|University of Vermont
Department of Biology
Our research addresses the signal transduction pathways of chemical sensing in Paramecium and mice. Most recently, we are extending our work to mouse olfaction through a collaboration with the laboratory of Rona Delay Below are some synopses of our lab members' research projects.
Dr. Judith Van Houten
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.
Dr. Junji Yano
" 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.
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.
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.
Cassie Jacobs is an accelerated masters student, who is finishing her work on 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.
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.
I am curious to know how adenylyl cyclase communicates with various chemosensory signal pathways in Paramecium. Cyclic AMP (cAMP) acts as a second messenger and is generated upon activation of adenylyl cyclase. Many studies show that cAMP levels in Paramecium are elevated by potassium conductance. Recently, a novel group of proteins with ion channel homology and adenylyl cyclase domains at N and C- terminus have been reported in Paramecium. It has been reported that this protein exists in ciliary membranes and contributes to ciliary movements in response to stimuli. We are excited to find that 16 similar genes exist in Paramecium from Paramecium Genome project. Currently I am interested in studying the change in chemosensory behavior by silencing this gene using RNAi methods. Future research may be to reveal the puzzle of how adenylyl cyclase co-ordinate with various other chemosensory signal transduction pathway 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.
Yunfeng (Charles) Pan
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.
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.
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.