Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse
- Stanford University, United States
- Children's Hospital of Philadelphia Research Institute, United States
- Perelman School of Medicine, University of Pennsylvania, United States
Figures
Figure 1
STIM1 and Orai1 accumulate in puncta in the actin-depleted zone of the immune synapse.
(A) TIRF images of Jurkat cells stimulated on anti-CD3 coated coverslips in 0.8 mM Ca2+o. mCh-STIM1 (green) and Orai1-EGFP (red) puncta accumulate in the center of the synapse over time. Images …
Figure 2 with 1 supplement
Synaptic ER tubules extend from the ADZ and are moved centripetally by actin.
(A) TIRF images of a Jurkat cell coexpressing GFP-actin (red) and ER-mCh (green), after spreading on an anti-CD3-coated coverslip. (B) Magnification of the boxed regions in A depicting an extending …
Figure 2—figure supplement 1
The ER extends at the tips of dynamic microtubules that move radially toward the lamella/lamellipod border.
(A) TIRF images of a Jurkat cell stimulated on anti-CD3 coated coverslips expressing EB1-EGFP (red) and ER-mCh (green). Magnified view of the boxed region shows an ER tubule moving peripherally …
Figure 3
Calcium influx organizes synaptic actin and promotes retrograde flow.
(A) TIRF images of a Jurkat cell expressing F-tractin-P-tdTom after spreading on anti-CD3 in 2 mM Ca2+o (left), 3.25 min after Ca2+o removal (center), and 1 min after readdition of 2 mM Ca2+o …
Figure 4
Calcium influx promotes ER corralling.
(A) A cell expressing F-tractin-P-tdTom (red) and ER-GFP (green) on an anti-CD3 coverslip is shown in 0 Ca2+o and after readdition of 2 mM Ca2+o. Peripheral ER tubules in the TIRF images are sparse …
Figure 5 with 1 supplement
Intracellular calcium reduces the density of F-actin at the synapse.
Jurkat T cells expressing F-tractin-P-tdTom and loaded with fura-2 were stimulated on anti-CD3-coated coverslips. (A) Pseudocolor image of F-tractin-P-tdTom intensity in a cell exposed sequentially …
Figure 5—figure supplement 1
Ca2+ influx through CRAC channels reduces F-actin density at the synapse.
(A) Jurkat cells expressing F-tractin-P-tdTom were allowed to spread on anti-CD3 coated coverslips in 2 mM Ca2+o, followed by perfusion with 100 µM 2-APB to block Ca2+ influx through Orai1. 2-APB …
Figure 6 with 2 supplements
Calcium accelerates actin depolymerization and centripetal velocity at the synapse.
Jurkat T cells expressing F-tractin-P-tdTom and PAGFP-actin were stimulated on anti-CD3 coated coverslips and TIRF images of F-tractin-P-tdTom (A, left) were used to identify regions in the …
Figure 6—figure supplement 1
Actin filament velocity and half-life at the synapse.
Jurkat cells were stimulated on anti-CD3 coated coverslips and PAGFP-actin was photoactivated in small regions within the lamellipod and lamella. (A-D) Fluorescence intensity profiles of …
Figure 6—figure supplement 2
Calcium influx alters actin organization and density independently of myosin activity.
(A) TIRF images of Jurkat cells expressing F-tractin-P-tdTom pretreated for 30 min with 50 µM blebbistatin, then stimulated on anti-CD3 coated coverslips in 2 mM Ca2+o and blebbistatin (left) …
Figure 7
Calcium restricts actin polymerization to the distal edge of the synapse.
(A, B) Two Jurkat T cells expressing F-tractin-P-tdTom and PAGFP-actin were stimulated on anti-CD3 coated coverslips in 0.5 mM Ca2+, and PAGFP-actin was photoactivated in the ADZ regions indicated …
Figure 8
Effects of calcium on actin dynamics and retrograde flow at the synapse.
(A) Retrograde actin flow at the immune synapse (yellow arrows) continually removes extended ER tubules (purple) from the periphery, thereby concentrating the ER in the ADZ. An expanded view of the …
Videos
Video 1
STIM1 and Orai1 accumulate in puncta in the center of the synapse.
Time-lapse TIRF movie of a Jurkat cell expressing mCh-STIM1 (left) and Orai1-EGFP (center) stimulated on an anti-CD3-coated coverslip. A merge of the STIM1 (green) and Orai1 (red) channels is shown …
Video 2
STIM1 puncta accumulate in the ADZ of the synapse.
Time-lapse TIRF movie of a Jurkat cell expressing mCh-STIM1 (left) and GFP-actin (center) stimulated on an anti-CD3-coated coverslip. A merge of the STIM1 (green) and actin (red) channels is shown …
Video 3
ER tubules extend from the ADZ into the lamella and are moved centripetally by actin.
Time-lapse TIRF movie of a Jurkat cell expressing GFP-actin (left) and ER-mCh (middle) stimulated on an anti-CD3-coated coverslip. A merge of the actin (red) and ER (green) channels is shown at …
Video 4
ER tubules extend toward the synapse edge on the tips of microtubules.
Time-lapse TIRF movie of a Jurkat cell expressing ER-mCh (left) and EB1-EGFP (middle) stimulated on an anti-CD3-coated coverslip. A merge of the ER (green) and EB1 (red) channels is shown at right. …
Video 5
EB1 moves radially in the ADZ but parallel to the cell edge at the lamella/lamellipod border.
Time-lapse TIRF movie of a Jurkat cell expressing F-tractin-P-tdTom (left) and EB1-EGFP (middle) stimulated on an anti-CD3-coated coverslip. A merge of the actin (red) and EB1 (green) channels is …
Video 6
Calcium influx organizes synaptic actin and promotes retrograde flow in Jurkat cells.
Time-lapse TIRF movie of a Jurkat cell expressing F-tractin-P-tdTom after spreading on an anti-CD3 coverslip in 2 mM Ca2+o, followed by perfusion with 0 Ca2+o and 2 mM Ca2+o. Images acquired every 5 …
Video 7
Calcium influx organizes synaptic actin and promotes retrograde flow in primary human T lymphoblasts plated on anti-CD3 and ICAM-1.
Time-lapse spinning disk confocal movie of a human T lymphoblast expressing Lifeact-GFP after spreading on anti-CD3 and ICAM-1 Fc in 0.5 mM Ca2+o (left), 1.3 min following perfusion with 0 Ca2+o …
Video 8
Calcium influx organizes synaptic actin and promotes retrograde actin flow in primary human T lymphoblasts plated on anti-CD3 without ICAM-1.
Time-lapse spinning disk confocal movie of a primary human T lymphoblast expressing Lifeact-GFP after spreading on anti-CD3 in 0.5 mM Ca2+o (left), 4.3 min following perfusion with 0 Ca2+o (center) …
Video 9
Calcium-dependent retrograde actin flow corrals the ER in the ADZ.
Time-lapse TIRF movie of a Jurkat cell expressing ER-GFP (left) and F-tractin-P-tdTom (center) that had spread in 2 mM Ca2+o before perfusion with 0 Ca2+o. Video begins with the cell in 0 Ca2+o and …
Video 10
Calcium increases actin depolymerization and centripetal velocity at the synapse.
Time-lapse TIRF movie of a Jurkat cell expressing F-tractin-P-tdTom and PAGFP-actin. Bars of PAGFP-actin are photoactivated in the lamella and lamellipod of the same cell in 2 mM Ca2+o (left) and 0 …
Video 11
Calcium restricts actin polymerization to the distal edge of the synapse.
Time-lapse TIRF movie of a Jurkat cell expressing F-tractin-P-tdTom and PAGFP-actin. Monomeric PAGFP-actin is photoactivated in the ADZ of two different cells in 2 mM Ca2+o (left) and 0 Ca2+o …
Video 12
Calcium promotes localization of Abi1 to the distal edge of the synapse.
Time-lapse TIRF movie of a Jurkat cell expressing F-tractin-P-tdTom and EGFP-Abi1 stimulated on anti-CD3 coated coverslip in 0.5 mM Ca2+o, followed by perfusion with 0 Ca2+o and 2 mM Ca2+o. Images …
Tables
Table 1
Effects of calcium on actin dynamics at the primary T cell immune synapse
| Anti-CD3 | Anti-CD3 + ICAM-1 | |||||
|---|---|---|---|---|---|---|
| 0.5 Ca2+ | 0 Ca2+ | 2 Ca2+ | 0.5 Ca2+ | 0 Ca2+ | 2 Ca2+ | |
| Velocity (nm/s) | 243 ± 8 (9) | 145 ± 6 (9) | 208 ± 8 (9) | 167 ± 4 (11) | 94 ± 3 (11) | 162 ± 5 (11) |
| Lamellipod width (µm) | 3.0 ± 0.2 (9) | 1.5 ± 0.1 (9) | 2.3 ± 0.2 (9) | 2.8 ± 0.1 (10) | 1.1 ± 0.1 (10) | 2.3 ± 0.2 (8) |
-
[Ca2+]o indicated in mM in the order in which the solutions were applied (see text). Means ± SEM; number of cells indicated in parentheses. Velocities are from a total of 104-133 measurements from kymographs made at 3 different locations per cell
Table 2
Effects of calcium on actin dynamics at the Jurkat cell immune synapse
| Lamellipod | Lamella | |||
|---|---|---|---|---|
| 2 Ca2+ | 0 Ca2+ | 2 Ca2+ | 0 Ca2+ | |
| Half-life (s) | 6.5 ± 0.4 (10) | 8.0 ± 0.4 (10) | 6.4 ± 0.4 (19) | 7.5 ± 0.5 (19) |
| Velocity (nm/s) | 58 ± 6 (10) | 34 ± 5 (10) | 36 ± 6 (18) | 13 ± 3 (18) |
-
[Ca2+]o indicated in mM. Means ± SEM; number of cells indicated in parentheses.
