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>Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles ofChaetopterusoocytes and HeLa cells
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Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles ofChaetopterusoocytes and HeLa cells
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机译：Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles ofChaetopterusoocytes and HeLa cells
AbstractThe incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing 3HGTP‐labeled or dichlorotriazinylamino fluorescein (DTAF)‐labeled tubulin. Metaphase HeLa cells or spindle‐containing “minicells” fromChaetopterusoocytes were lysed in a microtubule‐assembly buffer plus 0.5 Nonidet P‐40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and 3HGTP. After different periods of incubation, mitotic spindles were isolated in 2 M‐glycerol‐containing assembly buffer and separated from unbound counts by centrifugation through a 4 M‐glycerol cushion;3H counts per mg protein increase linearly for 8–12 min and then reach a plateau or steady state in bothChaetopterusoocytes and HeLa cells. Addition of 4 mM CaCl2blocks or reverses incorporation. Little or no 3HGTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture.To measure the loss rate of 3HGTP‐tubulin from mitotic spindles, cells were incubated in tubulin plus 3HGTP for 30 min, and a 20‐fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for3H content. 3HGTP is lost from spindles at a rate of about 16/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of 3HGTP‐tubulin in spindle microtubules of these lysed‐cell models.The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF‐tubulin into mitotic spindles of these lysed cell types. A short pulse (2–5 min) appears to label microtubules (MTs) near metaphase chromosomes and longer exposures label the entire spindle.The rates of incorporation and turnover that we see by 3HGTP and fluorescent tubulin incorporation in situ are faster than those observed with brain MTs at steady state in vitro but are in the range of the rates of spindle fiber formation in prophase, and sp
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