Our research addresses the signal transduction pathways of chemical sensing in Paramecium and mice.
My interests center on chemoreception using Paramecium, a single-celled animal, as a model. These cells are like little swimming neurons and, like our neurons that detect odors or tastes, they respond to stimuli by membrane electrical changes. We approach sensing of chemical stimuli on several levels: membrane biochemistry to identify receptors and "signal transduction" components that turn a chemical stimulus signal into an electrical one; molecular genetics to clone genes for receptors and other proteins in chemoreception and to make predictable changes in the gene and protein sequences; measurements of calcium and calcium metabolism by fluorescence and isotopic methods; measurements of internal, second messengers such as cyclic nucleotides; electrophysiology to characterize membrane electrical changes; motion analysis to digitize normal and mutant swimming.
YanoSan is the molecular biology master of the laboratory. He deftly creates vectors for transformation of Paramecium and turns them green with Green Fluorescent Protein. He targets plasma membrane calcium pumps by over-expression and antisense down-regulation. He also uses bioinformatics and molecular cloning to identify the genes for enzymes in the glycosylphosphatidylinositol anchor synthesis pathway.
Cyclic-AMP is a well recognized chemoattractant for the single-cell eukaryote Paramecium tetraurelia. Decidedly a food cue, cAMP causes cell hyperpolarization inducing smooth and fast forward swimming. A protein of ~48kDa has been purified and antibodies against it specifically inhibit chemoresponse to cAMP. However, the gene for the receptor has not been cloned.
Six sequences from the P. tetraurelia Genome have been identified as cAMP receptor candidates based on amino acid homology to the Dictyostelium CARS, which are seven transmembrane G protein coupled cyclic AMP chemotaxis receptors. Similar sequences are found in the annotated Tetrahymena genome as well.
These sequences have been used to design RNA interference (RNAi) feeding constructs to down-regulate the expression of these putative receptor proteins. These RNAi-fed cells are used in T-maze assays to quantify the reduction of cAMP chemoattraction. Semi-quantitative RT-PCR shows the reduction of transcript levels. To date, we have shown significant reduction in chemoresponse to cAMP with one of the RNAi constructs.
These tests will help to characterize as well as identify the cAMP receptors in Paramecium tetraurelia.
I use techniques such as RNA to research Intraflogellar Transport and its role in ciliogenesis and ciliary protein trafficking
I am interested in studying the role of Glycosyl-phosphatidylinositol (GPI) anchored proteins in Chemosensory Signal Transduction in Paramecium tetraurelia. GPI anchors represent a unique method of binding proteins to the plasma membrane. This anchor includes ethanolamine phosphate, trimannoside, glucosamine and inositol phospholipid and is added to the carboxyl terminus of the protein as a post-translational modification.
GPI-anchored proteins include a wide range of proteins such as cell surface receptors, cell adhesion molecules, cell surface hydrolases and protozoan and mammalian antigens. In Paramecium, GPI-anchored proteins make up the largest class of ciliary surface proteins. GPI-anchored proteins are the major constituents in lipid rafts and are postulated to play a significant role in chemosensory cascades.
My research is focused on characterizing two putative full-length GPI-anchored folate receptors. In addition I am also working on 4 putative glutamate binding proteins and their role in glutamate uptake in Paramecium tetraurelia.
Sukanya Mujumder is exploring the function of the Pawn-A gene by using RNAi to down regulate its mRNA. Sukanya can make Pawn phenocopies using RNAi. These cells do not swim backward, just like the Pawn-A mutants. This work sets the stage for a study of the function of the Pawn-A protein in calcium channel function.
The Plasma Membrane Calcium ATPase (PMCA or Ca2+ pump) is ubiquitous expressed protein that transports Ca2+ out of cell by hydrolysis of ATP. PMCAs are essential in control of Ca2+ concentration in cytosol, maintaining the normally low intracellular free Ca2+ ([Ca2+]i) and countacting the elevated [Ca2+]i generated by Ca2+ signaling. In paramecium, it has been found that 3 isoforms are expressed (PMCA2, 3, and 4). PMCA is a multiregulated transporter. The activity of the PMCA can be regulated by calmodulin, protein kinases (PKA, PKC), protease, acidic phospholipids, oligomerization and differential reactions with PDZ domain-containing anchoring and signaling proteins. All the three PMCA isoforms in paramecium contain the PDZ-binding domain. And PMCAs play roles in both depolarization and hyperpolarization that affects chemoresponses of paramecium. But the result on regulation of PMCA in paramecium is limited. So I am trying to identify the interacting partners of PMCA, and further to clarify the regulatory relations and mechanism between PMCAs and their interacting proteins
I am interested in studying the role of Plasma Membrane Calcium ATPases (PMCAs) as a calcium extrusion mechanism in mouse olfactory sensory neurons (OSNs). When an odorant binds to the G protein-coupled receptor on the OSN, it activates an adenylate cyclase resulting in an increase in cAMP which elicits opening of Cyclic Nucleotide Gated (CNG) channels. Ca2+ enters through the CNG channels which activates a chloride current leading to cell depolarization. The receptor potential finally leads to the generation of action potentials conveying the chemosensory information to the olfactory bulb. Following the depolarization, calcium concentration must be restored to resting levels for the OSN to be able to detect new odors. It is believed that Na+/Ca2+ exchanger is the only mechanism responsible for calcium clearance. We postulate that PMCAs are also major players in this process. I am using calcium imaging techniques on PMCA2 knock-out and wild type mice to see the extent to which PMCAs are involved in calcium clearance after excitation.
Research: Significance of BBS proteins in paramecium
I am presently a second year Ph.D. student working under the supervision of Dr. Judith Van Houten. My research is focused on the Lipid Rafts of Paramecium cellular membrane. These lipid rafts are small, highly organized microdomains, enriched in 3-hydroxysterols and sphingolipids and are resistant to cold non-ionic detergents like Triton X-100. Some signaling proteins associated with them are the GPI-anchored proteins mainly with some other transmembrane proteins, G proteins and doubly acylated tyrosine kinases. A low buoyant density in different density gradients characterizes them. The rafts float around in small regions in the membrane and are clustered together when they are turned On to play an active role in signal transduction.
Paramecium, a ciliate, is an important model for studying Ca2+ signaling and chemoreception signal transduction. We have evidence for the existence of GPI-anchored surface antigens A and B all over the membrane. The plasma membrane calcium pumps (PMCA) isoforms 2, 3 and 4 were partially associated with rafts in the cell surface membrane but are absent in the ciliary membrane. cAMP receptor, a transmembrane protein, is also found to associate with lipid rafts. Other GPI-anchored proteins like the PCMs and the Folate binding proteins are consistently found in Paramecium membrane. I am using Optiprep for density gradient to characterize raft fractions in the ciliary membrane. I would also like to quantify the proteins, gangliosides and sterols present. Cholesterol depletion studies using Methyl Beta Cyclodextrin and designing an antibody against 5’nucleotidase, a good GPI anchored marker protein of lipid rafts, are parts of my future work.
Atreyi Ghatak is working with mice that have no plasma membrane calcium pump-2 (PMCA 2)courtesy of Dr. G. Shull. These knockout mice clear calcium from their olfactory sensory neurons more slowly than wild type neurons (see Sam Ponissery above), and Atreyi is examining the behavior of the mice to determine whether the knock outs have changes in their ability to smell and respond to odor. She is using fear conditioning with various odorants as the conditioned stimulus.
I am interested in calcium image and taste cells
Cassie Jacobs an Accelerated Masters Graduate, studied the glutamate chemoreceptor in Paramecium. Cassie has used RNAi to show that a particular gene pGluR1 functions in chemoresponse to L-glutamate and to no other stimuli that she tested. She has used a GFP-fusion protein to demonstrate that the protein from her gene is in the cell body membrane and cilia. In addition, she has found that RNAi to G-protein beta also inhibits glutamate chemoresponse, perhaps providing a link between the receptor and the adenylyl cyclase that must be activated by glutamate to generate a large increase in intracellular cAMP.
Paramecium tetraurelia can detect micromolar chemical changes in their environment and modify swimming behavior accordingly. The swimming behavior is tightly regulated through the plasma membrane Ca2+ ATPase (PMCA). My research project is focused on the PMCA (Plasma Membrane Ca2+ ATPase) and its functional role in such a chemoresponse. In Paramecia there are three currently known PMCAs (PMAC2, PMCA3, PMCA4) I have cloned and additional three novel PMCAs (PMCA5, PMCA6, PMCA7) and shown them to be highly homologous to the known PMCAs.
I am performing functional analysis of the PMCA isoforms via RNA-mediated interference (RNAi) of gene expression. Through RNAi, I will observe loss-of-function phenotypes for the PMCAs. Continuous backward swimming behavior is a simple measure of abnormally high intracellular calcium from non-functional PMCAs. This swimming behavior is my indication of successful RNAi.
Also, through immunodection confocal microscopy I am specifically and individually describing localization patterns of the PMCAs to further give insight of their functionality. Deconvolution microscopy allows additional spatial differentiation of specific PMCA isoforms perhaps assigning a positional reliance on function.