Giovannucci, David R.

Imbery, John

Khuder, Sura

Slama, James T.

Weiss, Amanda

Elsevier - Publisher Connector 

Acidic Calcium Stores Contribute to Secretory Activity Following Elevation of Camp in the Salivary Gland 

104a Sunday, February 8, 2015neither the lipid targets for this protein nor the residues responsible for binding
are well characterized. Our study attempts to identify the respective roles of (a)
phosphoinositides such as phosphatidylinositol (4,5)-bisphosphate (PIP2) and
(b) anionic backgroundlipids such as phosphatidylserine (PS) in binding of
the granuphilin C2A domain to liposomes. Affinities are measured using
protein-to-membrane FRET and titration with the competitive inhibitor inositol
(1,2,3,4,5,6)-hexakisphosphate (IP6). Granuphilin C2A binds to liposomes
containing phosphatidylcholine, dansyl-phosphatidylethanolamine, and PS
and/or PIP2. Decreased FRET is measured as the protein is displaced from lipid
upon titration with IP6. Granuphilin C2A has similar affinity for liposomes con-
taining either 24% PS or 2% PIP2, but affinity increases ~100-fold in the pres-
ence of both target lipids. Binding site(s) for the two lipids are being probed
using site-directed mutagenesis and NMR. Overall, granuphilin demonstrates
the capability to bind to background anionic lipids and phosphoinositides inde-
pendently, but the presence of both lipids greatly increases the binding affinity
of this protein. This suggests that granuphilin may serve as a coincidence de-
tector to target vesicles to sites of secretion on the plasma membrane.
522-Pos Board B302
The Membrane Bending Action of the Syt-1 C2AB Studied on Supported
Lipid Bilayers
Lauren P. MacConnachie1, Neo C. Poyiadji2, Tejeshwar C. Rao1,
Arun Anantharam1.
1Biological Sciences, Wayne State University, Detroit, MI, USA, 2Wayne
State University, Detroit, MI, USA.
Calcium-triggered exocytosis is vital to the cellular and physiological homeo-
stasis of higher organisms. The broad rules of exocytosis, including its regula-
tion by SNARE proteins and Ca2þ, have now been known for several years.
However, there is a gap in our knowledge with respect to the molecular events
that regulate fusion pore expansion and concomitant content dispersal. Within
this context, the studies focus on the Ca2þ-binding Synaptotagmin (Syt) family
of proteins. Syt actions during exocytosis are often tied to its ability to bend and
deform membranes. But very little real-time evidence exists that Syt bends
membranes or that membrane bending is an essential part of its function. In
this project, we utilized synthetic membranes and purified Syt proteins to
answer a specific question: Does Syt bend membranes in a Ca2þ-dependent
manner? To answer this question, we used a combination of in vitro ap-
proaches, including recombinant Syt C2AB proteins, supported lipid bilayers
(SLBs), and a curvature-sensitive optical technique called polarized total inter-
nal reflection fluorescence microscopy (pTIRFM). Although the results are pre-
liminary, we find that purified Syt proteins added to supported lipid bilayers
(SLBs) do create localized membrane deformations in the presence of Ca2þ.
Future work will investigate how the bending process is regulated, whether it
differs between synaptotagmin isoforms, and ultimately, how it relates to the
function of synaptotagmin in living cells undergoing exocytosis.Calcium Signaling I
523-Pos Board B303
Computational System Analysis of Ca2D Signaling in the Pancreatic Beta-
Cells
Leonid E. Fridlyand, Louis H. Philipson.
Medicine, University of Chicago, Chicago, IL, USA.
The Ca2þ ion serves as a ubiquitous second messenger in pancreatic beta-cells.
Beta-cells are equipped with sophisticated machinery to precisely regulate
Ca2þ signal in a localization-specific manner to regulate insulin secretion.
Based on our mathematical models of beta-cell electrophysiology, Ca2þ oscil-
lations, cAMP signaling, metabolism and mitochondrial functions we present
an updated complex computational model for beta-cells to clarify the mecha-
nisms underlying intracellular Ca2þ responses to varying stimuli. The system
employs a Hodgkin-Huxley-type ionic model for plasma membrane action
potentials with equations for key currents and pumps including Naþ/Ca2þ ex-
changers, TRP and store-operating Ca2þ channels. The model incorporates
modern data for Ca2þ extrusion/uptake systems in the endoplasmic reticulum.
The role of mitochondria in regulation of Ca2þ signaling is also evaluated. The
values of most of the model parameters were inferred from available experi-
mental data. This model simulates and explains the Ca2þ dynamics under a
wide range of experimental conditions, including changes in islet electrical
activity due to ion channel blockade and action of several specific G-protein re-
ceptor agonists. Our analysis of the dynamic data provides evidence for a
pivotal role of store-operated Ca2þ channels in maintenance of physiological
cytoplasmic Ca2þ concentration even at low glucose levels while voltage-
dependent calcium channels are blocked. The role of endoplasmic reticulum,
TRP and store-operated Ca2þ channels in cytoplasmic Ca2þ oscillations wasalso investigated. This computational systems approach provides a framework
to analyze the regulatory mechanisms related to release of calcium stores in
beta-cell insulin secretion.
524-Pos Board B304
Acidic Calcium Stores Contribute to Secretory Activity Following
Elevation of Camp in the Salivary Gland
John Imbery1, Sura Khuder1, Amanda Weiss1, James T. Slama2,
David R. Giovannucci1.
1Neuroscience, University of Toledo, Toledo, OH, USA, 2Medicinal
Chemistry, University of Toledo, Toledo, OH, USA.
Calcium signaling in response to autonomic neural input drives fluid and pro-
tein secretion within salivary glands. Previous research focused primarily on
the canonical IP3-mediated calcium release from ER stores. However, other
important reservoirs for calcium release exist such as lysosomes, endosomes,
and secretory granules. Whether and how these acidic stores are activated re-
mains largely undefined in terms of their contributions to global calcium sig-
nals, protein exocytosis and saliva production in salivary tissue. To address
this question we used lysotracker red, a fluorescent dye that labels acidic organ-
elles. Confocal images demonstrated that mouse parotid acini had abundant
stores that were apically located and whose pH gradients were dissipated by
treatment with the vacuolar H-ATPase inhibitor bafilomycin A1. In contrast,
parotid ductal cells displayed only a punctate and dispersed or perinuclear
labeling. The functional effect of bafilomycin treatment was tested using live
cell imaging methods. Bafilomycin treatment resulted in a two-thirds diminish-
ment of peak calcium release amplitudes evoked by a threshold level of carba-
chol stimulation. The diminishment in peak amplitude and in the rate of rise of
the evoked calcium signal was only evident in cells where cAMP was first
elevated by either forskolin or the beta-adrenergic agonist isoproterenol. There
is evidence that acidic store calcium release can be triggered by the dinucleo-
tide second messenger NAADP. Consistent with this, NED19, a NAADP re-
ceptor antagonist, diminished by half the peak calcium response. This data
indicates that calcium release from acidic stores in mouse parotid acinar cells
is cAMP-dependent and may be mediated by NAADP. In addition, bafilomycin
and NED19 treatment significantly reduced exocytotic secretory activity, even
without cAMP elevation, supporting the idea that acidic stores may contribute
to vesicle fusion and protein release.
525-Pos Board B305
Mechanics Regulates ATP-Stimulated Calcium Response in Fibroblast
Cells
Josephine Lembong1, Benedikt Sabass2, Bo Sun3, Matthew E. Rogers4,
Howard A. Stone2.
1Chemical and Biological Engineering, Princeton University, Princeton, NJ,
USA, 2Mechanical and Aerospace Engineering, Princeton University,
Princeton, NJ, USA, 3Physics, Oregon State University, Corvallis, OR, USA,
4Firmenich Inc., Plainsboro, NJ, USA.
Cells constantly sense their chemical and mechanical environment. Here we
study the effect of mechanics on the ATP-induced calcium response of fibro-
blast cells in experiments that mimic various tissue environments. We find
that in two-dimensional configurations, closely packed cell colonies cultured
on a soft polyacrylamide gel (elastic modulus E ¼ 690 Pa) have more cells ex-
hibiting calcium oscillations than colonies on a rigid substrate (E ¼ 36000 Pa).
Cells on soft substrates also show a higher number of oscillations following a
stimulation, with a slower decay of calcium level relative to those on rigid sub-
strates. Inhibition of gap junctions results in a decrease of the oscillation period
and reduced correlation of calcium responses, which suggests additional
complexity of signaling upon cell-to-cell contact. Moreover, the frequency of
calcium oscillations is independent of the rigidity of the substrate but depends
on ATP concentration and cell-cell contact. In additional experiments with
cells embedded in a three-dimensional hydrogel matrix, where cell-to-cell con-
tact is absent, more calcium responses are observed when cells are encapsulated
in a softer hydrogel (E ¼ 85 Pa). When cell contractility is obstructed by inhi-
bition of myosin, we observe less responsive and fewer oscillating cells. Over-
all, our observations highlight the influence of mechanical environment on
chemical sensing in fibroblast cell colonies.
526-Pos Board B306
Calcium Movement in Cardiac Mitochondria
Liron Boyman1,2, George S.B. Williams1,2, Aristide C. Chikando1,
Ramzi J. Khairallah3,4, Sarah Kettlewell5, Christopher W. Ward4,1,
Godfrey L. Smith5, Joseph P.Y. Kao1,2, W.J. Lederer1,2.
1BioMET, University of Maryland, Baltimore, MD, USA, 2Department of
Physiology, University of Maryland School of Medicine, Baltimore, MD,
USA, 3Department of Cell and Molecular Physiology, Loyola University
Chicago, Chicago, IL, USA, 4University of Maryland School of Nursing,


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Acidic Calcium Stores Contribute to Secretory Activity Following Elevation of Camp in the Salivary Gland

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