A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles.

Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset of Xenopusextract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems.

Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin.

A comparative analysis of spindle morphometrics across metazoans.

Cell division in all eukaryotes depends on function of the spindle, a microtubule-based structure that segregates chromosomes to generate daughter cells in mitosis or haploid gametes in meiosis. Spindle size adapts to changes in cell size and shape, which vary dramatically across species and within a multicellular organism, but the nature of scaling events and their underlying mechanisms are poorly understood. Cell size variations are most pronounced in early animal development, as egg diameters range from tens of microns up to millimeters across animal phyla, and decrease several orders of magnitude during rapid reductive divisions. During early embryogenesis in the model organisms X. laevis and C. elegans, the spindle scales with cell size [1, 2], a phenomenon regulated by molecules that modulate microtubule dynamics [3-6], as well as by limiting cytoplasmic volume [7, 8]. However, it is not known to what extent spindle scaling is conserved across organisms and among different cell types. Here we show that in a range of metazoan phyla, mitotic spindle length decreased with cell size across an ∼30-fold difference in zygote size. Maximum spindle length varied, but linear spindle scaling occurred similarly in all species once embryonic cell diameter reduced to 140 µm. In contrast, we find that the female meiotic spindle does not scale as closely to egg size, adopting a more uniform size across species that most likely reflects its specialized function. Our analysis reveals that spindle morphometrics change abruptly, within one cell cycle, at the transition from meiosis to mitosis in most animals.