Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.

Abstract

Abstract. Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle.We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure. MOST investigations of mitotic spindle morphogenesis in animal cells have emphasized the role of microtubule organizing centers, the centrosome, and the kinetochore. The extent to which centrosomes and kinetochores determine when and where microtubules will be positioned in a cell is only beginning to be understood (7,12,13,15). Although the importance of organizing centers is apparent, it is unlikely that all specificities for microtubule arrays are controlled by them. For example, while the mechanisms involved in microtubule length determination in vivo remain unknown, it is clear that microtubule length is controlled by something beyond tubulin concentration (1). Recent evidence suggests that chromosomes have an active role in spindle morphogenesis in addition to the role of their kinetochores in the origin of kinetochore microtubules. Chromosomes stabilize or promote the assembly of microtubules (7, 10, 17) and remarkably, the presence of chromosomes activates otherwise inactive centrosomes (7) and pericentriolar material (11). An understanding of how centrosomes, kinetochores, and chromosomes interact to organize a functional spindle apparatus will be essential if mechanisms of spindle morphogenesis and chromosome movement are to be resolved.Cultured Drosophila melanogaster primary spermatocytes (6) are well-suited for micromanipulation experiments bearing on spindle formation. In contrast to most higher eucaryotes, several layers of membranes surround the spindle and separate it from the cytoplasm (24). In addition, the membrane layers are circumscribed by a dense layer of mitochondria. Outside of this mitochondria-membrane-spindle complex, the cytoplasm is relatively free of large cytoplasmic organelles. Therefore, when bivalents are detached fro...

Functional role of centrosomes in spindle assembly and organization

“…Spindle assembly assays using Xenopus egg extracts have demonstrated that chromatin can serve as a template for MT nucleation and stabilisation in vitro [Heald et al, 1996]. Moreover, chromosomes in the meiotic cytoplasm of insect spermatocytes have been shown to stabilize MTs [Church et al, 1986;Zhang and Nicklas, 1995a;Fuge, 1999]. Such observations are compatible with a model of anastral spindle assembly, according to which chromosomes are the source of spindle MTs [Karsenti et al, 1984;Steffen et al, 1986].…”

Contribution of Noncentrosomal Microtubules to Spindle Assembly in Drosophila Spermatocytes

“…It is also consistent with the active role of chromosomes in spindle organisation. For instance, it has been reported that bivalents micromanipulated away from the spindle in Drosophila spermatocytes induce the assembly of anastral minispindles in the cytoplasm (Church et al 1986). Likewise, the removal of chromosomes before NEB has been shown to inhibit spindle assembly in grasshopper spermatocytes (Zhang and Nicklas 1995), although the phenotype of fusolo mutants, recently described, seems to argue otherwise (Bucciarelli et al 2003).…”

Metaphase Spindle Assembly

“…These experiments indicated that chromatin—even in the absence of a kinetochore—was sufficient to generate a signal that can promote microtubule formation. In parallel, micromanipulation studies in grasshopper spermatocytes revealed that a single chromosome can induce the formation of a mini-spindle [15]. Electron microscopy analyses indicated that only a small fraction (~4%) of the total microtubules were kinetochore-associated, leading to the proposal that the chromosome—and not just the kinetochore—contributes to microtubule formation.…”

“…For example, when a microneedle is used to “cut” the spindle, it recovers its bipolar shape [12]. What is even more remarkable is that when two spindles are close enough to interact, they can fuse to form a single spindle of the same size as the original individual spindles [13,14,15]. One might assume that spindle size and shape are somehow intrinsically set by the biochemical components, and therefore such repair and fusion are possible.…”