Owen P. Hamill, PhD - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

9318255 McBride Recently, a pressure-clamp technique has been developed (1) which is used in conjunction with patch-clamp techniques to study the kinetic properties of single and whole-cell mechanosensitive (MS) channel currents. The pressure-clamp technique is based on the balancing of negative and positive pressures to achieve the desired pressure at the membrane patch. Apart from its use in the activation of MS channel currents it can also provide a convenient means of applying defined and reproducible pressure/suction protocols for tight seal formation necessary in all patch-clamp experiments. The main goals of this project are to improved the existing prototype and make it into a convenient unit accessible to researchers studying MS channels as well as to patch-clampers in general. The main improvements center around efforts to reduce the response time of the clamp from its present value of 10 ms to the sub-millisecond time range. Another related improvement involves miniaturization of the system which will make the clamp a more compact and convenient package. Another aspect of the proposal involves determining the dynamics of membrane movement during the application of rapid pressure/suction steps. For instance, does membrane movement precede or is it even necessary for MS channel activation? This will be addressed by high resolution (both temporal and spatial) video microscopy. We will simultaneously monitor the pressure, voltage, current, membrane capacitance and membrane movement during the pressure/suction step. y, a pressure-clamp technique has been developed (1) which is used in conjunction wit h patch-clamp tech ! ! ! F 3 Times New Roman Symbol & Arial 1 Courier D " h E E = abstract Deseree King, BIR Deseree King, BIR

DESCRIPTION (provided by applicant): In its early stages prostate cancer stays in the prostate and is not life-threatening, but without treatment spreads to other parts of the body and eventually causes death. A major challenge is thus to recognize and target the molecular mechanisms allowing prostate tumor cells to spread. Our long-term goal is to identify and characterize mechanosensitive and Ca2+-dependent mechanisms that regulate tumor cell migration and invasiveness. Our research is based on the emerging concept that pathophysiologically enhanced cell motility is a critical step in the metastatic cascade leading to migration and invasion by tumor cells. This project focuses on the mechanosensory Ca2+-permeable cation channels (MscCa) that transduce membrane stretch into a Ca2+ influx, thus providing feedback between cell extension and Ca2+-dependent mechanisms that generate traction forces and regulate cell adhesions. We hypothesize that specific MscCa properties acquired during tumor cell transformation allow it to coordinate migration and invasiveness. We will test this hypothesis via two specific aims comparing the functional and molecular expression of MscCa in normal human prostate epithelial and in prostate tumor cell derivatives showing transformed motility and metastatic potential vs. their parent cell lines. Aim 1 will use patch-/pressure-clamp techniques and confocal immunofluorescence microscopy to compare MscCa's properties, (including conductance, gating dynamics, membrane surface distribution, and interaction with other Ca2+-sensitive channels) between "normal" and transformed" cells. Aim 2 will use time-lapse video microscopy and fluorescent Ca2+ imaging to measure fluctuations in intracellular [Ca2+] in order to determine how MscCa and other Ca2+-sensitive channels regulate cell migration. Cell migration plays a critical role in the pathogenesis of prostate cancer, so identifying and characterizing the MscCa, a rate-limiting regulatory molecule in cell migration, should introduce an important new therapeutic target for pharmacological and genetic treatments that are urgently required to prevent the spread and mortality of prostate cancer.