The mitotic spindle must function in cell types that vary greatly in size, and its dimensions scale with the rapid, reductive cell divisions that accompany early stages of development. The mechanism responsible for this scaling is unclear, because uncoupling cell size from a developmental or cellular context has proven experimentally challenging. Here we combined microfluidic technology with Xenopus egg extracts to characterize spindle assembly within discrete, geometrically defined volumes of cytoplasm. Reductions in cytoplasmic volume, rather than developmental cues or changes in cell shape, were sufficient to recapitulate spindle scaling observed in Xenopus embryos. Thus, mechanisms extrinsic to the spindle, specifically a limiting pool of cytoplasmic component(s), play a major role in determining spindle size.

Cells assemble mitotic spindles during each round of division to insure accurate segregation of their duplicated genome. In animal cells, stereotypical spindles have two poles, each containing one centrosome, from which microtubules are nucleated. In contrast, many cancer cells often contain more than two centrosomes and form transient multipolar spindle structures with more than two poles. In order to divide and produce viable progeny, the multipolar spindle intermediate must be reshaped into a pseudo-bipolar structure via a process called centrosomal clustering. Pseudo-bipolar spindles appear to function normally during mitosis, but they occasionally give rise to aneuploid and transformed daughter cells. Agents that inhibit centrosomal clustering might therefore work as a potential cancer therapy, specifically targeting mitosis in supernumerary centrosome-containing cells.

The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule-dependent phenomena such as cell motility, cell division, and mitosis. Here we describe a novel set of fluorescent protein fusions designed specifically to visualize microtubules in living systems using fluorescence microscopy. Each fusion contains a fluorescent protein module linked in frame to a modified phospho-deficient version of the microtubule-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the microtubule-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, microtubule dynamics were not grossly perturbed by the presence of Tau-based fluorescent protein fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based fluorescent protein fusions as minimally perturbing tools to accurately visualize microtubules in living systems.

The maize ligule is a fringe of epidermis-derived tissue, which arises from the preligule band (PLB) at a boundary between the blade and sheath. A hinge-like auricle also develops immediately distal to the ligule and contributes to blade angle. Here, we characterize the stages of PLB and early ligule development in terms of topography, cell area, division orientation, cell wall rigidity, and auxin response dynamics. Differential thickening of epidermal cells and localized periclinal divisions contributed to the formation of a ridge within the PLB, which ultimately produces the ligule fringe. Patterns in cell wall rigidity were consistent with the subdivision of the PLB into two regions along a distinct line positioned at the nascent ridge. The proximal region produces the ligule, while the distal region contributes to one epidermal face of the auricles. Whereas the auxin transporter PIN1 accumulated in the PLB, observed differential auxin transcriptional response did not underlie the partitioning of the PLB. Our data demonstrate that two zones with contrasting cellular properties, the preligule and preauricle, are specified within the ligular region prior to ligule outgrowth.