The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell-cell or cell-extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus-centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.image quantification ͉ micropattern ͉ cell standardization

Cells display a large variety of shapes when plated in classical culture conditions despite their belonging to a common cell type. These shapes are transitory, since cells permanently disassemble and reassemble their cytoskeleton while moving. Adhesive micropatterns are commonly used to confine cell shape within a given geometry. In addition the micropattern can be designed so as to impose cells to spread upon adhesive and nonadhesive areas. Modulation of the pattern geometry allows the analysis of the mechanisms governing the determination of cell shape in response to external adhesive conditions. In this study, we show that the acquisition of cell shape follows two stages where initially the cell forms contact with the micropattern. Here, the most distal contacts made by the cell with the micropattern define the apices of the cell shape. Then secondly, the cell borders that link two apices move so as to minimise the distance between the two apices. In these cell borders, the absence of an underlying adhesive substrate is overcome by stress fibres forming between the apices, which in turn are marked by an accumulation of focal adhesions. By inhibiting myosin function, cell borders on nonadhesive zones become more concave, suggesting that the stress fibres work against the membrane tension in the cell border. Moreover, this suggested that traction forces are unevenly distributed in stationary, nonmigrating, cells. By comparing the stress fibres in cells with one, two, or three nonadherent cell borders it was reasoned that stress fibre strength is inversely proportional to number. We conclude that cells of a given area can generate the same total sum of tractional forces but that these tractional forces are differently spaced depending on the spatial distribution of its adherence contacts.

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We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts), or actin, but are arrested when both Mts and actin filaments are disrupted.As the centrioles replicate at the G 1 /S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated ␥ -tubulin, and nucleate similar number of Mts in Mt repolymerization experiments, during G 1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.

During rodent cortex development, cells born in the medial ganglionic eminence (MGE) of the basal telencephalon reach the embryonic cortex by tangential migration and differentiate as interneurons. Migrating MGE cells exhibit a saltatory progression of the nucleus and continuously extend and retract branches in their neuritic arbor. We have analyzed the migration cycle of these neurons using in vitro models.We show that the nucleokinesis in MGE cells comprises two phases. First, cytoplasmic organelles migrate forward, and second, the nucleus translocates toward these organelles. During the first phase, a large swelling that contains the centrosome and the Golgi apparatus separates from the perinuclear compartment and moves rostrally into the leading neurite, up to 30 m from the waiting nucleus. This long-distance migration is associated with a splitting of the centrioles that line up along a linear Golgi apparatus. It is followed by the second, dynamic phase of nuclear translocation toward the displaced centrosome and Golgi apparatus.The forward movement of the nucleus is blocked by blebbistatin, a specific inhibitor of nonmuscle myosin II. Because myosin II accumulates at the rear of migrating MGE cells, actomyosin contraction likely plays a prominent role to drive forward translocations of the nucleus toward the centrosome. During this phase of nuclear translocation, the leading growth cone either stops migrating or divides, showing a tight correlation between leading edge movements and nuclear movements.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones.

The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.