Life Sciences Commons^™

Mechanisms Underlying Serotonergic Excitation Of Callosal Projection Neurons In The Mouse Medial Prefrontal Cortex, Emily K. Stephens, Arielle L. Baker, Allan T. Gulledge

Dartmouth Scholarship

Serotonin (5-HT) selectively excites subpopulations of pyramidal neurons in the neocortex via activation of 5-HT_2A (2A) receptors coupled to G_q subtype G-protein alpha subunits. G_q-mediated excitatory responses have been attributed primarily to suppression of potassium conductances, including those mediated by K_V7 potassium channels (i.e., the M-current), or activation of non-specific cation conductances that underlie calcium-dependent afterdepolarizations (ADPs). However, 2A-dependent excitation of cortical neurons has not been extensively studied, and no consensus exists regarding the underlying ionic effector(s) involved. In layer 5 of the mouse medial prefrontal cortex, we tested potential mechanisms of serotonergic excitation …

Calcium-Mediated Actin Reset (Caar) Mediates Acute Cell Adaptations, Pauline Wales, Christian Schuberth, Roland Aufschnaiter, Johannes Fels, Ireth Garcia-Aguilar, Annette Janning, Christopher D. Dlugos, Marco Schaefer-Herte, Christoph Klingner, Mike Waelte, Julian Kuhlmann, Ekaterina Menis, Hockaday Kang Hockaday Kang, Kerstin C. Maier, Wenya Hou, Antonella Russo, Henry N. Higgs

Dartmouth Scholarship

Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in …

Plant Calcium Content: Ready To Remodel, Jian Yang, Tracy Punshon, Mary Lou Guerinot, Kendal D. Hirschi

Dartmouth Scholarship

By identifying the relationship between calcium location in the plant cell and nutrient bioavailability, the plant characteristics leading to maximal calcium absorption by humans can be identified. Knowledge of plant cellular and molecular targets controlling calcium location in plants is emerging. These insights should allow for better strategies for increasing the nutritional content of foods. In particular, the use of preparation-free elemental imaging technologies such as synchrotron X-ray fluorescence (SXRF) microscopy in plant biology may allow researchers to understand the relationship between subcellular location and nutrient bioavailability. These approaches may lead to better strategies for altering the location of calcium …

Lapg, Required For Modulating Biofilm Formation By Pseudomonas Fluorescens Pf0-1, Is A Calcium-Dependent Protease, Chelsea D. Boyd, Debashree Chatterjee, Holger Sondermann, George A. O'Toole

Dartmouth Scholarship

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro …

The Role Of Cax1 And Cax3 In Elemental Distribution And Abundance In Arabidopsis Seed, Tracy Punshon, Kendall Hirschi, Jian Yang, Antonio Lanzirotti, Barry Lai, Mary Lou Guerinot

Dartmouth Scholarship

The ability to alter nutrient partitioning within plants cells is poorly understood. In Arabidopsis (Arabidopsis thaliana), a family of endomembrane cation exchangers (CAXs) transports Ca²⁺ and other cations. However, experiments have not focused on how the distribution and partitioning of calcium (Ca) and other elements within seeds are altered by perturbed CAX activity. Here, we investigate Ca distribution and abundance in Arabidopsis seed from cax1 and cax3 loss-of-function lines and lines expressing deregulated CAX1 using synchrotron x-ray fluorescence microscopy. We conducted 7- to 10-μm resolution in vivo x-ray microtomography on dry mature seed and 0.2-μm resolution x-ray …

Structural Characterization Of A Conserved, Calcium-Dependent Periplasmic Protease From Legionella Pneumophila, Debashree Chatterjee, Chelsea D. Boyd, George A. O'Toole, Holger Sondermann

Dartmouth Scholarship

The bacterial dinucleotide second messenger c-di-GMP has emerged as a central molecule in regulating bacterial behavior, including motility and biofilm formation. Proteins for the synthesis and degradation of c-di-GMP and effectors for its signal transmission are widely used in the bacterial domain. Previous work established the GGDEF-EAL domain-containing receptor LapD as a central switch in Pseudomonas fluorescens cell adhesion. LapD senses c-di-GMP inside the cytosol and relays this signal to the outside by the differential recruitment of the periplasmic protease LapG. Here we identify the core components of an orthologous system in Legionella pneumophila. Despite only moderate sequence conservation at …

The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith

Dartmouth Scholarship

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and …

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which …

Analysis Of Microtubule Sliding Patterns In Chlamydomonas Flagellar Axonemes Reveals Dynein Activity On Specific Doublet Microtubules, M. J. Wargo, Mark A. Mcpeek, Elizabeth F. Smith

Dartmouth Scholarship

Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca2+ conditions. InChlamydomonas, high Ca2+ induces changes in waveform which are predicted to result from regulating dynein

activity on specific microtubules. Our …

Regulation Of Flagellar Dynein By Calcium And A Role For An Axonemal Calmodulin And Calmodulin-Dependent Kinase, Elizabeth F. Smith

Dartmouth Scholarship

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonasmutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at …

On The Mechanism Of Parathyroid Hormone Stimulation Of Calcium Uptake By Mouse Distal Convoluted Tubule Cells, F A. Gesek, A. Friedman

Dartmouth Scholarship

PTH stimulates transcellular Ca2+ absorption in renal distal convoluted tubules. The effect of PTH on membrane voltage, the ionic basis of the change in voltage, and the relations between voltage and calcium entry were determined on immortalized mouse distal convoluted tubule cells. PTH (10(-8) M) significantly increased 45Ca2+ uptake from basal levels of 2.81 +/- 0.16 to 3.88 +/- 0.19 nmol min-1 mg protein-1. PTH-induced 45Ca2+ uptake was abolished by the dihydropyridine antagonist, nifedipine (10(-5) M). PTH did not affect 22Na+ uptake. Intracellular calcium activity ([Ca2+]i) was measured in cells loaded with fura-2. Control [Ca2+]i averaged 112 +/- 21 nM. …