PURIFICATION OF TUBULIN AND ASSOCIATED HIGH MOLECULAR WEIGHT PROTEINS FROM PORCINE BRAIN AND CHARACTERIZATION OF MICROTUBULE ASSEMBLY <i>IN VITRO</i>*

139

111

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies , J. Cell Biol., 99:2165-2174Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186 which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.

Section: Preparation Of Dichlorotriazinylaminofluorescein-tubulinmentioning

confidence: 99%

Section: Preparation Of Dichlorotriazinylaminofluorescein-tubulinmentioning

confidence: 99%

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes.

Soltys¹,

Borisy²

1985

139

111

“…Microtubnle protein was purified from bog brain by two cycles of in vitro assembly and disassembly in 0.1 M Na-PIPES buffer (adjusted to pH 6.94 at 23°C) by the method of Borisy et al (2) with the following modifications: (a) brain cortex was homogenized at 0°C with an equal volume of buffer containing 5 mM fl-mercaptoethanol; and (b) before the first polymerization, and for this step only, glycerol was added to the extract to a final concentration of 2.7 M. During cycles of microtubule purification samples were centrifuged at 37,000 g (max) for 30 rain at 30°C to pellet microtubules or at 5°C to remove protein aggregates. Pellets of microtubules that had been purified by two cycles of in vitro assembly-disassembly (H2P) were frozen in liquid nitrogen and stored at -80°C.…”

Section: Preparation Of Microtubule Protein and Mapsmentioning

confidence: 99%

Identity and Origin of the ATPase activity associated with neuronal microtubules. I. The ATPase activity is associated with membrane vesicles.

Murphy¹,

Hiebsch

Wallis

1983

Microtubule protein purified from brain tissue by cycles of in vitro assemblydisassembly contains ATPase activity that has l~een postulated to be associated with microtubule-associated proteins (MAPs) and therefore significant for studies of microtubule-dependent motility. In this paper we demonstrate that >90% of the ATPase activity is particulate in nature and may be derived from contaminating membrane vesicles. We also show that the MAPs (MAP-l, MAP-2, and tau factors) and other high molecular weight polypeptides do not contain significant amounts of ATPase activity. These findings do not support the concept of "brain dynein" or of MAPs with ATPase activity.

“…Microtubule protein was purified from porcine brain as described previously (1,10). Equal volumes of purified microtubule protein in polymerization buffer (100 mM PIPES, 1 mM EGTA, 0.1 mM MgCl, I mM GTP, pH 6.7) were added to test samples in extraction medium .…”

Section: Preparation Of Microtubule Protein and Polymerization Onto Cmentioning

confidence: 99%

Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle.

Kuriyama

Borisy²

1981

187

The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes . However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity . Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome . Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared . In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle . Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle .