Pulses of Ca2+ coordinate actin assembly and exocytosis for stepwise cell extension

Abstract

Many eukaryotic cells grow by extending their cell periphery in pulses. The molecular mechanisms underlying this process are not yet fully understood. Here we present a comprehensive model of stepwise cell extension by using the unique tip growth system of filamentous fungi. Live-cell imaging analysis, including superresolution microscopy, revealed that the fungus Aspergillus nidulans extends the hyphal tip in an oscillatory manner. The amount of F-actin and secretory vesicles (SV) accumulating at the hyphal tip oscillated with a positive temporal correlation, whereas vesicle amounts were negatively correlated to the growth rate. The intracellular Ca²⁺ level also pulsed with a positive temporal correlation to the amount of F-actin and SV at the hyphal tip. Two Ca²⁺ channels, MidA and CchA, were needed for proper tip growth and the oscillations of actin polymerization, exocytosis, and the growth rate. The data indicate a model in which transient Ca²⁺ pluses cause depolymerization of F-actin at the cortex and promote SV fusion with the plasma membrane, thereby extending the cell tip. Over time, Ca²⁺ diffuses away and F-actin and SV accumulate again at the hyphal tip. Our data provide evidence that temporally controlled actin polymerization and exocytosis are coordinated by pulsed Ca²⁺ influx, resulting in stepwise cell extension.

Keywords: Aspergillus; actin; calcium; filamentous fungi; oscillation.

Fig. 1.

Stepwise growth of hyphae. (A) DIC images of growing hyphae. Wild-type A. nidulans was grown in minimal media at room temperature. The elapsed time is given in seconds. (Scale bar, 10 μm.) (B) Kymograph of the growing hypha in A along the growth axis. Total 600 s. (Scale bar, 5 μm.) (C) The length traveled by the hyphal apex over a 20-s interval in the growing hypha shown in A is shown in total period of 600 s. (D) Sequence of PALM images of mEosFPthermo-ChsB reconstructed by moving window binning (500 frames each with a shift increment of 50 frames or 2.5 s, total 140 s). (Scale bar, 1 µm.) (E) Overlay of cell profiles from a series of six PALM images expressing hypha. Larger extensions of the apical membrane were indicated by arrows. (Scale bar, 300 nm.) (F) Fluorescence image sequence of F-actin and the SV visualized by GFP-TpmA and mCherry-ChsB, respectively, in the growing hypha. The elapsed time is given in seconds. (Scale bar, 2 μm.) (G) Kymographs of F-actin (green) and SV (red) along the growth axis in the growing hypha in F. Total 180 s. (H) Fluorescence intensity of F-actin (green) and SV (red) along the apex of the growing hypha between dotted lines in G. (I) Normalized cross-correlation of F-actin signal (GFP-TpmA) and SV signal (mCherry-ChsB).

Fig. S1.

Stepwise growth of hyphae. (A) Kymograph of the growing hypha along the growth axis (Left) and enhanced image (Right). Total 60 min. (Scale bar, Left, 10 μm.) (B) The length of the interval traveled by the hyphal apex along the line in overlay of cell profiles from a series of six PALM images expressing hypha (Fig. 1E). Larger extensions of the apical membrane were indicated by arrows. (Scale bar, 300 nm.) (C) Cross-correlation between GFP-TpmA and mCherry-ChsB. To account for the effect of photobleaching during imaging (3 min), the baseline of each signal was defined as a line with negative slope, based on the average value of first and second half of the signal. The cross-correlation was then calculated based on the corrected signal. (D) Fluorescence intensity of secretory vesicles (red) along the apex of the growing hypha between dotted lines in (Fig. 1G). The length of the interval traveled by the hyphal apex was measured every 20 s. (E) The position of Spitzenkörper was aligned every 3 s from Movie S3.

Fig. 2.

Negative correlation between SV accumulation and the cell-extension rate in N. crassa. (A) Fluorescence image sequence of SV visualized by CHS-1-GFP in growing hypha of N. crassa. The elapsed time is given in seconds. (Scale bar, 10 μm.) (B) Kymograph of SV accumulation along the growth axis in the growing hypha in A. Total 300 s. (Scale bar, 10 μm.) (C) Fluorescence intensity of SV along the apex of the growing hypha between dotted lines in B. Arrows and numbers indicate the time of peaks. (D) Distribution of interval between two peaks. n = 30 in 3 hyphae. (E) Fluorescence intensity of the SV at the apex (green) and growth rate (gray) every 2 s. The growth rates were calculated by distance of tip position between 2 s. (F) Normalized cross-correlation of SV signal (CHS-1–GFP) and cell-extension rate.

Fig. 3.

Oscillation of Ca²⁺ influx correlated with F-actin and the SV. (A) Fluorescence image sequence of R-GECO. The elapsed time is given in seconds. (Scale bar, 5 μm.) (B) Kymograph along the hypha shown in A. Total 120 s. (Scale bar, 5 μm.) (C) Fluorescence intensity around the tip of hypha in B is plotted. Arrows and numbers indicate the time of Ca²⁺ peaks. (D) Distribution of intervals between two peaks. n = 36 in 10 hyphae. (E) Fluorescence images and kymographs along the growth axis of F-actin (GFP-TpmA; green) and Ca²⁺ (R-GECO; red). Total 180 s. (Scale bar, 2 μm.) (F) Fluorescence intensity of F-actin (green) and Ca²⁺ (red) along the apex of the growing hypha between dotted lines in E. (G) Normalized cross-correlation of F-actin signal (GFP-TpmA) and Ca²⁺ signal (R-GECO). (H) Fluorescence images and kymographs along the growth axis of SV (BglA-GFP; green) and Ca²⁺ (R-GECO; red). Total 180 s. (Scale bars, 1 μm.) (I) Fluorescence intensity of SV (green) and Ca²⁺ (red) along the apex of the growing hypha between dotted lines in H. (J) Normalized cross-correlation of SV signal (BglA-GFP) and Ca²⁺ signal (R-GECO).

Fig. S2.

Intracellular Ca²⁺ level by R-GECO. (A) Scheme of the Ca²⁺ biosensor, R-GECO. (B) Fluorescence image sequence of R-GECO. The elapsed time is given in seconds. (Scale bar, 5 μm.) (C) The time-lapse signal intensity of R-GECO at different points from the tip shown in B. (D) Fluorescence image sequence of R-GECO in the three hyphae. Arrows indicate hypha showing the R-GECO signal with different timing. The elapsed time is given in seconds. (Scale bar, 10 μm.)

Fig. S3.

Effect of CaCl₂ on the oscillation of R-GECO. (A and B) Fluorescence image of R-GECO in the hypha growing in the media without CaCl₂ (A) and 1 μM CaCl₂ + 10 mM EGTA (B). (Scale bars, 10 μm.) Kymographs (Lower) along the hyphae. Total 240 s, every 2 s. (Scale bars, 1 μm.) (C, Upper) Hyphal morphology of the cchA-deletion strain. (Scale bar, 20 μm.) (Lower) Colonies of wild-type, midA-deletion and cchA-deletion strain grown on the minimal media plate for 3 d. (D and E) Fluorescence image of R-GECO and GFP-TpmA, in the cchA-deletion hyphae growing in the media with 1 μM CaCl₂ (D), and in the wild-type hypha growing in the media with 10 mM CaCl₂ (E). (Scale bars, 5 μm.) (Lower) Kymographs along the hypha. Total 240 s, every 2 s. (Scale bars, 1 μm.) (F) Fluorescence image of R-GECO and GFP-TpmA in the wild-type hypha growing in the media with 1 μM CaCl₂ after the treatment of calcium ionophore A23187 (Sigma) 5 μg/mL for 30 min. (Scale bars, 5 μm.)

Fig. 4.

Deletion of Ca²⁺ channel. (A) Hyphal morphology of the midA deletion strain. (Scale bar, 20 μm.) (B) Fluorescence images of GFP-TpmA and R-GECO in the midA-deletion hyphae growing in the media with 1 μM CaCl₂. (Scale bar, 10 μm.) (C) The length traveled by the hyphal apex over a 20-s interval in the wild-type and midA-deletion strain. Total period of 600 s. (D) Fluorescence images of GFP-TpmA and R-GECO in the midA-deletion hyphae growing in the media with 10 mM CaCl₂ (Upper) and time course of R-GECO (Left). (Scale bars, Upper and Right, 5 μm.) Kymographs along the hyphae in B and D. Total 240 s, every 2 s. (Scale bar, Bottom, 1 μm.)

Fig. 5.

Scheme of oscillation in fungal tip growth coordinated by Ca²⁺ influx.