Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells - PubMed (original) (raw)

Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells

Roy Cohen et al. J Immunol. 2009.

Abstract

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

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Figures

Figure 1. Visualization of Ca2+ dynamics in individual RBL mast cells

A) Cells were plated at low density on MatTek dishes, and individual cells were stimulated using a pulled capillary (arrow; 0.5-1 μm diameter), filled with stimulating solution (as specified for each experiment) that was puffed at the cell (5psi/10sec pulse) from a distance within 100 μm. B) Image of RBL cell expressing GCaMP2 and sensitized with anti-DNP IgE, stimulated with a puff of 1.7 μg/ml Ag (DNP-BSA). Middle panel shows time line analysis of the stimulated cell and changes in Ca2+ concentration occurring over time in the 7 pixel wide line segment defined in the left panel. Changes in Ca2+ concentration are indicated by relative changes in brightness, where brighter colors represent higher Ca2+ concentration. Right panel expands early time points of the time line analysis and highlights our general observation that Ca2+ elevation initiates in cellular protrusions and propagates through the cell body. C) Traces of GCaMP2 intensity changes over time as calculated for the same cell imaged in B (inset). Black and red traces represent the changes in Ca2+ concentration in cell body (yellow ROI) or cell protrusion (red ROI), respectively.

Figure 2. Dependence of stimulated Ca2+ waves and oscillations on extracellular Ca2+and stimulus

A) Representative cell sensitized with IgE and stimulated with 1.7μg/ml Ag in the absence of extracellular Ca2+. Right panel shows time line analysis for early time points and indicated segment in left panel image. B) Histogram showing percentage of cells responding to stimulus with Ca2+ waves as averaged over multiple experiments for 0mM Ca2+ (n=29), 2mM Ca2+ (n=80); 10mM Ca2+ (n=28). Bar height shows total percentage of waves; orange portion represents the percentage of waves originating in protrusions (PRTS). C) Average number of oscillations within 2 min of stimulation under specified conditions. D) Representative traces of cells sensitized with anti-DNP IgE and stimulated with puff from pipette containing 1.7 μg/ml DNP-BSA (Ag), 1μM thapsigargin (TG), 20μM A23187 or BSS buffer only (vehicle). E) Percentage of cells responding to stimulus with Ca2+ waves averaged over multiple experiments and represented as in (B) with indicated conditions: Ag (n=80); TG (n=16); A23187 (n=29). F) Average number of oscillations within 2 min of stimulation under specified conditions. The first oscillation corresponds to the originating Ca2+ wave (dotted line). Error bars correspond to standard error of the mean (SEM).

Figure 3. Ca2+ dynamics in individual rat BMMCs

A) Confocal image of a representative rat BMMC sensitized with anti-DNP IgE and loaded with Fluo5F (left panel), and time line analysis of indicated ROI for this cell stimulated at t=0 with a puff of 1.7 μg/ml DNP-BSA (right panel). Changes in Ca2+ concentration in response to stimulation are depicted, where brighter colors represent higher Ca2+ concentration. Time line analysis highlights the Ca2+ elevation initiating in the tip of the cellular protrusion and propagating through the cell body. B) Traces of Fluo5F intensity changes over time as calculated for the same cell imaged in (A). Black and red traces in right panel represent changes in Ca2+ concentration in cell body (yellow ROI) and in cell protrusion (red ROI), respectively, in the left panel.

Figure 4. Stimulation of RBL cells by contact with Ag-coated micropipette

A and B) Representative cells expressing GCaMP2, sensitized with IgE and stimulated with DNP-BSA conjugated micropipettes. A) Micropipette (indicated by yellow arrowhead) contacting the cell at the tip of a protrusion elicits a train of spatially restricted Ca2+ puffs, each traveling no more than 30μm along the protrusion. B) Contact stimulation at the cell body results repetitive Ca2+ puffs in the cell body that sometimes propagate as a wave to the protrusion. Left panels show images with ROIs defined; Right panels show Ca2+ concentration changes in ROIs of corresponding color. Black arrow indicates initiation of contact between micropipette and cell. C) Histogram showing percentage of cells responding with local Ca2+ puffs only (light blue) or more global Ca2+ elevation (dark blue) due to contact with DNP-BSA-conjugated micropipettes in the presence (n=28) or absence (n=18) of extracellular Ca2+, or with unmodified BSA-conjugated micropipettes (n=18).

Figure 5. Effects of inhibitors on intracellular Ca2+ waves and oscillations

A) Percentage of cells from multiple experiments showing a measurable Ca2+ response to stimulation by 1.7 μg/ml Ag in the presence of the following reagents in the extracellular buffer: 1-5 μM U73122 (n>18 each), 20mM 2-APB (n=23), 8μM D-sphingosine (n=26); 8μM DM-sphingosine (n=31), 10μM nifedipine (n=15), 1 or 10 μM GdCl3 (Gd3+; n>20 each) B) Percentage of cells responding to stimulus with Ca2+ waves as averaged over multiple experiments with indicated conditions. Bar height shows total % waves; orange portion represents % waves originating in protrusions. C) Average number of oscillations within 2 min of stimulation under specified conditions. The first oscillation corresponds to the originating Ca2+ wave (dotted line). Error bars correspond to SEM.

Figure 6. Participation of TRPC channels in Ag stimulated initiation of the Ca2+ response

RBL cells were co-transfected with GCaMP2 and shRNA plasmids targeting TRPC1, 3, 5, 7 or mock sequence (for each condition n>29). Cells sensitized with anti-DNP IgE were stimulated with puff from pipette containing 1.7 ng/ml DNP-BSA in BSS. A) Percentage of cells from multiple experiments showing a measurable Ca2+ response corresponding to elevation of intracellular Ca2+ cells. Dark blue bars include cells that only include Ca2+ puffs in the count, and light blue bars exclude this population. B) Average lag time before initiation of Ca2+ wave with specified shRNA. Error bars correspond to SEM. *P < 0.05, **P < 0.01 vs. cells expressing shGFP. C) Percentage of cells responding to stimulus with Ca2+ waves with indicated shRNA. Bar height shows total % waves; orange portion represents % waves originating in protrusions. D) Model for initiation of Ca2+ mobilization in extended protrusions. Ag crosslinks IgE/FcεRI complexes, activating Ca2+ influx via TRPC1 channels to cause local increase in cytoplasmic Ca2+ at the tip of a cell protrusion. This potentiates IP3-mediated Ca2+ release from the nearby ER store that propagates via Ca2+-induced Ca2+ release to generate a Ca2+ wave that initiates Ca2+ oscillations and cell activation. Two possible mechanisms for wave initiation at these protrusions are indicated.

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Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells - PubMed (original) (raw)