Micronuclear division and microtubular stability in the ciliate Colpoda steinii

Abstract

The first microtubules which appear in the prophase micronucleus of Colpoda steinii are located beneath the nuclear envelope and not connected to the chromosomes. Most microtubules of the metaphase spindle are connected to the tapered tips of the micronucleus and terminate singly at the chromosomes surrounded by a conical, RNA-containing kinetochore which disappears upon cold treatment. During anaphase, an interzonal stembody is formed which is maximally stretched at telophase before the daughter micronuclei are pinched off from its ends. The macronucleus, which also stretches parallel to the micronuclear stembody, has fewer microtubules which insert at the inner nuclear envelope but are not attached to the chromatin. Based upon the effects of depolymerizing factors different classes of microtubules can be distinguished. Kinetochore microtubules are sensitive to cold and vinblastine (VLB). In 2.5×10−5 M VLB their number is drastically reduced and the interzonal microtubules of early anaphase, which are also highly sensitive to nocodazole, become completely disassembled. The cross-bridged microtubules of the fully formed stembody of late anaphase display the highest resistance to depolymerization. They show signs of partial disassembly only after prolonged cold exposure and withstand higher concentrations of VLB or nocodazole than other micronuclear microtubules. Microtubules in the elongating macronucleus are fairly insensitive to cold but are depolymerized by 5×10−5 M VLB while 1.66×10−5 M nocodazole, which leaves only traces of stembody microtubules, merely reduces their number and length. All microtubules are fairly resistant to colchicine since high concentrations (5×10−2 M) are required to prevent assembly while fully formed stembodies are unaffected. Macronuclear microtubules are depolymerized at this concentration. Nocodazole, which depolymerizes all premetaphase microtubules at 6.6×10−6 M, leads to multipolar metaphase spindles with numerous microtubules, even at 1.66×10−5 M, an effect ascribed to the activity of the nuclear envelope as a microtubule organizing centre. At twice this concentration multipolar spindles are no longer found and the remaining microtubules show no apparent order. A stabilizing influence of the micronuclear envelope is indicated by the fact that whenever remnants of microtubules are found after depolymerizing treatments, they are located in its vicinity.