Rapid Analysis of Signaling in Adherent Cells

Univ Of North Carolina Chapel Hill, Chapel Hill NC

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Abstract

DESCRIPTION (provided by applicant): Application of microanalytical techniques for chemical separations has had a profound impact on biological investigation, and recent advances for chemical analysis of single cells hold great promise for understanding cellular physiology in health and disease. An emerging area in the molecular analysis of cellular function is the study of the activation of signal transduction proteins within individual cells. The current grant proposes to develop a platform that will greatly enhance the throughput for measurements of enzyme activity in single, adherent cells. State-of-the-art microfabrication methods and mass-produced, printed-circuit technology will be integrated to develop arrays of electrically addressable cell positions. This array in conjunction with capillary electrophoresis (CE) will provide a unique approach for subsecond cell lysis and sampling. This rapid sampling component will be combined with a new buffer-delivery strategy for CE and with electronics control systems to produce a semi-automated platform enabling rapid, serial sampling and chemical separation of the contents of individual, adherent, mammalian cells. The system will be used to perform quantitative biochemical assays of the activation of signal transducing kinases in single cells. Three kinases (protein kinase C, protein kinase B, and calcium/calmodulin-activated kinase II) which regulate a diverse array of processes will be targeted; however, the device and method are applicable to most other cellular kinases. A multidisciplinary team of investigators with expertise in analytical chemistry, microfabrication, and cell biology will collaborate to produce and validate the device which will mount on the stage of a microscope so that cells can be studied prior to measurement of their kinase activation status. A critical part of the development process will be the conduct of careful controls to insure cell health and viability prior to cell sampling. The integrated device and method will enable analyses of adherent cells in the numbers needed to quantitatively characterize signaling responses after physiologic stimulation. In the final phase of the grant, the assays will be performed on populations of single cells to test a fundamentally important concept in the field of cellular signal transduction: the existence of kinase substates during signal transduction in mammalian cells.