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Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin.

Publication ,  Journal Article
Inoué, S; Borisy, GG; Kiehart, DP
Published in: The Journal of cell biology
July 1974

Purified tubulin solutions stabilized and augmented the birefringence (BR) of isolated Chaetopterus spindles. Tubulin was extracted from pig brain tissue in cold PEG buffer (0.1 M piperazine-N-N'-bis[2-ethane sulfonic acid], 1 mM ethylene bis-[oxyethylenenitrilo]tetraacetate, [EGTA], 2.5 mM guanosine triphosphate, [GTP], pH 6.94, at 25 degrees C), and purified by two cycles of a reversible, temperature-dependent assembly-disassembly procedure. The spindle BR of the meiotic metaphase-arrested oocytes of Chaetopterus decreased linearly at a rate of 1.5 nm/min when perfused with PEG buffer without tubulin. In this hypotonic, calcium-chelating solution, the cell lysed within 1.5 min, and after a brief, transient rise, the BR disappeared in ca. 4 min from the time of buffer application. Cells perfused with tubulin in PEG buffer also showed BR decay at the same rate until cell lysis. Immediately upon cell lysis the spindle BR increased, initially at ca. 2.3 nm/min and then more slowly until the BR attained or exceeded intact cell values. Spindle and asters grew considerably larger than those in intact cells. From the kinetics of the transient BR increase after lysis, we infer that, initially, Chaetopterus cytoplasmic tubulin contributes to increased BR; further augmentation required added pig brain tubulin and most probably reflects the addition and incorporation of heterologous porcine tubulin into the spindle and asters. Isolated, augmented spindles depolymerized rapidly at 6 degrees C. Upon return to 23 degrees C, spindle BR returned slowly in tubulin-PEG. The BR of the isolates also decayed in solutions containing calcium ions 2.5 mM in excess of the EGTA. However, the isolates did not respond, or responded very slowly, to 1 mM colchicine or Colcemid and to dilution of tubulin with PEG solution. Microinjection into Chaetopterus oocytes of tubulin-PEG, but not PEG alone, enhanced spindle and aster BR which reversibly disappeared upon chilling the cell.

Duke Scholars

Published In

The Journal of cell biology

DOI

EISSN

1540-8140

ISSN

0021-9525

Publication Date

July 1974

Volume

62

Issue

1

Start / End Page

175 / 184

Related Subject Headings

  • Time Factors
  • Swine
  • Ovum
  • Nerve Tissue Proteins
  • Mitosis
  • Methods
  • Insecta
  • Female
  • Evaluation Studies as Topic
  • Drug Stability
 

Citation

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ICMJE
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Inoué, S., Borisy, G. G., & Kiehart, D. P. (1974). Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. The Journal of Cell Biology, 62(1), 175–184. https://doi.org/10.1083/jcb.62.1.175
Inoué, S., G. G. Borisy, and D. P. Kiehart. “Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin.The Journal of Cell Biology 62, no. 1 (July 1974): 175–84. https://doi.org/10.1083/jcb.62.1.175.
Inoué S, Borisy GG, Kiehart DP. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. The Journal of cell biology. 1974 Jul;62(1):175–84.
Inoué, S., et al. “Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin.The Journal of Cell Biology, vol. 62, no. 1, July 1974, pp. 175–84. Epmc, doi:10.1083/jcb.62.1.175.
Inoué S, Borisy GG, Kiehart DP. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. The Journal of cell biology. 1974 Jul;62(1):175–184.

Published In

The Journal of cell biology

DOI

EISSN

1540-8140

ISSN

0021-9525

Publication Date

July 1974

Volume

62

Issue

1

Start / End Page

175 / 184

Related Subject Headings

  • Time Factors
  • Swine
  • Ovum
  • Nerve Tissue Proteins
  • Mitosis
  • Methods
  • Insecta
  • Female
  • Evaluation Studies as Topic
  • Drug Stability