Spontaneous Calcium Bursts Organize the Apical Actin Cytoskeleton of Multiciliated Cells

Abstract

Motile cilia perform crucial functions during embryonic development and in adult tissues. They are anchored by an apical actin network that forms microridge-like structures on the surface of multiciliated cells. Using Xenopus as a model system to investigate the mechanisms underlying the formation of these specialized actin structures, we observed stochastic bursts of intracellular calcium concentration in developing multiciliated cells. Through optogenetic manipulation of calcium signaling, we found that individual calcium bursts triggered the fusion and extension of actin structures by activating non-muscle myosin. Repeated cycles of calcium activation promoted assembly and coherence of the maturing apical actin network. Inhibition of the endogenous inositol triphosphate-calcium pathway disrupted the formation of apical actin/microridge-like structures by reducing local centriolar RhoA signaling. This disruption was rescued by transient expression of constitutively active RhoA in multiciliated cells. Our findings identify repetitive calcium bursts as a driving force that promotes the self-organization of the highly specialized actin cytoskeleton of multiciliated cells.

Keywords: RhoA; Xenopus; actin; calcium signaling; cilia; cytoskeleton; optogenetics.

Figure 1

Calcium bursts in MCCs cause apical contraction and changes to the apical actin network: (A) Calcium bursts occur regularly in MCCs during the development of the Xenopus epidermis (arrows). Stills of a movie recorded over 10 min (20 frames/minute) of a stage 21–22 embryo with actin (RFP-Utrophin, magenta) and calcium (GCaMP6s, green) labels. Scale bar 20 µm. (B) Setup for experimental induction of calcium bursts in MCCs. Targeted activation of BACCS by blue light causes Ca²⁺-influx through Orai channels. Xenopus illustrations © Natalya Zahn (2022), xenbase.org RRID:SCR_003280 [28]. (C–E) Effects of stimulated calcium bursts on MCCs in a representative cell. (C) Graph of GCaMP fluorescence and apical surface of an MCC during an optogenetically induced calcium burst. (D,E) Maximum intensity projections of apical actin labeled by RFP-Utrophin in a developing MCC before and after an induced calcium burst. (D’,E’) Skeletonized actin derived from the confocal images. Scale bars 5 µm.

Figure 2

Actin network assembly in response to calcium bursts in MCCs: (A) Scheme: At stages 21–22, MCCs expressing BACCS components were recorded for 180 s. BACCS was activated to induce a calcium burst, resulting in apical contraction (green arrows) Apical area and the apical actin network were quantified and compared between a pre- and a post-burst timepoint. Xenopus illustrations © Natalya Zahn (2022) [28]. (B–D) Changes to the apical area and the actin network (number of skeletons, average branch length) in MCCs with and without calcium burst. (B) Untreated embryos, (C) Blebbistatin, (D) SMIFH2. Cells were recorded for 180 s and a calcium burst was induced after 12 s. Magenta lines show values for individual cells without a calcium burst, green lines for those with an induced calcium burst; end-points indicate the pre- and post-burst values. Statistical test: Wilcoxon matched pairs signed rank test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s. not significant. For significant changes, the median change is included in the graph. Mean and SD values are provided in Table S1. (E) Comparison of changes to the apical area and the actin network (number of skeletons, average branch length) between MCCs with and without calcium activity both for untreated, Blebbistatin-treated, and SMIFH2-treated embryos. Graphs show the changes presented in (B–D) in percent. Each data point represents an individual cell. Box and whiskers indicate quartiles. Dashed line indicates 0% (no change). Averages and SD: Apical Area (Untreated, no burst: +1.2 ± 3.5%; calcium burst: −19.3 ± 10.0%; Blebbistatin, no burst: −1.7 ± 3.9%; calcium burst: −7.5 ± 6.1%; SMIFH2, no burst: −0.3 ± 4.4%; calcium burst: −18.3 ± 8.1%). Skeleton number (Untreated, no burst: +8.1 ± 10.5%; calcium burst: −23.9 ± 15.8%; Blebbistatin, no burst: +1.6 ± 17.5%; calcium burst: −8.7 ± 11.5%; SMIFH2, no burst: +2.2 ± 10.1%; calcium burst: −18.9 ± 17.1%). Average branch length (Untreated, no burst: −5.7 ± 6.8%; calcium burst: +11.7 ± 10.0%; Blebbistatin, no burst: −4.1 ± 11.8%; calcium burst: +6.0 ± 9.0%; SMIFH2, no burst: −1.2 ± 11.0%; calcium burst: +2.2 ± 7.1%). Statistical test: 2-way ANOVA + Tukey’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; non-significant comparisons are not shown; 53 cells from 30 untreated embryos; 41 cells from 14 Blebbistatin-treated embryos; 39 cells from 18 SMIFH2-treated embryos.

Figure 3

Non-muscle myosin II and recurring calcium bursts drive apical actin network development: (A–C) Maximum intensity projections of apical actin in MCCs of embryos treated with (A) DMSO or (B) 50 µM Blebbistatin from stage 19 to 30. (A’,B’) show actin labeled by phalloidin in magenta, and GFP-centrin-labeled basal bodies in green. Scale bars 5 µm. (A’’,B’’) show the skeletonized actin network. (C) Quantification of the apical actin network of MCCs in embryos treated with DMSO or Blebbistatin. Number of skeletons (separate actin structures) per µm² (DMSO 0.36 ± 0.09; Blebbistatin 0.53 ± 0.11). Average branches per skeleton (DMSO 5.5 ± 2.2; Blebbistatin 3.6 ± 1.0). Length of the apical actin network normalized to surface area (DMSO 1.25 ± 0.15 µm⁻¹; Blebbistatin 1.13 ± 0.15 µm⁻¹). Error bars show mean and SD; 39 cells from 12 to 13 embryos analyzed per condition. Statistical test: Mann–Whitney Test (D,E) Effect of repeated induction of calcium bursts. (D) Experimental setup: Embryos expressing BACCS and Orai were grown in darkness or with 10 s pulses of 450 nm light every 4 min from stage 19 to 30. Xenopus illustrations © Natalya Zahn (2022) [28]. (E) Quantification of MCC apical actin: Number of skeletons per µm² (Dark 0.44 ± 0.09; Blue 0.33 ± 0.09). Average branches per skeleton (Dark 5.2 ± 1.6; Blue 7.7 ± 3.0). Length of the apical actin network normalized to surface area (Dark 1.30 ± 0.15 µm⁻¹; Blue 1.40 ± 0.15 µm⁻¹). Error bars show mean and SD; 24–27 cells from 8 to 9 embryos analyzed per condition. Statistical test: Mann–Whitney Test.

Figure 4

Inhibition of IP3R and PLC leads to truncation of the apical actin network and microridges: (A–C) Maximum intensity projections of apical actin in MCCs of embryos treated with (A) DMSO, (B) 2-APB, or (C) U73122 from stage 19 to 30. (A’–C’) show actin labeled by phalloidin in magenta, and RFP-centrin-labeled basal bodies in green. (A’’–C’’) show the skeletonized actin network. Scale bars 5 µm. (D,E) Scanning electron micrographs of MCCs on the surfaces of embryos treated with (D) DMSO or (E) 2-APB. Microridges were manually labeled red in magnified insets (dashed circles). Scale bar 5 µm. (F) Scheme illustrating the interactions of small molecule inhibitors 2-APB and U73122 with the IP₃-calcium pathway. IP₃, inositol 1,4,5-triphosphate; IP3R, inositol triphosphate receptor; PIP₂, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PM, plasma membrane; ER, endoplasmic reticulum membrane. (G–J) Quantification of apical surface area and the apical actin network of MCCs in embryos treated with DMSO or 2-APB. (G) Apical surface area (DMSO: 208.5 ± 55.4 µm²; 2-APB: 282.4 ± 71.0 µm²). (H) Number of skeletons (separate actin structures) per µm² (DMSO: 0.36 ± 0.08; 2-APB: 0.57 ± 0.11). (I) Average branches per skeleton (DMSO: 6.2 ± 1.7; 2-APB: 3.3 ± 0.8). (J) Length of the apical actin network normalized to surface area (DMSO: 1.30 ± 0.13 µm⁻¹; 2-APB: 1.03 ± 0.11 µm⁻¹). (K–N) Quantification of apical surface area and the apical actin network of MCCs in embryos treated with DMSO or U73122. (K) Apical surface area (DMSO 254.3 ± 51.2 µm²; U73122 344.9 ± 64.5 µm²). (L) Number of skeletons (separate actin structures) per µm² (DMSO 0.41 ± 0.07; U73122 0.62 ± 0.11). (M) Average branches per skeleton (DMSO 4.1 ± 1.1; U73122 2.4 ± 0.9). (N) Length of the apical actin network normalized to surface area (DMSO 1.12 ± 0.11 µm⁻¹; U73122 0.80 ± 0.29 µm⁻¹). Error bars show mean and SD; 21–24 cells from 6 to 8 embryos analyzed per condition. Statistical test: Mann–Whitney Test.

Figure 5

(A–C) Polarization of ciliary rootlets in MCCs treated with (A) DMSO and (B) 2-APB. Ciliary rootlets labeled by GFP-clamp are in green, and basal bodies labeled by RFP-centrin are in magenta (Maximum intensity projections). Scale bar 5 µm. (C) Rootlet angle circular standard deviation in MCCs (DMSO 25.4° mean, 24.3° median; 2-APB 48.2° mean, 38.0° median). Statistical test: Mann–Whitney test (D) Ciliary Gliding Assay. Stage 30 embryos treated with DMSO, 2-APB, or U73122 starting stage 19. Red traces indicate distance moved within 20 s. Scale bar 1 mm. In the graph, bars indicate the median (DMSO: 4.61 mm/min; 2-APB: 0.21 mm/min; 0.49 mm/min); 19–21 embryos analyzed in two independent experiments. Statistical Test: Kruskal–Wallis Test + Dunn’s multiple comparisons test.

Figure 6

Inhibition of IP3R disrupts the apical actin network by interfering with local activation of RhoA: (A–D) Active RhoA (GFP-rGBD, green, A’,B’) localizes to the anterior rootlet (mCherry-clamp, magenta) of basal bodies in MCCs of control embryos treated with DMSO from stage 19 to 24 (Maximum intensity projections). Boxed areas are magnified in (A’’,A’’’,B’’,B’’’). Yellow outlines in magnified areas indicate basal body rootlets. Scale bars 5 µm. (C) Graph comparing the average fluorescence intensity (a.u.) within the yellow outlines in (A’’’,B’’’) between DMSO and 2-APB treatments. (DMSO: 1043 ± 410; 2-APB: 548 ± 237). (D) Rootlet/cell background ratio of GFP-rGBD fluorescence compared between DMSO and 2-APB treatments. (DMSO: 1.67 ± 0.14; 2-APB: 1.48 ± 0.14); 24–30 cells from 8 embryos analyzed per condition. Statistical test: Mann–Whitney Test. (E,F) Maximum intensity projections of apical actin in MCCs expressing (E) GFP-RHOA^Q63L or (F) GFP-RHOA^T19N. (E’,F’) show actin labeled by phalloidin in magenta, and RFP-centrin-labeled basal bodies in green. Scale bars 5 µm. (G–J) Quantification of the apical surface area and the apical actin network of MCCs expressing GFP-RHOA^Q63L or GFP-RHOA^T19N. (G) Apical area (RHOA^Q63L 239.5 ± 49.7 µm²; RHOA^T19N 272.83 ± 64.1 µm²). (H) Number of skeletons (separate actin structures) per µm² (RHOA^Q63L 0.38 ± 0.10; RHOA^T19N 0.52 ± 0.10). (I) Average branches per skeleton (RHOA^Q63L 4.9 ± 2.1; RHOA^T19N 3.0 ± 0.9). (J) Length of the apical actin network normalized to surface area (RHOA^Q63L 1.09 ± 0.15 µm⁻¹; RHOA^T19N 0.91 ± 0.15 µm⁻¹). Error bars show mean and SD; 24–27 cells from 8 embryos analyzed per condition. Statistical test: Mann–Whitney Test.

Figure 7

Apical actin truncation due to IP3R inhibition is rescued by constitutively active RhoA: (A–H) Maximum intensity projections of apical actin and quantification thereof in MCCs treated with DMSO or 2-APB and expressing constitutively active RHOA^Q63L. Scale bars 5 µm. (E) Apical surface area (DMSO control: 263.3 ± 78.2 µm²; 2-APB control: 402.1 ± 112.2 µm²; DMSO RHOA^Q63L: 255.5 ± 65.6 µm²; 2-APB RHOA^Q63L: 254.5 ± 79.7 µm²). (F) Number of skeletons (separate actin structures) per µm² (DMSO control: 0.44 ± 0.11; 2-APB control: 0.84 ± 0.17; DMSO RHOA^Q63L: 0.46 ± 0.10; 2-APB RHOA^Q63L: 0.46 ± 0.12). (G) Average branches per skeleton (DMSO control: 4.3 ± 1.7; 2-APB control: 1.9 ± 0.6; DMSO RHOA^Q63L: 4.1 ± 1.1; 2-APB RHOA^Q63L: 4.2 ± 1.7). (H) Length of the apical actin network normalized to surface area (DMSO control: 1.12 ± 0.13 µm⁻¹; 2-APB control: 0.75 ± 0.20 µm⁻¹; DMSO RHOA^Q63L: 1.09 ± 0.16 µm⁻¹; 2-APB RHOA^Q63L: 1.07 ± 0.18 µm⁻¹). Error bars show mean and SD; 31–51 cells from 10 to 16 embryos analyzed per condition. Statistical test: 2-way ANOVA + Tukey’s multiple comparisons test. **** p < 0.0001. (I) Model depicting the role of calcium, myosin, and RhoA in the maturation of the apical actin network. Green arrows symbolize the apical contraction following a calcium burst. Apical actin is depicted skeletonized in pink.