AfCS > AfCS laboratories > Microscopy Laboratory

ALLIANCE FOR CELLULAR SIGNALING

MICROSCROPY LABORATORY

Stanford University
Palo Alto, California

Overview

The Alliance for Cellular Signaling (AfCS) Microscopy Laboratory uses confocal, epifluorescence, and total internal reflection microscopy to acquire information about the subcellular localization of signaling proteins and changes in their localization that occur in response to activation of signaling pathways.

One of the primary goals of the lab is to develop quantitative analytical procedures to measure the extent of co-localization of signaling proteins with subcellular markers (as a function of time) in a receptor-stimulated macrophage cell model. To implement this strategy, the lab is generating, fluorescently-tagged markers for subcellular structures, such as the plasma membrane, Golgi, nucleus, nuclear membrane, and several vesicular structures. These will be used as references to compare expression patterns with members of an extensive collection of fluorescently-tagged signaling proteins in Gateway vectors.

The Microscopy Laboratory is also focusing on signaling proteins that undergo translocation within the cell, since such events can be used as readouts of the time-dependence of signaling processes. One objective is to understand the chemical and structural basis of cellular localization and translocation.

As the Alliance efforts continue, cellular imaging will play an increasing role in attempts to order components in signaling pathways using perturbation strategies such as RNAi, pharmacology and dominant negatives and to quantify the flux of information through these pathways. Our lab is implementing single cell imaging screens as a part of this effort.

Collaborators

The Microscopy Laboratory is collaborating with Cell Signaling Technology (use and development of antibodies) www.cellsignal.com.

Staff

Alliance for Cellular Signaling
Microscopy Laboratory
975 California Avenue
Palo Alto, California 94304
Phone: 650-852-0381
Fax: 650-952-9584

Director	Tobias Meyer, Ph.D.
Lead Scientist	Nancy O'Rourke, Ph.D.
Lead Scientist	Grischa Chandy, Ph.D.
Research Assistant	James Whalen, Ph.D.
Research Assistant	Mary Verghese, Ph.D.
Research Assistant	Elizabeth Gehrig
Research Assistant	WeiSun Park
Research Assistant	Heather Bryan
Research Scientist	Won Do Heo, Ph.D.
Research Scientist	Jen Liou, Ph.D.

Slide Presentations from May 23-26, 2004, AfCS Annual Meeting

• "Biosensors: Translocations and FRET Analysis of Protein Interactions, Activities, and Conformations", Tobias Meyer
• "Single-cell Assays" [A] [B], Grischa Chandy, Nancy O'Rourke

Goals for Year 5 (9/04-8/05)

• FXM Single Cell Calcium and PIP3 Assays. We will continue to screen lentivirally-transformed RAW 264.7 cells that stably express shRNA against proteins targeted by the FXM project. To complement the lentiviral data, we will work on protocols for more transient knockdown of signaling proteins by transfecting duplex RNAi or transiently expressing hairpin constructs. In addition, we will employ appropriate chemical reagents to characterize the calcium and PIP3 responses further. Concurrent with these studies, we will continue our efforts to streamline data acquisition and analysis with the goal of increasing the throughput. We will collaborate with the AfCS data analysis group to develop models of the calcium and PIP3 signaling modules.
• Co-localization, and Translocation of PH Domains. Our work to characterize localization, translocation, and binding properties of 136 murine PH domains is largely complete. Our future efforts will turn to synthesizing these data to shed light on the structural properties of the PH domains that determine these characteristics. Where appropriate, PH domains will be developed into new biosensors to be used in signaling assays. We will also assay translocation and localization of the full-length proteins that contain the PH domains. In another set of co-localization studies, we will co-transfect the PH domains along with subcellular markers to verify the identity of the organelles where they reside.
• Generation of STK constructs Thus far, our lab has teamed up with the Molecular biology lab to generate constructs encoding in excess of 225 unique protein kinases. We generated K-to-R mutants for roughly half of those. We will complete the set of STK constructs that we are working on currently. We are in the process of mutagenizing another 100 of the newly cloned kinases to generate the KtoR variants of these.
• Localization, Colocalization and Translocation of Full Length Proteins. We will continue to evaluate the localization patterns of proteins encoded by the full-length constructs generated by the AfCS labs. We will choose a subset of proteins that have interesting localization patterns and compare their distribution with known markers. In addition, we will employ a targeted strategy of identifying proteins likely to translocate with certain stimuli and screen these with ligands or cocktails of stimuli to induce translocation.