Robust method for high-throughput surface patterning of deformable substrates.

We describe a simple and robust method for high-throughput surface patterning of deformable substrates such as silicone rubber films covered with a thin layer of protein and cell antifouling hydrogel (PLL-g-PEG). The irradiation with deep UV (<200 nm) of PLL-g-PEG-coated rubber substrates through a synthetic quartz photomask created micropatterns over a large area of the substrate. Incubation with proteins resulted in stable patterns with high feature resolution. RPE1 cells seeded on fibronectin patterns were constrained for days even after stretching. We also propose the crossbow feature as an interesting example allowing the stretching of normalized stress fibers.

Cell type-- dependent effects of Polo-like kinase 1 inhibition compared with targeted polo box interference in cancer cell lines.

Multiple critical roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. Plk1 contains two domains amenable for targeted interference: a kinase domain responsible for the enzymatic function and a polo box domain necessary for substrate recognition and subcellular localization. Here, we compare two approaches for targeted interference with Plk1 function, either by a Plk1 small-molecule enzyme inhibitor or by inducible overexpression of the polo box in human cancer cell lines. Inducible expression of the Plk1 polo box resulted in growth inhibition of RKOp27 human colon adenocarcinoma cells without obvious signs of mitotic abnormalities. A Plk1 kinase inhibitor in the same cell line arrested cells in mitosis with subsequent onset of apoptosis. Similarly, PC-3 human prostate cancer cells were growth inhibited on expression of the polo box. Prolonged expression of the polo box in these cells resulted in the occurrence of binucleated or multinucleated cells. In contrast, U2OS human osteosarcoma cells responded to overexpression of the polo box with a massive mitotic accumulation coinciding with the onset of apoptosis. Comparison of spindle formation revealed very similar mitotic abnormalities in polo box-overexpressing U2OS cells compared with U2OS cells treated with the Plk1 kinase inhibitor. We conclude that interference with polo box function and inhibition of Plk1 kinase activity can exert very similar phenotypic effects in certain cell lines but highly contrasting effects in others. This may point to subtle differences in the molecular machinery of mitosis regulation in cancer cells.

Comparative study and improvement of current cell micro-patterning techniques.

The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.

SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation.

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane-actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN-NuMA force generator complexes and hence for proper spindle orientation.

External forces control mitotic spindle positioning.

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.

Free Brick1 is a trimeric precursor in the assembly of a functional wave complex.

BACKGROUND: The Wave complex activates the Arp2/3 complex, inducing actin polymerization in lamellipodia and membrane ruffles. The Wave complex is composed of five subunits, the smallest of which, Brick1/Hspc300 (Brk1), is the least characterized. We previously reported that, unlike the other subunits, Brk1 also exists as a free form. PRINCIPAL FINDINGS: Here we report that this free form of Brk1 is composed of homotrimers. Using a novel assay in which purified free Brk1 is electroporated into HeLa cells, we were able to follow its biochemical fate in cells and to show that free Brk1 becomes incorporated into the Wave complex. Importantly, incorporation of free Brk1 into the Wave complex was blocked upon inhibition of protein synthesis and incorporated Brk1 was found to associate preferentially with neosynthesized subunits. Brk1 depleted HeLa cells were found to bleb, as were Nap1, Wave2 or ARPC2 depleted cells, suggesting that this blebbing phenotype of Brk1 depleted cells is due to an impairment of the Wave complex function rather than a specific function of free Brk1. Blebs of Brk1 depleted cells were emitted at sites where lamellipodia and membrane ruffles were normally emitted. In Brk1 depleted cells, the electroporation of free Brk1 was sufficient to restore Wave complex assembly and to rescue the blebbing phenotype. CONCLUSION: Together these results establish that the free form of Brk1 is an essential precursor in the assembly of a functional Wave complex.