Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item ARABIDOPSIS THALIANA GLUTAMATE RECEPTOR-LIKE 3.7 UNDERLIES ROOT MORPHOLOGY AND SIGNALING VIA MEMBRANE POTENTIAL HOMEOSTASIS(2021) Barbosa-Caro, Juan Camilo; Feijó, José A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plants perceive highly variable environments and biotic interactions through membrane receptors like the GLutamate Receptor-like (GLR) family, related to the ionotropic Glutamate Receptors that underlie information transmission in neurons. GLRs underpin information transduction and morphological adaptations in plants. However, mechanistic understanding is scarce. In Arabidopsis thaliana roots, we investigated how GLRs underlie amino acid-induced electric and Ca2+ excitability. We also assessed the contribution of GLR3.7 in root hair elongation. We present GLRs as mediators of a local, glutamate-induced electric and Ca2+ response in roots, with the same initiation kinetics of wound-induced Slow Wave Potentials (SWP). We identify GLR3.7 as mediator of root hair elongation through maintenance of membrane depolarization at the growing cell apex. These results propose a parallel between glutamate-triggered signals and SWP initial phase as local and chemically induced, and posit GLR3.7 as a possible contributor to Ca2+ homeostasis in root hair apical growth.Item DISCOVERY OF AN ESCHERICHIA COLI CHANNEL WITH HIGH VOLTAGE DEPENDENCE AND COOPERATIVITY.(2015) Lin, Shang-Hsuan; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Channels are essential for controlling the permeability of cellular membranes. The regulation of channel gating therefore plays an important role physiologically. Voltage-gating is one of the regulations that cells utilize wherein the change in transmembrane potential cause conformational changes in channels. Here a novel bacterial channel from Escherichia coli with remarkable voltage-gating properties is reported. When the channel-forming protein was reconstituted into a planar phospholipid membrane, two different types of channel activities were observed. Type A is weakly cation-selective, with a single channel conductance about 1.5 nS (in 1M KCl solution), corresponding to a pore size of 0.9 nm. High positive voltages cause step-wise closures. Type B is voltage-independent, with much larger and noisier conductance. When LaCl3 was added, Type B channels first showed a decrease in conductance, and the residual conductance became voltage-gated, indistinguishable from Type A channels. Under triangular voltage waves, more interesting voltage-gating behaviors were revealed. The single conducting unit seems to be composed of three channels, each with the identical 1.5 nS conductance (namely channel (1), channel (2), and channel (3)). Based on the voltages at which they close/reopen, and the sequence of their closure/reopening, a model was proposed as follows. All three channels are proposed to be molecularly identical but, channel (1) and channel (3) have the same orientation, which is opposite to that of channel (2). Altogether these three channels form the conducting unit in a linear array. The voltage sensor domain of each channel is proposed to take the form of a dipole moment. The interaction between dipole moments of the channels, each with an opposite orientation with its neighbor(s), leads to the impressively high cooperativity between channels. Although the physiological roles of these channels are not clear yet, the remarkably steep voltage dependence (n~14) rivals that of the channels in excitable membranes.Item Ceramide Channels and the Induction of Apoptosis: Structural Insights on Channel Formation and Regulation by Bcl-2 Family Proteins(2012) Perera, Meenu Nadisha; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A critical event in apoptosis is the release of intermembrane space proteins from mitochondria following mitochondrial outer membrane (MOM) permeabilization (MOMP). The Bcl-2 family of proteins regulates MOM integrity and includes pro- and anti-apoptotic members, like Bax and Bcl-xL respectively. Preceding MOMP, the MOM becomes enriched with the sphingolipid, ceramide, which can self-assemble to form ceramide channels, contributing to MOMP. Bax and ceramide channels were found to act synergistically in the generation of MOMP and a direct interaction between these was observed in phospholipid membranes. The apparent affinity of activated Bax for ceramide channels increases with ceramide channel size, consistent with an induced fit mechanism; Bax drives the enlargement of ceramide channels to an optimum fit for the Bax binding site. A ceramide channel specific inhibitor prevented the enhanced MOMP in the presence of Bax and ceramide indicating ceramide channels were the primary permeabilizing entity. Analogs with changes to all the major structural features of ceramide were used to assess the molecular basis of stability of ceramide channels. Methylation of the C1- hydroxy group abrogated channel formation in mitochondria. Methylation of the amide nitrogen or a change in chirality at C2, which influences the C1-hydroxy group orientation, greatly reduced channel-forming ability whereas other changes were well tolerated. Competition experiments between ceramide and analogs resulted in synergism or antagonism, depending on compatibility of the analog structure with the ceramide channel model. The results provide evidence for ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions. Analogs that retained channel-forming ability were used to assess the structural features of ceramide channels required for regulation by Bcl-2 family proteins. The stereochemistry of the ceramide head group and access to the amide nitrogen is indispensible for regulation by Bax, implicating the polar portion of the channel as the Bax binding site. Bcl-xL's ability to disassemble ceramide channels depends on the length of the hydrophobic chains of ceramide. Specific Bcl-xL inhibitors reveal that BclxL binds ceramide channels through its hydrophobic groove and this is supported by simulated docking. The opposite effects of pro-and anti-apoptotic proteins are achieved at different sites on the ceramide channel.Item VISUALIZATION OF CERAMIDE CHANNELS BY TRANSMISSION ELECTRON MICROSCOPY(2011) SAMANTA, SOUMYA; COLOMBINI, MARCO; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40 nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.