microtubules (MTs), and calcium/calmodulin (Ca2+/CaM) abolishes the bundling.

These images are darkfield micrographs - direct and rapid observations of MTs without fixation, fluorescent labeling, mounting, etc.

Using only purified components reconstituted in vitro:
Carrot MTs (a) were bundled by the carrot EF-1a homolog, pp50 (b); the universal regulatory complex, Ca2+/CaM dissipates such bundles.

At present (1996), this and related data are the most detailed functional characterization of a putative microtubule-associated protein in plants. Our subsequent in situ studies indicate that EF-1a is a bona fide, not putative, microtubule-associated protein.

Details:
a. Carrot MTs (10 然 taxol-stabilized from DEAE-purified carrot tubulin) visualized by dark-field microscopy. The fineness of the MTs indicates that they are not bundled.
b. When purified carrot EF-1a homolog (1.2 然) is added, the carrot MTs (5.1 然) unbundle. These bundles are very bright (to protect the SIT video camera, 5X filtration, relative to a, was required to obtain this image) and much longer than the unbundled MTs in a (note scale bars). The suspension of bundles shown here also contains 500 然 Ca2+, so Ca2+ does not inhibit these structures.
c. When CaM (8 然) is added to the suspension in b, the bundles dissipate, and individual MTs are difficult to image (microscopic conditions as in b).
Scale bar = 10 痠.


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©1996 Neil A Durso, III

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