A system for high-speed microinjection of adherent cells.

This paper reports on a semi-automated microrobotic system for adherent cell injection. Different from embryos/oocytes that have a spherical shape and regular morphology, adherent cells are flat with a thickness of a few micrometers and are highly irregular in morphology. Based on computer vision microscopy and motion control, the system coordinately controls a three-degrees-of-freedom microrobot and a precision XY stage, demonstrating an injection speed of 25 endothelial cells per minute with a survival rate of 95.7% and a success rate of 82.4% (n=1012). The system has a high degree of performance consistency. It is operator skill independent and immune from human fatigue, only requiring a human operator to select injection destinations through computer mouse clicking as the only operator intervention. The microrobotic system makes the injection of a large number of adherent cells practical for testing cellular responses to foreign molecules.

Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1.

Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein-protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined in vivo. We find that elimination of murine GRIP1 results in embryonic lethality. GRIP1(-/-) embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development.

Regulation of c-fos expression by static stretch in rat myometrial smooth muscle cells.

OBJECTIVE: We investigated whether mechanical stretch stimulates expression of the immediate early gene c-fos messenger RNA in rat myometrial smooth muscle cells. STUDY DESIGN: Freshly isolated myometrial smooth muscle cells (from virgin rats) were plated on flexible culture plates that were coated with either collagen I, laminin, elastin, or pronectin and were subjected to a static stretch with a computerized strain unit. Immunocytochemistry was used to define smooth muscle cell phenotype, and c-fos expression levels were assessed by Northern blotting. RESULTS: Myometrial smooth muscle cells maintained their phenotype for at least 7 days in culture. Stretch induced a dramatic increase in levels of c-fos messenger RNA, which peaked after 30 minutes. Stimulation of c-fos gene expression was dependent on the degree of stretch (maximum induction at 25% elongation). Interestingly, as little as 1 minute of stretch was sufficient to induce maximal c-fos expression at 30 minutes. The most dramatic induction of c-fos was observed in smooth muscle cells that were stretched on collagen I-coated plates. CONCLUSION: Myometrial smooth muscle cells can directly respond to mechanical stretch with a strength- and matrix component-dependent increase in c-fos messenger RNA. The time delay between the initial stimulus and the cumulative elevation of c-fos messenger RNA suggests a complex regulation from mechanoreception to c-fos gene expression.