In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

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32 Scopus citations

Abstract

Dogfish brain tubulin exhibits properties which differ from mammalian and chick brain tubulin. Polymerization of dogfish brain tubulin occurs optimally between 18 and 21 °C. The high molecular weight proteins which typically co-purify with tubulin are nearly absent as shown by SDS-polyacrylamide gel electrophoresis. A "dynein-like" protein occasionally co-purifies with dogfish brain tubulin, but its absence does not affect polymerization. At 0 °C, tubulin exists almost exclusively in the form of 6S dimers as revealed by analytical ultracentrifugation. Samples negatively stained and observed in the electron microscope have insignificant numbers of ring and spiral structures present. In some preparations, short thin filaments, approx. 200 nm in length, are seen. During polymerization, large numbers of coiled sheet polymers appear. These coiled ribbons are present throughout the early stages of polymerization, but not after polymerization reaches a plateau. The helical ribbons have an average width of 35 nm and contain approx. 8 protofilaments. The pitch angle of the helix is 33 ° and two or three turns are usually seen. The addition of 10 mM CaCl2 to a polymerized or polymerizing solution of tubulin induces the formation of rings which transform into stable coiled sheet polymers. Some of the coiled sheets form tubules when adjacent gyres of the spirals join together. The diameter of these "macrotubules" range between 50 and 100 nm. The data suggest that ring structures are not obligate intermediates of polymerization and bring into question the role of the microtubule-associated proteins.

Original languageEnglish (US)
Pages (from-to)139-151
Number of pages13
JournalExperimental Cell Research
Volume111
Issue number1
DOIs
StatePublished - Jan 1978
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology

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