Dr. Junji Yano, Research Assistant Professor
Yano San 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.
Recently, I am interested in the role of plasma membrane ATPase (PMCA) on the regulation of ciliary calcium, which affects the frequency and direction of ciliary beat. When the cells are depolarized and get the action potential, the influx of calcium into cilia through the ciliary voltage-gated calcium channels (CaV) causes the reversal of ciliary beat, and the cells swim backward. The cells start the forward swimming by the return of intarciliary calcium to the resting level. However, it is not clear where and how the intraciliary calcium is removed. Twenty three PMCA genes found in P. tetraurelia are subdivided into 9 paralog groups. It is unknown each PMCA has the different localization and/or function in Paramecium cells. Using the proteomics, RNAi, expression of tagged-proteins, and immunoprecipitation (IP), I found PMCA2a and 2b, one of paralog groups, are localized in the cilia and necessary for removing intraciliary calcium after the action potential. I also identified the voltage-gated calcium channel alpha 1 subunit (CaV1a, b, and c) responsible for the influx of calcium into cilia at the action potential. I showed the PMCA2s interact with or are closely located to the CaV1s, by the reciprocal IP and the analysis of the detergent insoluble ciliary proteins using an iodixanol density gradient. Thus, it is suggested the PMCA2 responsible for removing calcium after the activation of CaV1 functions as a regulator of local calcium.
Glutamate and acetate attract Paramecium cells and hyperpolarize their membrane potential. The hyperpolarized cells swim smoothly and increase the swimming speed. The calcium/calmodulin controls many cytoplasmic events such as activities of potassium channel and PMCA. The PMCA is suggested to be involved in these chemoresposes, as the decrease of cytoplasmic calcium increases the swimming speed. The down-regulation of calmodulin by the expression of antisense calmodulin RNA and antisense oligo-deoxyribonucleotides disturbed the chemoresponse to acetate. The hyperpolarization by acetate and glutamate initiates the outward current of potassium through calcium activated potassium channel. Calmodulin may primarily regulate the potassium channel. The overexpression of PMCA1, another paralog group, did not showed the clear data. Studies of the signal transduction pathway show that cyclicAMP rapidly increases with glutamate stimulation and that kinase activity is needed for this attraction. PKA in particular increases the activity of calcium ATPase. Collectively, the data suggest that a PMCA maintains the hyperpolarization induced by glutamate. I am now examining the role of PMCA1 for the chemoresponse to glutamate, using RNAi.
Cullen Ross, Lab Research Technician
I am a recent graduate from the University of Vermont with my major in Biochemistry and a minor in Astronomy. I am super excited to be a part of Dr. Van Houten’s lab! I was originally born in Corvallis, Oregon but eventually moved here to Burlington, Vermont when I started high school. My hobbies primarily include going for bike rides, crafting homebrewed beer, playing Dungeons and Dragons, and hanging out with my kitten Ollie. I hope to some day move onto grad school either studying yeast cells for brewing beer or focus on cancer research. Feel free to stop by room 224 and say hi!
Ashikun Nabi, Ph.D. Graduate Student
In our lab, I am focusing on understanding the roles of transition zone proteins beyond their primary function of maintaining the ciliary gatting. I use RNA interference, immunofluorescence, various biochemical approaches and Mass spectrometry for my research.