Cell Biology Annual Report 2010-11 (FY 2011) - Department of Cell ...

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CBP Faculty Research Summaries Faculty Research Interests Cell Biology and Physiology Annual Report Meir Aridor, Ph.D. Associate Professor The endoplasmic reticulum (ER) is the first compartment of the secretor pathway. Plasma membrane receptors, ion channels, hormones and secreted enzymes are few examples of proteins which are being processed and sorted for vesicular transport in the ER. Mistakes in sorting lead to the development of variety of diseases, ranging from hemochromatosis, cystic fibrosis or hereditary emphysema to Pelizaeus-Merzbacher or Alzheimer’s neurodegeneration. Viruses such as the cytomegalovirus, HIV-1 Epstein-Barr and many others manipulate ER sorting to evade immune surveillance, a specialized function of the compartment. Dr. Aridor is utilizing a variety of molecular biochemical, biophysical and cellular techniques to unravel the molecular basis of ER sorting. Catherine J. Baty, D.V.M., Ph.D. Research Assistant Professor Our laboratory has begun focusing on lymphatic endothelial function. We have developed a 3 dimensional tissue culture system to study potential mechanisms of lymphatic failure. Despite the fact that the lymphatic vessels were identified hundreds of years ago, limited understanding exists of lymphatic development, function, and disease. Greater understanding of the structure and function of lymphatic endothelium will provide plausible new candidate genes for mutation screening in families with hereditary lymphedema. Such studies will ultimately reveal specific therapeutic targets appropriate both for those suffering from primary lymphedema and the greater population of patients with secondary lymphedema. Carol A. Bertrand, Ph.D. Research Assistant Professor The primary research interests of the lab focus on the regulation of airway surface liquid (ASL) pH and mucin secretion in epithelia, and the involvement of ion channels in modulating the process. Both bicarbonate and mucin contribute to the pH of the ASL, which varies considerably in disease from acidic in CF to alkaline in chronic bronchitis. Current work centers on the biosynthesis and activity of chloride channels and anion exchangers that complement and may regulate the CFTR chloride channel, as well as the apical membrane permeability to bicarbonate. In addition, ongoing effort is devoted towards the development and refinement of methods for performing electrophysiology and live cell fluorescence microscopy. Michael B. Butterworth, Ph.D. Assistant Professor 18 Dr. Butterworth’s research interest is in the regulation of epithelial channels by vesicle trafficking and recycling. Research is focused into two broad areas. First, ongoing studies aim to characterize the mechanisms that underlie channel regulation by membrane trafficking in the mammalian kidney. Three renal transporters, namely the epithelial sodium channel (ENaC), potassium channel (ROMK) and aquaporin water channels are investigated. The work aims to map the intracellular itinerary of these channels and identify protein mediators that regulate
CBP Faculty Research Summaries channel surface density. In separate, but related studies, primary human bronchiolar epithelial cells are used to characterize ENaC regulation in the human distal airway, in particular mechanisms which may contribute to disease states like cystic fibrosis. By comparing ENaC regulation in two distinct systems, areas of common and divergent regulation have been established. The second research focus investigates the regulation of ENaC by microRNAs (miRNA). miRNAs are small RNAs that pair to the mRNA of protein coding genes to direct their posttranscriptional repression. Channel density in epithelial cells is determined to a large extent by steroid hormone signaling. The regulation of miRNAs by these hormones and impact of changes in miRNA expression on channel regulation is being studied. Daniel C. Devor, Ph.D. Professor Intermediate (KCa3.1 or IK) and small (KCa2.3 or SK3) conductance, calcium-activated potassium channels play critical roles in a host of physiological processes, including the endothelial derived hyperpolarizing factor (EDHF) response which is critical to the maintenance of vascular tone and hence blood pressure regulation, the maintenance of a hyperpolarized membrane potential across the basolateral membrane of polarized epithelia required for transepithelial fluid secretion as well as being intimately involved in the afterhyperpolarization in nerves and a host of other processes. Thus, an understanding of the physiological and pharmacological regulation of these channels as well as their assembly, trafficking and gating is crucial to the development of novel therapies based on targeting these channels. The long-term goals of my lab are to obtain a detailed molecular understanding of these channels in order to unravel the mechanisms involved in their assembly, trafficking, regulation and gating as well as to define the physiological role these channels play using C. elegans as a model system. In light of these goals, we have several ongoing projects designed to further our understanding of these channels. Cell Biology and Physiology Annual Report First, Mark Bailey, a graduate student in the lab, is carrying out patch-clamp studies designed to elucidate the role of S6 in the gating of KCa3.1. In these studies, we are employing PCMBS to probe the cysteines in S6 and evaluate their role in gating. PCMBS has advantages over MTS reagents in both the site of the reactive moiety as well as the size of the molecule such that a larger perturbation in local molecular space is achieved. By using PCMBS in combination with a mutagenesis approach we have demonstrated that side chains pointing away from the pore, and toward S5, are critical to the coupling between Ca2+ binding to the calmodulin binding domain and channel gating. In collaboration with Dr. Michael Grabe, of the Biological Sciences Department at the University of Pittsburgh, we are modeling the gating kinetics of KCa3.1 to extract the rate constants being affected by both PCMBs as well as mutations in this region of the channel. In the future, we plan to probe S5 by conducting a tryptophan scan of the region across from the cysteines in S6 to further our understanding of how S5-S6 interactions modulate the coupling between increasing Ca2+ and channel gating. We have also identified critical amino acids in the S4-S5 linker region of both KCa3.1 and the related family member KCa2.3 which, when mutated to increase side-chain volume, result in a shift in apparent Ca2+ affinity. These results suggest this region of the channel is similarly involved in the coupling between Ca2+ binding to calmodulin in the cytoplasmic C-terminus and subsequent gating. A combination of patch-clamping, mutagenesis and modeling will be employed to definitively define the role of this region of the channel in the coupling between Ca2+ and gating. 19
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Page 3 and 4: Table of Contents Table of Contents
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CBMP Graduate Students Christopher
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CBP Faculty Roster CBP FACULTY ROST
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CBP Faculty Honors, Recognition and
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CBP Business Plan - Executive Summa
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CBP Business Plan - Budget Cell Bio
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Cell Biology and Physiology Annual
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