Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

Abstract

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

Sea Urchin Embryo as a Model Organism for the Rapid Functional Screening of Tubulin Modulators

“…Several studies also suggest that tubulins originating from diverse animal sources contain highly conserved amino acid sequence (1). In addition, purified pig brain tubulin injected into the sand dollar Clypeaster japonicus eggs co-polymerizes with native tubulin and incorporates into mitotic spindles during several cell cycles (36). Our tests of tubulin destabilizing drugs showed that the effective concentrations resulting in cleavage alteration/arrest and embryo death before hatching were close to the reported IC 50 values for a panel of a human tumor cell lines (Table 2).…”

Acetylated α‐tubulin in spermatogenic cells of the crane fly Nephrotoma suturalis: Kinetochore microtubules are selectively acetylated

“…However, not all spindle microtubules are equivalent in terms of their stability. Whereas asters and nonkinetochore microtubules are especially labile and appear to turn over rapidly [Inoue and Sato, 1967;Inoue and Ritter, 1975;h u e et al, 1975;Salmon and Begg, 1980;Salmon et al, 1984a;Saxton et al, 1984;Hamaguchi et a]., 1985Hamaguchi et a]., , 1987Mitchison et al, 19861, kinetochore microtubules are generally much more stable, both in terms of resistance to depolymerizing agents [see, e.g., Salmon and Begg, 1980;Salmon et al, 1984b] and in terms of their turnover rates [Mitchison et al, 19861. The reason for the relative stability of the kinetochore microtubules is not clear. It has been suggested that the kinetochore microtubules are more stable because they are capped at the kinetochore and pole [Salmon et al, 1976;Mitchison et al, 1986;Cassimeris et al, 1987).…”

Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol.

“…Fluorescently labeled tubulin has recently been proved to be useful for the investigation of tubulin dynamics in the mitotic apparatus in living cells (7,18,20,25). Evidence has been presented that labeled tubulin which had been injected into living cells was quickly incorporated into the mitotic apparatus and that photobleaching of the mitotic apparatus was quickly followed by uniform redistribution of fluorescence there.…”