Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.[Keywords: Centrosomes; mitosis; actin; adhesion; cancer; cell cycle] Supplemental material is available at http://www.genesdev.org.

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.

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.

We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen-Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.

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.

Light-induced molecular adsorption of proteins (LIMAP) allows for quantitative sub-micrometer-resolution printing of multiple biomolecules. Surface-bound gradients are patterned within minutes over an entire glass cover-slip. LIMAP is used to perform selective immuno-assays, to dynamically control the adhesion of individual cells, and to achieve hierarchical co-cultures instrumental for tissue engineering.

Novel ester-functionalized polypyrrole−silica nanocomposite particles were prepared by oxidative copolymerization of pyrrole and N-succinimidyl ester pyrrole (50/50% initial concentrations), using FeCl3 in the presence of ultrafine silica nanoparticles (20 nm diameter). The N-succinimidyl ester pyrrole monomer was prepared in aqueous solution using 1-(2-carboxyethylpyrrole) and N-hydroxysuccinimide in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The resulting nanocomposites (N-succinimidyl ester polypyrrole−silica) are raspberry-shaped agglomerates of silica sol particles “glued” together by the insoluble poly(pyrrole-co-N-succinimidyl pyrrole). The N-succinimidyl ester polypyrrole−silica particles were characterized in terms of their size, density, copolymer content, and polydispersity. Scanning electron microscopy and disk centrifuge sedimentometry confirmed that the nanocomposite particles had narrow size distributions. X-ray photoelectron spectroscopy analysis indicated a silica-rich surface and a high surface concentration of N-succinimidyl ester groups. These nanoparticles exhibited good long-term dispersion stability. The chemical stability of the ester functions in aqueous media after several weeks of storage was monitored by FTIR spectroscopy. The functionalized nanocomposites were tested as bioadsorbents of human serum albumin (HSA). The very high amount of immobilized HSA determined by UV−visible spectroscopy is believed to be due to covalent binding. Incubation of the HSA-grafted nanocomposite with anti-HSA resulted in immediate flocculation, an indication that they are alternative candidates for visual diagnostic assays.

Adsorption of human serum albumin (HSA) onto conducting polypyrrole powders, doped with chloride (PPyCl), dodecyl sulfonate (PPyDS), and tosylate (PPyTS), has been monitored in 0.1 M phosphate buffer saline (PBS) and pH 7.4 using UV-visible spectroscopy in conjunction with the depletion method. The decreasing trend of adsorption was PPyTS > PPyDS > PPyCl and was interpreted in terms of hydrophobic interactions. Electrochemically synthesized PPyCl, PPyDS, and PPyTS films were used as model surfaces for contact angle measurements. Both static, advancing, and receding water contact angle (θW) suggested that the PPyTS is the most hydrophobic polymer among the three under test. The simple measure of θW permitted qualitative interpretation of the adsorption trend in terms of hydrophobic protein-PPy interactions. The van Oss-Good-Chaudhury (VOGC) method was further used to determine the dispersive, acidic, and basic components of the surface free energy (γS d , γS + , and γS -, respectively) of the conducting polypyrroles. These components show that polypyrrole generally behaves as a strong Lewis acid. The three surface free energy components were subsequently used to assess the absolute hydrophobicity of the substrates (∆G1W1), that is, the PPy-PPy interaction in water, the trend of which is that of protein adsorption. More importantly, the VOGC theory permitted determination of ∆G1W2, the free energy of protein-PPy in water, that is, the extent of hydrophobic interaction forces. The decreasing trend of ∆G1W2 values (absolute) was found to be PPyTS > PPyDS > PPyCl. This is a quantitative evidence for the role of hydrophobic interactions at the protein-PPy interface. In the case of PPyTS and PPyDS, the acid-base force contribution was much more important than the van der Waals one. In contrast, for the HSA-PPyCl system, the van der Waals forces predominantly contributed to ∆G1W2.