David A. Zacharias
Assistant Professor of Neuroscience (Ph.D. Mayo Graduate School, 1996)

daz@whitney.ufl.edu

Cellular Neurobiology and Signal Transduction

A very large fraction of proteins localized specifically to neuronal synapases are modified by lipids. My lab studies how lipid modifications of proteins regulate their function and participation in signal transduction. Specific projects include: discovery and characterization of the enzymes that regulate protein palmitoylation and characterization of the lipid microdomain that is the preferred residence for prenyl adducts.

My lab also studies the mechanisms by which selective serotonin reuptake inhibitors (SSRIs) modulate communication within and between neurons. SSRIs comprise a class of compounds that is therapeutically useful in treatment of many psychiatric disorders including anxiety, depression and Tourette's Syndrome. It is also well known that different SSRIs have different efficacies for producing a particular desired therepeutic outcome as well as a wide variety of off-target activity. The goal of my lab is to define components of the off-target activity and to determine the relevance of this activity to the variability in therapeutic outcome among the various SSRIs.

Finally, I am developing a cnidarian cell-culture model as a tool to understand how corals and anemones regulate the expression level and diversity of endogenous GFP-like proteins.

Current Projects

Regulation of Protein Lipid Modifications

Figure 1. Confocal image of MDCK cells expressing mYFP that is lipid modified. Note the strong presence of the fluorophore on the plasma membrane.

Figure 2. Two rat hippocampal neurons expressing lipid modified YFP. The lipid modifications cause them to be concentrated 1) at sites of cell:cell contact, presumably synapses and 2) in the growth cones of growing branches.

Figure 3. Lipid modifications being studied in my lab: A. Prenylation and B. Acylation. Each class of modification targets proteins (to which they are attached) to unique subcellular locales. This ability is likely due to their varying chain length, degree of saturation and their physical position on the proteins. Both forms of prenylation occur via stable thioether bonds on the final cysteine of a “CAAX” box at the C-terminus of a protein. Myristoylation occurs via a stable amide bond to the N-terminal glycine of a protein while addition of palmitate occurs most commonly via a labile thioester bond to the side chain of a free, reactive cysteine on the cytoplasmic side of the PM.

Many proteins are concentrated on the plasma membrane (PM) (Figure 1), trapped in specialized subcellular regions, like synapses (figure 2) and caveolae, by virtue of their lipid modifications (figure 3). Thio-acylation or S-palmitoylation, a common form of lipid modification, is unique in that it is reversible and dynamic, suggesting a modulatory role in signal transduction similar to phosphorylation. Recent data indicate that proper, dynamic regulation of the palmitoylation of PSD-95, an abundant scaffolding protein in the synapse, is critical for synaptic organization and function, linking palmitoylation to complex processes such as learning, memory and disease. In support of this position, it is known that mutations in one gene regulating S-palmitoylation result in a severe neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis or ICNL. Additionally, a candidate gene for the regulation of S-palmitoylation is linked to schizophrenia.

Biochemical characterization of the enzymes responsible for S-palmitoylation (palmitoyl thio-acyl transferases, S-PATs) has been difficult and controversial; recent data from experiments in yeast add substantial weight to the argument that such enzymes exist. To date, functional genomics discovery programs in vertebrate systems similar to those in yeast have been expensive and time consuming. My lab is addressing this issue by combining a novel form of gene-trapping in vertebrate cell cultures, with a fully automated readout in a high-throughput microscopy format (for a description of the microscope, go to http://www.q3dm.com/htmlsite/products_EIDAQ.php. Using this assay system we are testing directly and functionally the hypothesis that S-PATs exist in vertebrates. Should this prove to be the case, as we suspect that it will, we will be ideally positioned to elucidate the entire enzymatic pathway for protein S-palmitoylation by quantitatively analyzing millions of cells from tens of thousands of “trapped” cell lines.

We are developing this system so that it can also be used as an experimental tool that can be easily extended to screens for other genes that regulate the subcellular distribution and concentration of proteins enabling numerous applications in basic and therapeutic research.

The high-throughput microscopy system has the capability to quantify virtually any change in cellular morphology whether it is a change in the shape of the cell itself or redistribution of a fluorescence-labeled molecule within the cells. For a description of the types of algorithms we (Q3DM and myself) have worked with and are developing see http://www.q3dm.com/htmlsite/technology_
applications.php.

Serotonin-related signal transduction

My lab also studies the mechanisms by which selective serotonin reuptake inhibitors (SSRIs) modulate communication within and between neurons. SSRIs comprise a class of compounds that is therapeutically useful in treatment of many psychiatric disorders including anxiety, depression and Tourette's Syndrome. It is also well known that different SSRIs have different efficacies for producing a particular, desired therepeutic outcome as well as a wide variety of off-target activities. The goal of my lab is to define components of the off-target activity and to determine the relevance of this activity to the variability in therapeutic outcome among the various SSRIs. The first family of proteins we will analyze is GPCRs. HTM will allow us to test simultaneously the activity of any number of SSRI (or any other drugs) on the activity of hundreds of GPCRs (see application note from Q3DM). This approach is often referred to as reverse pharmacology and is a strategy used in the pharmaceutical industry to discover new targets for old drugs and/or to fine tune drug-target interactions and the downstream consequences of the binding event. The classes of drugs and targets can be mixed and matched in any number of ways. The high-throughput nature of the system will allow us to rapidly deconvolute the signaling pathways associated with receptors that we find interesting.

Personnel

David A. Zacharias, Assistant professor
Sonia Planey, Post Doctoral Associate
Terri Seron, Post Doctoral Associate
Jun Zhang, Post Doctoral Associate
Carolina Ceballos, Laboratory Technician

Selected Publications

Moran, Timothy J., Casey Laris, Emma Palfreyman, Michael Theileking, Ivana Mikic, David Zacharias, Scott Callaway, Jeffrey Price, Edward Hunter (In press) NF beta nuclear translocation validation and performance in high throughput cell imaging. Assay and Drug Development Technologies.

Zacharias, David A., and Roger Y. Tsien. (In press) Biochemistry and Mutagenesis of the Green Fluorescent Protein. In: Green Fluorescent Protein: Properties, Applications and Protocols. Eds. Chalfie and Kain.

Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. and Tsien, R.Y. (2003) Genetically targeted chromophore-assisted light inactivation. Nature Biotechnology 21: 1505-1508. View PDF file.

Zacharias, D.A. (2002) Sticky caveas in an otherwise glowing report: oligomerizing fluorescent proteins and their use in cell biology. Science's STKE.
www.stke.org/cgi/content/full/OC_sigtrans;2002/131/pe23 . View PDF file.

Campbell, R.E., Oded T., Palmer, A. Steinbach, P. Baird, G. Zacharias, D. and Tsien, R. (2002) A monomeric red fluorescent protein. PNAS 99: 7877-7882. View PDF file.

Zacharias, D.A., Violin, J.D., Newton, A.C. and Tsien, R.Y. (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296: 913-916. View PDF file.

Strehler, E.E. and Zacharias, D.A. (2001) Role of alternative splicing in generating isoform diversity among plasma membrane calcium pamps. Physiol. Rev 81: 21-50. View PDF file.

Chan, F.K-M., Siegel, R.M., Zacharias, D., Swofford, R., Holmes, K.L., Tsien, R.Y. and Lenardo, M.J. (2001) Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein. Cytometry 44: 361-368. View PDF file.

Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. and Tsien, R.Y. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 276: 29188-29194. View PDF file.

Baird, G.S., Zacharias, D.A., Gross, L., Hoffman, R., Baldridge, K., and Tsien, R.Y. (2000) Biochemistry, mutagenesis and chromophore of dsRED, a red fluorescent protein from coral. PNAS 97: 11984-11989. View PDF file.

Siegel, R.M., Fredricksen, J.K., Zacharias, D.A., Chan, F. K-M., Johnson, M., Lynch, D. Tsien, R.Y. and Lenardo, M.J. (2000) Fas preassociation is essential for transmembrane signaling and dominant inhibition by pathogenic mutations. Science 288: 2354-2357. View PDF file.

Siegal et al 2000 Science STKE.pdf

Zacharias Baird and Tsien 2000 Curr Opin Neuro.pdf

Baird Zacharias Tsien 1999 PNAS Circular Permutations.pdf

Zacharias and Kappen 1999 BBA.pdf

Toutenhoofd et al 1998 BBA.pdf

Zacharias Zemarco Strehler MBR 1997.pdf

Schenone et al 1996 JNeuroimm.pdf

Zacharias Dalrymple Strehler 1995 MBR.pdf

Zacharias et al Development 1993.pdf

Zacharias and Strehler 1996 Current Biology.pdf