Biochemical and Pharmacological Studies

Biochemical and Pharmacological Studies David Holowka Alice Wagenknecht-Wiesner Roy Cohen Lavanya Vasudevan Stephanie Hammond Norah Smith Nat Calloway Jinmin Lee
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	David Holowka Large-scale organization of intracellular membranes relevant to receptor-mediated Ca2+ mobilization with Lavanya Vasudevan and Stephanie Hammond

Ca2+ is an important intracellular second messenger. In mast cells, Ca2+ mobilization is necessary for degranulation and the consequent release of mediators of allergies such as histamine. Ca2+ mobilization is regulated spatially and temporally, and it is triggered in mast cells by a decrease in Ca2+ levels in intracellular stores that activate Ca2+ influx via store-operated Ca2+ channels. One hypothesis for the mechanism of Ca2+ entry is spatial coupling between the Ca2+ entry channels on the plasma membrane and Ca2+ release channels on the endoplasmic reticulum (ER). Results In recent studies using immunocytochemistry, we have observed spatially localized membrane subpopulations that are associated with the inner leaflet of the plasma membrane and appear to play a role in Ca2+ mobilization. These membrane pools, or ‘PIP plaques’, detected by a monoclonal anti-PIP2 antibody, are sensitive to detergent treatment after fixation (see A). Cellular components that localize to these subdomains include phosphatidylinositol 4,5-bisphosphate (PIP2) and phospholipase Cg (see B), as well as SERCA 2 ATPase and voltage operated Ca2+ channels. Future Studies We are developing labeling strategies to characterize these plaques in live cells and Ca2+ imaging methods to monitor localized Ca2+ mobilization with high spatial and temporal resolution. We are also utilizing biochemical approaches to isolate and characterize the membranes localized to the plaques and molecular genetic approaches to understand functional roles for plaque components.

(above) A) RBL mast cells were fixed with 4% formaldehyde and labeled with anti-PIP2 mAb in the presence or absence of 0.1% Triton X-100, followed by RITC-goat anti-mouse g2b. B) Cells were fixed as in (A) and labeled with anti-PIP2 mAb and anti-PLCg1 mAb in the absence of TX-100, followed by RITC-goat anti-mouse g2b and FITC-goat anti-mouse g1 antibodies.


Alice Wagenknecht-Wiesner Transmembrane Sequences are Determinants of Immunoreceptor Signaling with Julie Gosse

	What are the structural features critical for signal initiation by antigen-stimulated IgE receptor FceRI? We investigated the role of the transmembrane (TM) sequence of the IgE receptor in its signaling. To look at the structural features of immunoreceptors critical for lipid raft-dependent signaling we prepared and characterized single-chain receptors that contain the essential structural features of the high affinity IgE receptor, FceRI. Our constructs contain extracellular human FceRI for IgE binding, a variable TM region, and the ITAM-containing cytoplasmic tail of the T cell receptor z subunit. We selectively evaluated expression, morphological changes, raft localization, phosphorylation, calcium response, and degranulation due to crosslinking the chimeric IgE receptors in RBL mast cells stably transfected with these chimeric receptors.
Stimulation of tyrosine phosphorylation, calcium mobilization, degranulation and lipid raft association are strongly dependent on the chimeric receptor TM sequences, and these responses are highly correlated to crosslink-dependent association with detergent-resistant lipid rafts. Those with TM domains from non-raft proteins (aPz, a45z) gave very small responses. For the chimera aFz, mutation of a TM cystein abolishes robust signaling an lipid raft association. In addition, TM disulfide-mediated dimerization of azz enhances signaling.

	Roy Cohen Studying Calcium Mobilization in RBL-2H3 Mast Cells with the Kotlikoff Group

Calcium mobilization is central in many cellular and physiological processes. Two closely associated pathways are responsible for the increase in cytosolic calcium concentration: ionic influx through specific channels at the plasma membrane and mobilization of calcium ions from intracellular stores. Using single cell stimulation and high rate calcium imaging of conventional calcium indicators or genetically encoded calcium sensors, we study calcium dynamics in RBL-2H3 mast cells.
Three different patterns of calcium mobilization were resolved in these cells: 1. Calcium waves - initiating at one or two regions of the cell, often at the tips of extended cell protrusions, and advance throughout the cytoplasm to generate general increases in intracellular calcium. 2. Calcium oscillations – repetitive elevations in calcium concentration that follow the initial calcium increase and demonstrate distinct dynamics depending on the type of stimulation and experimental conditions. 3. Calcium puffs – localized and transient increases in calcium concentration that do not necessarily evolve into large-scale increases in cytosolic calcium. Figure 1 (right). Imaging calcium dynamics in RBL-2H3 cell expressing the genetically encoded calcium sensor GFP based protein- GCaMP2. IgE sensitized cell stimulated with DNP-BSA under confocal microscopy (left top panel). A virtual line scan analysis demonstrates the oscillations in cytosolic calcium concentration (right top panel).Signal intensity was calculated and plotted over time (lower panel). The DNP-BSA pulse as well as calcium wave phase and oscillations are indicated.
Figure 2 (above).Calcium puff in RBL-2H3 cell expressing - GCaMP2.As in fig 1, cells were sensitized with IgE and than stimulated with DNP-BSA under the microscope (left top panel). The increase in calcium concentration is demonstrated by a virtual line scan analysis (right top panel). A transient increase in calcium concentration –”calcium puff” is indicated by arrowhead.The puff event indicated in the signal intensity plot (lower panel).	Movie and Figure 3 (below).Calcium wave in RBL-2H3 cell expressing the genetically encoded calcium sensitive GFP-based protein- GCaMP2. Summary of calcium wave movie. As in fig 1, cells were sensitized with IgE and stimulated with DNP-BSA under the microscope (left panel). The increase in calcium concentration was plotted using a virtual line scan analysis (right top panel).
Further characterization of the cellular machinery that regulates localized calcium elevation will be in the aim for future studies, using a combination of fast imaging and molecular and biochemical methods to examine the possible function of calcium heterogeneity in spatial regulation of downstream events such as degranulation.

Lavanya Vasudevan Regulation of Phosphatidylinositol 4,5 bisphosphate in RBL-2H3 mast cells

Phosphatidylinositol 4,5 bisphosphate (PIP2)is a minor constituent of eukaryotic cell membranes. It participates in processes such as calcium mobilization, actin polymerization and trafficking, and, is a precursor for three important second messengers (Inositol 1,4,5 trisphosphate, diacylglycerol, and phosphatidylinositol 3,4,5 trisphosphate). Rapid turnover of PIP2 upon activation of the cell is brought about by two classes of enzymes: The family of the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5kinase-I) that synthesize PIP2, and, the 5' phosphatases which cleave PIP2. The regulation of PIP2 by these enzymes is poorly understood in mast cells. My research involves over expressing or knocking down selected kinases and phosphatases in RBL-2H3 mast cells to study their regulation of PIP2 function. Figure 2 (above). Quantitation of stimulated ruffling. More than 600 cells per sample from two independent experiments were scored based on morphology of FITC-phalloidin-labelled cells. Bars represent percentage of cells that show antigen-stimulated ruffling for untransfected RBL-2H3 cells, two mutant clones (MUT) and three WT clones (WT). Error is standard deviation.	Figure 1 (below). Expression of mutant PIP5kinase-Iα suppresses stimulated cell ruffling. Cells expressing either endogenous levels of the kinase (2H3), mutant PIP5kinase-Iα (M31 and M37) or WT PIP5kinase-Iα (WT43, WT46 and WT49) were stimulated with antigen, fixed, and labeled with FITC phalloidin.


	Stephanie Hammond Quantification of Dynamic Processes in Live Cells

Regulation of the endocytotic pathway is necessary to maintain the protein and lipid composition of the plasma membrane. Stimulation alters the trafficking rates of recycling endosomal vesicles and can quickly bring proteins to the plasma membrane and alter lipid composition in a stimulus-dependent manner. In RBL cells, recycling endosomes contain the ganglioside GM1 and can be detected by binding of cholera toxin subunit B (CTxB) to GM1. We are using three-color confocal microscopy to quantify changes in trafficking upon stimulation and to further characterize the regulation of recycling endosomal trafficking.

Figures (above). Calcium ionophore stimulation increases GM1 trafficking. Newly trafficked GM1 from the internal pool of recycling endosomes is detected by binding of Alexa488-CTxB. The fluorescence at the plasma membrane is quantified and the rate of GM1 trafficking of stimulated cells is compared to the basal level of trafficking of unstimulated cells. Each sample represents the average of 19-21 cells collected over 4 experiments.


Norah Smith Cytokine Trafficking and the Role of Recycling Endosomes with David Holowka

Figure 1. Time courses for stimulated trafficking. D-sphingosine and DMS inhibit antigen-stimulated FITC-CTxB/GM1 trafficking, whereas TMS does not. Prior to stimulation by antigen (4 g/ml DNP-BSA), 2 M cytochalasin D was added to FITC-CTxB labeled cells in the presence or absence of 7.6M sphingosines.	Proteins and lipids undergo trafficking to and from the plasma membrane via a spatially organized pool of intracellular membranes termed recycling endosomes. FITC-labeled cholera toxin B (FITC-CTxB) bound to the ganglioside GM-1 is used to monitor IgE receptor stimulated trafficking of these endosomes to the plasma membrane in RBL-2H3 mast cells. Certain sphingosine derivatives, including D- or L-sphingosine and N,N dimethyl sphingosine (DMS), effectively inhibit this endosomal trafficking response stimulated by multivalent antigen (Figure 1). We plan to utilize the inhibitory effects of these compounds to investigate the role of recycling endosomes play in mast cells.
Cytokine secretion provides important immuno-modulatory signals that regulate and determine the type of immune response. We have preliminary evidence that certain cytokines secreted by RBL-2H3 mast cells may use recycling endosomes as a mechanism for traffic. Figure 2 shows the partial co-localization of the cytokine IL-4 with the recycling endosome marker, CTxB.	Figure 2. IL-4 is localized to a perinuclear structure and co-localizes with recycling endosome marker CTxB. Cells fixed after 3 hr stimulation in the presence of Alexa-555 CTxB (red). After fixation, cells were labeled with anti-IL4 followed by an Alexa-488 secondary antibody (green).


	Nat Calloway Store Operated Calcium Influx: Dynamics of STIM1 and Orai1 proteins

Calcium is normally stored in the endoplasmic reticulum in high concentrations and various cell signaling events can lead to the release of calcium from these stores as part of a signaling cascade. Release from calcium stores then triggers the ubiquitous process of store operated calcium influx (SOC) in which a sustained influx of calcium from the extracellular space maintains high intracellular calcium after stores are depleted. STIM1 is a protein located in the ER membrane that senses the intraluminal calcium concentration, and after calcium has been released from stores, STIM1 translocates and concentrates at regions close to the plasma membrane, where it appears to interact with the calcium channel CRACM1/Orai1 to mediate channel activation.Figure 1 shows the distribution of Orai1 (green) and STIM1 (red) in a resting state. Thapsigargan (TG) causes the passive release of calcium by blocking the refilling of the calcium stores. As shown in Figure 2, thapsigargan causes STIM1 and Orai1 to colocalize into discrete puncta at the plasma membrane (Figure 2).

Figure 1.	Figure 2.