Biochemical and Pharmacological Studies

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

Background 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 structures 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 concentrate to these subdomains include phospholipase Cg, as well as SERCA 2 ATPase and voltage operated Ca2+ channels. Recent evidence indicates a novel pathway for Ca2+ influx that may depend upon these plaques. 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 Dynamics Using High-resolution Live Cell Imaging in collaboration with the Kotlikoff Group

Calcium elevation is central to many cellular and physiological processes where the calcium ions function as second messengers. Two closely associated pathways are responsible for the increase in cytosolic calcium concentration: ionic influx trough specific plasma membrane channels and mobilization of calcium ions from intracellular stores. The aim of this project is to study the molecular and cellular mechanisms that regulate these two pathways and by that adjust the cytosolic calcium levels. Combining single cell stimulation with high speed calcium imaging using both conventional calcium dyes and genetically encoded calcium sensors (GCaMP2; Tallini et. al. PNAS 2006) we find a non-homogenous calcium increase in RBL mast cells, that under some conditions is preceded by local and transient calcium “sparks”. Moreover, visualizing the initial increase of calcium concentration with high rate imaging revealed a calcium wave that initiates from usually one or two regions within the cell that develops into a general cytosolic increase in calcium concentration.
Figure (above). Antigen stimulation of a living RBL mast cell expressing the genetically encoded calcium sensor – GCaMP2 reveals a wave-shaped calcium increase. The calcium wave initiates from the upper side of the cell and progresses to the other side of the cell as shown in the virtual line scan of fluorescence intensity along the Y axis of the cell (yellow line in left image) presented as an intensity plot (right side). Images were taken every 60 milliseconds for 30 seconds.	Figure (above). Thapsigargin stimulation of a single RBL mast cell expressing the genetically encoded calcium sensor – GCaMP2 reveals a local calcium “spark” in the lower side of the cell (arrowhead, right panel) that precedes the more general cytosolic calcium increase. Virtual line scan of fluorescence intensity was measured along the Y axis of the cell (designated by the yellow box, left image) and presented as an intensity plot (upper right) and a relative intensity curve (lower right). Images were taken every 75 milliseconds for 60 seconds.


Jinmin Lee Characterizing the molecular basis of mast cell motility and directed migration

Mast cells are derived from hematopoietic progenitor cells but do not normally circulate in mature form. Instead, mast cell differentiation and maturation takes place in vascularized tissues or serosal cavities, in which the mast cells will ultimately reside. Although this homing of mast cell progenitors is relatively well studied, little is known about the fully matured mast cell motility and migration inside the tissue site. The goal of my research is to characterize the function and regulation of mast cell motility and directed migration in mucosal tissue. The RBL-2H3 mast cell line has structural and functional characteristics of fully differentiated mucosal mast cells. We hypothesize that the motility characteristics of RBL cells will be comparable to interepithelial migration characteristics of fully differentiated mucosal mast cells in the gut. Our recent studies revealed that RBL-2H3 mast cells exhibit elongated morphological protrusions and undergo novel cell polarization and motility. These phenomena depend on several factors, including Ca2+ homeostasis (Figure 1). Using these observations as a starting point, we will investigate the molecular basis of unique motility properties of mast cells, utilizing real-time video microscopy and molecular and biochemical methods. z
(above) Figure 1. Phase contrast images of RBL-2H3 cells with elongated protrusions (a), RBL-2H3 cells before (b) and after (c) 90min treatment with 8mM EGTA. Arrows indicate protrusions before (b) and after (c) retraction in response to EGTA treatment.





Norah Smith Recycling Endosome involvement in the intracellular trafficking of cytokines in RBL-2H3 cells

Figure 1 is currently not available. Figure 1 (above). RBL-2H3 mast cells labeled with either FITC-CTxB or FITC-TfR. Fluorescence changes were monitored in response to addition of cytochalasin D and DNP-BSA (Ag).	Proteins and lipids undergo trafficking to and from the plasma membrane via a spatially organized pool of intracellular membranes termed recycling endosomes. FITC-cholera toxin B (FITC-CTxB) bound to the ganglioside GM-1 or FITC-a-transferrin receptor (FITC-TfR) is used to monitor stimulated trafficking of these endosomes to the plasma membrane in RBL-2H3 mast cells (figure 1).
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. We are monitoring the process of STIM1 interacting with Orai1 with confocal fluorescence microscopy and fluorescence resonate energy transfer (FRET) analyses. 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). By monitoring changes in fluorescent intensity during this process, we can observe the transfer of excitation energy from mGFP-tagged CRACM1/Orai1 (donor) to mRFP-tagged STIM1 (Figure 3). This FRET data provides direct, real-time evidcnce for stimulated interactions between these components.

Figure 1.	Figure 2.

Kirsten L. Elzer

Electrostatic protein-lipid interactions in plasma membrane biogenesis of key receptors

Phosphatidylinositol (PI) and its phosphorylated derivatives, termed phosphoinositides, have been shown to possess distinct biological functions due, in part, to their unique localizations throughout the cell. Phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 is highly localized to the inner leaflet of the plasma membrane, phosphatidylinositol 4-phosphate (PI4P) is enriched at the Golgi complex, and phosphatidylinositol 3-phosphate (PI3P) is most abundant in endosomal membranes. To investigate functionally relevant interactions between these lipid species and the receptor for immunoglobulin E on mast cells, we mutated basic amino acid residues in the cytoplasmic juxtamembrane sequence of the gamma subunit of a chimeric IgE receptor (Figure 1), and we observed inhibition of receptor trafficking from the endoplasmic reticulum (ER) to the plasma membrane (Figure 2). Similarly, biogenic trafficking of this receptor was inhibited by co-expression of ER-targeted polybasic MARCKS effector domain. We hypothesize that these observations reflect an interaction between the polybasic sequence of the receptor and ER-localized PI4P that is important for ER to Golgi trafficking of newly synthesized receptors. We are currently testing this hypothesis and its general relevance to plasma membrane biogenesis using site-specific mutagenesis, quantitative imaging of PI4P, flow cytometry, and pharmacologic perturbation of enzymes critical for this process.

(above) Figure 1. Wildtype and mutant constructs. The juxtamembrane sequence is underlined, basic amino acids are in bold, and mutated residues are shown in red.

(above) Figure 2. Expression of wild type and mutant constructs in CHO cells. Live cells transiently expressing wt and mutant were labeled with Alexa488-IgE, then fixed, permeabilized, and labeled with anti-human FcRI antibody followed by Alexa568 anti-rabbit IgG. Images were taken using a Leica SP2 TCS confocal microscope. This figure illustrates loss of IgE binding to γγ at the cell surface in MutB1,3,4 and partial (MutB1, MutB3) or complete (MutB1,3,4) retention in the ER.

Kari Midthun

Studying ER Heterogeneity in RBL-2H3 Mast Cells

Previous work in this laboratory established a method for cell fractionation, by sucrose gradient equilibrium centrifugation following lysis by nitrogen cavitation, and found evidence for separation of the endoplasmic reticulum (ER) into two distinct fractions: a light membrane fraction enriched in certain channel proteins, and a more conventional heavy ER fraction containing a variety of ER markers (Hammond, Ph.D. Thesis, 2008). The distribution of ER-associated proteins observed suggests that the light ER fractions may represent a region specialized for calcium mobilization and coupling between the ER and the plasma membrane.

A current focus has been to determine whether the distribution of ER–associated proteins changes under cell stimulation conditions. STIM1, an ER membrane protein that senses intraluminal calcium levels, oligomerizes in response to calcium depletion from the ER and redistributes to mediate ER-plasma membrane coupling and activation of store-operated calcium entry (SOCE). STIM1 interaction with the plasma membrane-localized calcium channel, Orai1 (also known as CRACM1), mediates this channel activation that is initiated by IgE receptor stimulation or by thapsigargin, which induces calcium loss from the ER by inhibiting the ER ATPase calcium pump. To characterize the location of STIM1 in the ER and to evaluate whether this changes upon activation of SOCE, we compared the distribution of STIM1 by sucrose gradient fractionation before and after activation of SOCE by thapsigargin. As shown in Figure 1, STIM1 is localized primarily to the heavy ER region of the gradient in the absence of stimulation, but a substantial percentage redistributes to the lighter ER region following treatment with thapsigargin. This redistribution suggests a change in ER environment for a population of STIM1 that may be relevant to its plasma membrane coupling. Future experiments will test whether this change is selective for STIM1 or reflects a more global change in ER structure.

(above) Sucrose gradient fractionations of RBL cells were lysed by nitrogen cavitation and analyzed using Western Blotting to visualize STIM1 protein distribution across the gradients. When stimulated with thapsigargin, a significant increase in the abundance of STIM1 in the Light ER fractions is seen, suggesting a redistribution of STIM1 in ER membrane subdomains.