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Published on: June 12, 2013
Confocal fluorescence microscopy for studying thapsigargin-induced bivalent-cation entry into B cells
Insights
Thapsigargin triggers calcium (Ca2+) entry into B cells, accumulating in both cytoplasm and nucleus. Barium (Ba2+) and manganese (Mn2+) ions suggest this nuclear Ca2+ originates from the cytoplasm.
Area of Science:
- Cell Biology
- Immunology
Background:
- Thapsigargin is a known inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase.
- Bivalent cations, such as Ca2+, play critical roles in cellular signaling pathways.
Purpose of the Study:
- To investigate the dynamics of thapsigargin-induced bivalent-cation entry into antigen-specific B cells.
- To determine the origin of intranuclear calcium (Ca2+) signals.
Main Methods:
- Confocal fluorescence microscopy using fluo-3 and fura-2 dyes.
- Stimulation of B cells with thapsigargin in the presence and absence of external Ca2+.
- Use of Ba2+ and Mn2+ as Ca2+ channel probes.
Main Results:
- Thapsigargin stimulation led to Ca2+ accumulation in both the cytoplasm and nucleus of B cells.
- In the absence of external Ca2+, nuclear and cytoplasmic Ca2+ levels returned to baseline.
- Subsequent addition of external Ca2+ re-established elevated Ca2+ levels in both compartments.
- Ba2+ and Mn2+ quenched fluorescence, indicating entry through Ca2+ channels and supporting cytoplasmic origin of nuclear Ca2+.
Conclusions:
- Thapsigargin-induced nuclear Ca2+ signals in B cells are likely derived from the cytoplasm, not internal nuclear stores.
- Bivalent cation entry into B cells involves pathways accessible to Ca2+, Ba2+, and Mn2+.
Abstract:
We studied thapsigargin-induced bivalent-cation entry into antigen-specific B cells (TP67.21) with a confocal fluorescence microscope. Confocal fluorescence images of fluo-3-loaded B cells showed that thapsigargin-stimulated Ca2+ signals were transferred not only to the cytoplasm but also to the nucleus. In the absence of external Ca2+ ions, the free Ca2+ concentrations both in the cytosol and in the nucleus declined to basal levels by 5 min after addition of thapsigargin. However, subsequent addition of Ca2+ in the external medium made the fluo-3 (fura-2) fluorescence intensity rise, reflecting the fact that Ca2+ accumulated again in the nucleus as well as in the cytoplasm. Then, we added Ba2+ and Mn2+ instead of Ca2+, because Ba2+ and Mn2+ are known to enter via Ca2+ channels. The addition of Ba2+ and Mn2+ in the external medium quenched the fluo-3 fluorescence both in the nucleus and in the cytoplasm of B cells. This suggested the possibility that the increase in intranuclear Ca2+ after thapsigargin stimulation may come from the cytoplasm, not from the nuclear stores.
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