ISSN:
0886-1544
Keywords:
Pipes
;
Hepes
;
calcium
;
VE-DIC microscopy
;
cytoplasmic extracts
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus-ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut-off 〈30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970210102
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