Calcium Signaling

Calcium Mediated Signaling and Cellular Dynamics David Holowka / Marcela Santos Deepti Gadi / Alice Wagenknecht-Wiesner Kate Corwith Kari Midthun
Back to Main Research page

Large-scale organization of intracellular membranes relevant to receptor-mediated Ca²⁺ mobilization
David Holowka	Marcela Santos

As with other receptors that activate Ca²⁺ mobilization to mediate functional responses, phosphoinositides play key roles in IgE receptor signaling. Among these roles, stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to produce inositol 1,4,5-trisphosphate (IP₃) is essential for initiation of store-operated Ca²⁺ entry (SOCE) and granule exocytosis. In addition, recent evidence from our laboratory highlights roles for PIP₂ in direct regulation of SOCE (1) and in granule fusion with the plasma membrane (2). Regulation of PIP₂ synthesis that is important for these processes is incompletely understood. We recently showed that two different isoforms of PIP5-kinase play different roles in IgE receptor-mediated Ca²⁺ mobilization (3), and these differential effects correspond to synthesis of two different pools of PIP₂ that fractionate with ordered and disordered lipid domains (1). Synthesis of PIP₂ by PIP5-kinases is tightly coupled to the production of the substrate for these enzymes, PI4-phosphate (PI4-P). In mammalian cells, this latter phosphoinositide is synthesized principally by two enzymes, PI4-kinase IIIα and PI4-kinase IIIα. The former is homologous to the yeast enzyme Stt4, which is localized to ER-plasma membrane junctions and is selectively inhibited by phenylarsine oxide (PAO; 4). Quercetin is a flavonoid derivative with a wide range of biological activities, including inhibition of a variety of ATPases and PI kinases (5). As shown in Figure 1, we find that both PAO and quercetin rapidly inhibit Ca²⁺ responses to antigen in RBL cells, but Ca²⁺ entry stimulated by thapsigargin, which bypasses the need for IP₃, is sensitive to rapid inhibition by quercetin but not by PAO. In addition, quercetin completely inhibits antigen-stimulated morphological changes in these cells, whereas PAO converts antigen-stimulated ruffling to less dramatic formation of stimulated filopodia (Figure 2). To account for these results, we hypothesize that quercetin directly inhibits PIP5-kinases (as well as PI4-kinases), whereas PAO inhibits PI4-kinase but not PIP5-kinase activity. Furthermore, our results provide evidence that synthesis of PI4P by PI4-kinase IIIα is dynamically coupled to PIP₂ synthesis and downstream signaling processes activated by IgE receptors.

Figure 1 Ca²⁺ responses to antigen (Ag) and thapsigargin (thaps) exhibit differential sensitivity to phenylarsine oxide (PAO) and quercetin. (justify) Ag-stimulated Ca²⁺ mobilization is rapidly and substantially inhibited by 1μM PAO or 10μM quercetin. (Right) Thaps-stimulated Ca²⁺ mobilization is not inhibited by 1μM PAO but is rapidly inhibited by quercetin added before or after PAO.

Figure 2 Antigen (Ag) stimulation of IgE-sensitized RBL mast cells causes dramatic cell ruffling that is completely inhibited by 20μM quercetin. In the presence of 2μM phenylarsine oxide (PAO), the morphological response to Ag appears as less dramatic cell filopodia.
1) Calloway et al., Stimulated association of STIM1 and Orai1 Is regulated by the balance of PIP2 between distinct membrane pools. Submitted for publication. 2) Gadi, D. et al., Sequestration of Phosphoinositides by Mutated MARCKS Effector Domain Alters Antigen-Stimulated Ca2+ Mobilization and Degranulation in Mast Cells. Manuscript in preparation. 3) Vasudevan et al., 2009.The b- and g- Isoforms of Type I PIP5K Regulate Distinct Stages of Ca2+ Signaling in Mast Cells. J Cell Sci. 122:2567. 4) Balla A., Balla T. 2006. Phosphatidylinositol 4-kinases: old enzymes with emerging functions. TRENDS in Cell Biology. 16:351. 5) Middleton et al. 2000. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacol. Rev. 52:673.


Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca²⁺ mobilization and degranulation in mast cells.

Deepti Gadi	Alice Wagenknecht-Wiesner

Previous evidence established that protein kinase Cβ (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the receptor for IgE, FcεRI, but the molecular basis has been unclear. We hypothesized that the polybasic effector domain (ED) of the abundant intracellular substrate for PKC known as MARCKS normally sequesters phosphoinositides (PIPs) at the inner leaflet of the plasma membrane until it is dissociated by phosphorylation mediated by activated PKC. Real time fluorescence imaging reveals synchronization between stimulated intracellular Ca²⁺ oscillations and oscillatory association of PKCβI-EGFP with the plasma membrane. MARCKS-ED tagged with mRFP undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that synchronizes with plasma membrane association of PKCβI, but MARCKS-ED dissociation is prevented by mutation of four serine residues in this sequence that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED show delayed onset of antigen-stimulated Ca²⁺ mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol trisphosphate production, is also substantially reduced by mutated MARCKS-ED, but store-operated Ca²⁺ entry is not inhibited. These results demonstrate the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, probably by sequestration of PIPs that mediate granule fusion at the plasma membrane.

Top PKCβI-EGFP in the cytosol (green) and mRFP-MARCKS-ED (red) at the plasma membrane prior to stimulation. Bottom PKCβI-EGFP (green) at the plasma membrane and mRFP-MARCKS-ED (red) in the cytoplasm following stimulation by antigen.

Local stimulation of RBL mast cells with antigen-coated beads
Kate Corwith

We recently showed that antigen-stimulated Ca²⁺ mobilization via IgE receptors in RBL-2H3 mast cells usually initiates as a fast Ca²⁺ wave that begins at the tip of an extended protrusion (Cohen et al. J. Immunol. 183:6478). At limiting doses of antigen, wave initiation depends on Ca²⁺ influx via TRPC1 and subsequently activates store-operated Ca²⁺ entry due to STIM1/Orai1 coupling that is essential for granule exocytosis in the allergic immune response. At these limiting doses of antigen, we find that granule exocytosis detected as real time imaging of FITC-dextran flashes occurs more frequently along protrusions with an initiating Ca²⁺ wave than in the cell body. Under these conditions, TRPC1 channels contribute significantly to the degranulation response, suggesting that the Ca²⁺ wave primes the extended protrusions to enhance the frequency of subsequent exocytotic events in this region.

To further investigate the spatial aspects of IgE signaling, we are locally stimulating RBL cells with 10μm antigen-coated polystyrene beads. Cells transfected with the genetically-encoded Ca2+ indicator GCaMP3 respond to stimulation by antigen-coated beads with Ca²⁺ waves initiating near bead placement, followed by both global oscillations and localized puffs near the bead. Further studies will investigate localization of other signaling components and degranulation under these conditions.

RBL-2H3 cell transfected with GCaMP3 and stimulated with a 10μm antigen-coated bead. Green fluorescence images show a Ca²⁺ wave originating at the cellular protrusion near the site of the bead and propagating to the cell body. This initial Ca²⁺ wave is followed by global Ca²⁺ oscillations and Ca²⁺ puffs near the site of the bead. The cell later phagocytosed the bead.

Studying ER Heterogeneity in RBL-2H3 Mast Cells

Kari Midthun

We are investigating the mechanism by which the endoplasmic reticulum (ER) protein stromal interaction molecule 1 (STIM1) associates with the calcium channel protein Orai1 on the plasma membrane to mediate store-operated calcium entry (SOCE). STIM1 oligomerizes in response to depletion of Ca²⁺ levels in the ER lumen, which initiates functional coupling to Orai1 forming the Ca²⁺ Release-Activated Ca²⁺ (CRAC) channel and initiating Ca²⁺ influx. This Ca²⁺ gating involves electrostatic interaction between a basic sequence in the Ca²⁺ activation domain of STIM1 and an acidic sequence in the C-terminus of Orai1 [Calloway et al. Biochem. 49: 1067-1071, 2010]. Using immunoprecipitation and Western Blotting analyses, we find that STIM1 strongly associates with a previously unidentified 20-25 kDa protein. This interaction is detected as a disulfide-bonded heterodimer that is formed during cell lysis, both before and after activation of SOCE by thapsigargin. Mutational analysis reveals that cysteine-437 in the Ca²⁺ activation domain of STIM1 participates in the formation of this 110 kDa heterodimer protein complex (Figure 1). An additional STIM1 disulfide complex is also observed under these conditions at ~ 260 kDa. Furthermore, cells expressing C437A mutant STIM1 show a substantial delay in stimulated Ca²⁺ entry, suggesting a possible functional role for the 20-25 kDa protein (Figure 2). Identification of the STIM1 binding partners detected in this study should provide new insights in the regulation of STIM1-mediated SOCE.

Figure 1: Western blots of STIM1 immunoprecipitated products from RBL cells previously transfected with WT-STIM1 or the C437A Mutant STIM1 construct. Both DNA constructs contain a Myc-His tag which allows for subsequent isolation and identification of transiently transfected STIM1 using an anti-Myc antibody. As shown, cells transfected with the C437A mutant construct and blotted with anti-Myc antibody do not contain a protein band at 110 kDa under non-reducing conditions, indicating that the interaction between STIM1 and the unidentified 20-25 kDa depends on this cysteine residue. Blotting these same immunoprecipitates with anti-STIM1 antibody reveals that endogenous STIM1 is still able to form the 110 kDa. Of note, the C437A mutation does not eliminate the high MW disulfide-bonded STIM1 complex.

Figure 2: Cos7 cells transfected with Orai1 and either WT STIM1 or C437A mutant STIM1 constructs were monitored for calcium response following addition of thapsigargin, an activator of SOCE. Expression of C437A mutant STIM1 causes ~2 minute average delay in calcium influx as compared to WT-STIM1 expressing cells. Error bars show SEM.