Martin Poenie, PhD, Stanford University - US grants

This proposal is part of an effort to understand how cells localize molecules and organelles to specific regions of the cytoplasm, which is referreed to as polarization. The observation that gradients of intracellular calcium ions ((Ca2+)i) can form in cells represents both an example, and perhaps, a clue to the solution of the problem. The cytotoxic T lymphocyte (CTL), is presently being used as a model system for thses studies. The CTL is important in itself as an important arm of the immune system, and an excellent system for the study of cell polarization. The CTL exhibits polarization of organelles in response to binding of appropriate target cells. At the time these movements are taking place, a calcium gradient also forms. Since calcium is an important regulatory element and is known to affect molecules invovled in cell motility, it is likley that the two are related. The aim of this project is to understand the mechanism of (Ca2+)i gradient formation and how Ca2+ might act locally to polarize the cell. A model has been formulated to explain the formation of a (Ca2+)i gradient which involves the participation of another important regulatory molecule, protein kinase C. It appears that protein kinase C is also locally activated in CTLs with a polarity opposite that of the calcium gradient. Since protein kinase C can in some cases act to lower (Ca2+)i it may be part of the mechanism for forming the (Ca2+)i gradient. This study will test the hypothesis that local activation of kinase C can activate calcium pumping systems and locally lower (Ca2+)i resulting in a (Ca2+)i gradient. Finally, since (Ca2+)i and protein kinase C can have reciprocal effects on the activity of some proteins, a Foundation is laid for polarity. This study will attempt to detect localized differences in the phosphorylation state of enzymes that are modulated reciprocally by (Ca2+)i and protein kinase C.

Dr. Martin Poenie will be recommended for a Presidential Young Investigator Award. His research interests are in the area of signal transducton across the cell plasma membrane and ionic regulation of cellular processes. He has been a leader in development of fluorescent indicators and instrumentation to image calcium ions in single living cells. The Presidential Young Investigator Award will support the further development of these techniques and their application to important problems of cellular regulation.

Several lines of evidence including vibrating calcium specific electrode measurements and calcium-imaging data support the hypothesis that calcium gradients are involved in setting up cell polarity. Of particular interest is the relationship between calcium gradients in sea urchin eggs which develop during pronuclear migration/fusion and the subsequent axis of cleavage. We also propose to study the role of calcium gradients in orienting motile cells such as macrophages and T lymphocytes. We propose to test this hypothesis directly, through the development of new optical tools including new long-wavelength, ratioable calcium indicators, probes for following the movements of the centrosome and methods for making localized gradients of calcium ions in cells. Included in the proposal is a proposed solution to the well recognized problems of dye loading and compartmentation seen with fura-2 and other ion indicators.

9318538 Poenie The goal of this project is to develop and apply new fluorescent probes that will ultimately make it possible to dissect the relationships between intracellular calcium, protein kinase C, and cytoskeletal organization that give rise to directional movements of cells and organelles. Three specific probes will be developed: 1) Calcium indicators based on the PE3 model which resist leakage and compartmentalization. The current generation of fluorescent calcium indicators such as fura-2, fluo-3, indo-1 and others are plagued by the problems of leakage and compartmentalization. The problem of leakage means that cells can only be reliably studied for a few minutes after they have been loaded. The problem of compartmentalization is that the dye may be sequestered in organelles either as the free anion or as the AM ester. This creates numerous apparent artifacts regarding the distribution of free calcium ions in the cell. At present, PE3 loads uniformly, but for many cells, little dye gets inside. Specific changes in the structure of these dyes will be made to rectify this problem. 2) Near-membrane calcium indicators will be developed based on the FFP18 model. FFP18, a fura-2 analog developed in this laboratory, has the potential to reveal differences between calcium ions near cell membranes as compared to the bulk cytosol. Because this indicator cannot diffuse freely through the cytosol it should not dissipate calcium gradients as fura-2 and other BAPTA-type indicators may. Improvements in FFP18 are needed, however, to realize the full potential of this type of indicator. FFP18 tends to form complexes or aggregates that distort its spectra and complicate calibrations. FFP18 analogs will be made that are negatively charged which should eliminate aggregation. Further, preliminary data indicate that calcium near membranes is higher than FFP18 can track. To solve this problem, FFP18 analogs with lower affinity for calcium will be synthesized. Analogs with higher selectivity for the plasma membrane will also be made. 3) New and improved fluorescent probes for protein kinase C (PKC) based on the fim-1 model will be developed. Fim-1 is a fluorescent probe that binds to protein kinase C. It exhibits good specificity for PKC when used with fixed and permeabilized cells. New fim-1 analogs will be developed that have brighter fluorescence and higher affinity for PKC, with the goal of obtaining a fluorescent probe for use in studying PKC in living cells. Fim-1 analogs that can indicate changes in PKC activation will also be developed. %%% The goal of this project is to develop and apply new fluorescent chemical probes that will ultimately make it possible to study the chemical signals that give rise to cellular organization and directional movements of cells and their internal structures. The signal molecules that will be tracked by these probes are calcium and a critical enzyme known as protein kinase C. A variety of evidence suggests that these signal molecules play major roles in controlling the dynamics of the cell's internal skeleton. These tools should allow direct visualization of where and when signals occur that direct the movements of cells.***

Visualizing microtubules and other cytoskeletal structures in most types of living cells has been a difficult problem. A new type of microscopy, called modulated polarization microscopy, which can routinely image individual microtubules and other cytoskeletal structures as well as vesicle movements along cytoskeletal tracks will be developed. Modulated polarization microscopy images cytoskeletal structures based on their birefringence but takes advantage of the fact that birefringence is sensitive to polarization angle. When birefringent structures are illuminated with plane polarized light of varying angle and viewed between crossed polarizers they oscillate in intensity. This behavior is not seen with structures that are not birefringent and provides a means for separating weakly birefringent elements from the cytoplasmic background. A prototype instrument has been built using Faraday rotators to modulate the plane of polarized light presented to the specimen. Images obtained using this microscope demonstrate its potential for imaging the cytoskeleton. An advanced version of the moldulated polarization microscope with greatly improved sensitivity, contrast, image quality, and resolution will be built. Improvements in the new microscope will be based on analysis of problems with the original prototype.

This proposal is aimed at understanding how alcohol activates T cell signaling pathways and how non-specific T cell activation could in turn lead to immunosuppression. We have used Jurkat cells stimulated by superantigen coated Raji cells as a ifiodel system for T cell activation and synapse formation. Our preliminary data indicates that 25-100 mM ethanol can cause Jurkat cells to adhere to Raji cells in the absence of superantigen. This adhesion depends on LFA-1 and is likely a consequence of triggering.T cell signaling pathways. Furthermore, ethanol induces the formation of a synapse that is essentially identical to that which forms when superantigen is present, a further indication that signaling pathways have been turned on. As a means of dissecting these signaling events, we propose to use the synapse and its components as molecular markers for T cell activation by ethanol. Our approach will be to use biochemical markers, mutant cells lines, and the three dimensional arrangement of key signaling proteins.at the synapse to monitor how ethanol intersects the normal T cell signaling system. Lipid rafts play an important role in T cell activation and it may be here that ethanol exerts it primary effects. To study this we will use fluorescence anisotropy measurements to determine the impact of ethanol on clustering of proteins lipid rafts. We will also analyze some of the consequences of activation such as T cell receptor downregulation to determine how ethanol modifies T cell responses after exposure. Having worked out the signaling pathway in Jurkat cells, we will look for similar activation events in murine T cells using a cloned allospecific cytotoxic T cell line (BM3.3). We will examine whether ethanol can cause adhesion and killing of target cells in the absence of antigen and we will determine if ethanol alters normal antigen-mediated target cell lysis. Jn an effort to abrogate the effects of ethanol, we will detemine whether inhibiting LFA-mediate adhesion or steps in the T cell activation pathway will block these effects.