1. Nuclear calcium signaling drives nuclear actin polymerization in T cells.
Nuclear calcium signaling drives nuclear actin polymerization in T cells. (A) Live-cell imaging of Ca2+ release (top, X-Rhod-1 AM) and nuclear actin polymerization (bottom) in Jurkat NLA cells upon contact with a TCR-stimulatory surface. Shown are still images at the indicated time after exposure to the stimulatory surface (see also movie S4). (B) Quantification of Ca2+ release triggered upon contact with a TCR-stimulatory surface in cells analyzed as in (A). Depicted are means ± SD of total and nuclear Ca2+ levels from eight cells analyzed. FC, fold change. (C) Single-cell analysis over 20 min after activation. Occurrence of actin polymerization at the PM (red) or the nucleus (green) as well as Ca2+ release (yellow) is shown. (D to G) Visualization of Ca2+ release and nuclear actin polymerization upon PMA/Iono stimulation in A3.01 NLA cells. (D and E) Stills of live-cell imaging movies of Ca2+ release of the same cell before (t = 0 s) and after (t = 44 s) stimulation with PMA/Iono. Dashed circles indicate the position of the nucleus. (F and G) Quantification of Ca2+ release (means ± SD from at least six cells each; intensities normalized to the frame before stimulation). Cells were treated with DMSO (D and F) or the Ca2+ chelator BAPTA-AM (E and G). (H) Relative occurrence of nuclear actin filaments in A3.01 NLA cells after treatment with the indicated stimuli and small-molecule inhibitors of Ca2+ signaling (means ± SD from three experiments with at least 30 cells evaluated each per condition). (I and J) Inhibition of nuclear CaM impairs the formation of nuclear F-actin filaments. Representative micrographs (I) and quantification of occurrence of nuclear or PM F-actin (means ± SD of three experiments with 30 cells evaluated each per condition) (J). A3.01 NLA (nuclear F-actin) or Jurkat NLA (F-actin ring) cells transiently expressing mCherry or the nuclear CaM inhibitor CaMBP4.mCherry were analyzed 58 s or 5 min after activation of a TCR-stimulatory surface, respectively. Asterisks indicate mCherry-positive cells. Statistical significance relative to the PMA/Iono (H) or mCherry (J) control was assessed by one-way analysis of variance (ANOVA) or one-sample t test, respectively. Scale bars, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments.
N. Tsopoulidis et al. Sci. Immunol. 2019;4:eaav1987
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