Biomedical subjects

Irena Lavelin

Publications and source records attributed to Irena Lavelin.

3 recordsLinked to original sources

PubMed2006

Development and application of automatic high-resolution light microscopy for cell-based screens.

Large-scale microscopy-based screens offer compelling advantages for assessing the effects of genetic and pharmacological modulations on a wide variety of cellular features. However, development of such assays is often confronted by an apparent conflict between the need for high throughput, which usually provides limited information on a large number of samples, and a high-content approach, providing detailed information on each sample. This chapter describes a novel high-resolution screening (HRS) platform that is able to acquire large sets of data at a high rate and light microscope resolution using specific "reporter cells," cultured in multiwell plates. To harvest extensive morphological and molecular information in these automated screens, we have constructed a general analysis pipeline that is capable of assigning scores to multiparameter-based comparisons between treated cells and controls. This chapter demonstrates the structure of this system and its application for several research projects, including screening of chemical compound libraries for their effect on cell adhesion, discovery of novel cytoskeletal genes, discovery of cell migration-related genes, and a siRNA screen for perturbation of cell adhesion.

Automation↗

PubMed2004

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton.

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.

Actins↗

PubMed2002

Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland.

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain.

Amino Acid Sequence↗