Chazeau A.,Institut Universitaire de France |
Chazeau A.,The Interdisciplinary Center |
Chazeau A.,University Utrecht |
Garcia M.,Institut Universitaire de France |
And 12 more authors.
Molecular Biology of the Cell | Year: 2015
The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin-coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. © 2015 Chazeau, Garcia, Czöndör, et al.
Degot S.,Cytoo Inc
Journal of visualized experiments : JoVE | Year: 2010
To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.
Cytoo Inc | Entity website
Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due toissues with running complex assays on large three-dimensional (3D) structures. To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned96-well plates with automated production of nine uniform spheroids per well ...
Cytoo Inc | Entity website
Head Quarter Minatec - BHT - Bt. 52 7 parvis Louis Nel 38040 Grenoble cedex 9 Phone : +33 (0)4 38 88 47 05
Cytoo Inc | Date: 2011-07-19
Laboratory equipment and instruments, namely, cell culture vessels for cell analysis, high content analysis and cell screening, for the life sciences research market and the pharmaceutical industry market; computer software for cell and molecular analysis, high content analysis and cell screening, other than for medical use.