Optical volume and mass measurements show that mammalian cells swell during mitosis.

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells.

Exploring the function of cell shape and size during mitosis.

Dividing cells almost always adopt a spherical shape. This is true of most eukaryotic cells lacking a rigid cell wall and is observed in tissue culture and single-celled organisms, as well as in cells dividing inside tissues. While the mechanisms underlying this shape change are now well described, the functional importance of the spherical mitotic cell for the success of cell division has been thus far scarcely addressed. Here we discuss how mitotic rounding contributes to spindle assembly and positioning, as well as the potential consequences of abnormal mitotic cell shape and size on chromosome segregation, tissue growth, and cancer.

Methods for two-dimensional cell confinement.

Protocols described in this chapter relate to a method to dynamically confine cells in two dimensions with various microenvironments. It can be used to impose on cells a given height, with an accuracy of less than 100 nm on large surfaces (cm(2)). The method is based on the gentle application of a modified glass coverslip onto a standard cell culture. Depending on the preparation, this confinement slide can impose on the cells a given geometry but also an environment of controlled stiffness, controlled adhesion, or a more complex environment. An advantage is that the method is compatible with most optical microscopy technologies and molecular biology protocols allowing advanced analysis of confined cells. In this chapter, we first explain the principle and issues of using these slides to confine cells in a controlled geometry and describe their fabrication. Finally, we discuss how the nature of the confinement slide can vary and provide an alternative method to confine cells with gels of controlled rigidity.

Perinucleolar relocalization and nucleolin as crucial events in the transcriptional activation of key genes in mantle cell lymphoma.

In mantle cell lymphoma (MCL), one allele of the cyclin D1 (Ccnd1) gene is translocated from its normal localization on chromosome 11 to chromosome 14. This is considered as the crucial event in the transformation process of a normal naive B-cell; however, the actual molecular mechanism leading to Ccnd1 activation remains to be deciphered. Using a combination of three-dimensional and immuno-fluorescence in situ hybridization experiments, the radial position of the 2 Ccnd1 alleles was investigated in MCL-derived cell lines and malignant cells from affected patients. The translocated Ccnd1 allele was observed significantly more distant from the nuclear membrane than its nontranslocated counterpart, with a very high proportion of IgH-Ccnd1 chromosomal segments localized next to a nucleolus. These perinucleolar areas were found to contain active RNA polymerase II (PolII) clusters. Nucleoli are rich in nucleolin, a potent transcription factor that we found to bind sites within the Ccnd1 gene specifically in MCL cells and to activate Ccnd1 transcription. We propose that the Ccnd1 transcriptional activation in MCL cells relates to the repositioning of the rearranged IgH-Ccnd1-carrying chromosomal segment in a nuclear territory with abundant nucleolin and active PolII molecules. Similar transforming events could occur in Burkitt and other B-cell lymphomas.

Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation.

Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of the limited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.