Programmed subcellular release to study the dynamics of cell detachment.

摘要

Cell detachment is central to a broad range of physio-pathological changes however there are no quantitative methods to study this process. Here we report programmed subcellular release, a method for spatially and temporally controlled cellular detachment and present the first quantitative results of the detachment dynamics of 3T3 fibroblasts at the subcellular level. Programmed subcellular release is an in vitro technique designed to trigger the detachment of distinct parts of a single cell from a patterned substrate with both spatial and temporal control. Subcellular release is achieved by plating cells on an array of patterned gold electrodes created by standard microfabrication techniques. The electrodes are biochemically functionalized with an adhesion-promoting RGD peptide sequence that is attached to the gold electrode via a thiol linkage. Each electrode is electrically isolated so that a subcellular section of a single cell spanning multiple electrodes can be released independently. Upon application of a voltage pulse to a single electrode, RGD-thiol molecules on an individual electrode undergo rapid electrochemical desorption that leads to subsequent cell contraction. The dynamics of cell contraction are found to have characteristic induction and contraction times. This thesis presents the first molecular inhibition studies conducted using programmed subcellular release verifying that this technique can be used to study complex signaling pathways critical to cell motility. Molecular level dynamics of focal adhesion proteins and actin stress fibers provide some insight into the complexities associated with triggered cell detachment. In addition to subcellular release, the programmed release of alkanethiols provides a tool for to study the spatially and temporally controlled release of small molecules or particles from individually addressable gold electrodes. Here we report on experiments which determine the dynamics of programmed release using fluorophore-terminated C6 or C11 thiols and the regeneration of individual electrodes using different molecules. Programmed release of small molecules has applications in drug delivery, while programmed subcellular release has the potential to study the full cascade of events that involve the extracellular matrix, actin rearrangement, and protein signaling pathways.