Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells

Abstract

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 μm s^-1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 μm s^-1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension.

Fig. 1. Superresolution imaging of Spitzenkörper dynamics.

(A) Localization image of a hypha with mEosFPthermo-ChsB clusters (500 frames). (B) Top: Image of the hyphal tip; bottom: ChsB clusters identified by cluster analysis. (C) Sequence of ChsB cluster images (clusters in different colors) rendered from images reconstructed by moving-window binning. (D) Time courses of total cluster areas. (E) Number of molecules. Lines are drawn in colors corresponding to the clusters in (C). Distributions of (F) cluster area and (G) number of molecules from all 80 identified clusters. (H) Sequence of images from cluster analysis. (I) Overlay of the corresponding tip profiles. Asterisks indicate large extensions of the apical membrane and shape change of the clusters. (J) Merged image of the image series in (H). Scale bars, 1 μm (A) and 300 nm (B, C, H, and J).

Fig. 2. Pulse-chase analysis of mEosFPthermo-ChsB in the hyphal tip region.

(A) Images of mEosFPthermo-ChsB before photoconversion (−1), with 405-nm light applied at the spot marked by the dashed line for 1 s (0) and after photoconversion. (B) Kymograph calculated from (A); arrows indicate anterograde and retrograde transport. The blue dashed line and the red asterisk mark the positions of the hyphal tip and the photoconversion locus, respectively; the red square indicates the photoconversion interval. (C) Image sequence from 4.6 to 7 s after photoconversion; arrows mark the transport processes in (B) in corresponding colors. (D) Kymographs of slow (red) and fast (blue) transport of mEosFPthermo-ChsB. Vertical scale bar, 2 μm; horizontal scale bar, 1 s. (E) Image sequences of the fast transport process marked by the blue arrow in (D) observed from 16.45 to 17 s. (F) Speed distribution of anterograde transport. (G) Speed of slow anterograde (red), fast anterograde (blue), and retrograde (green) transport (mean ± SD; n = 42, 7, and 9, respectively). Scale bars, 2 μm. The elapsed time is given in seconds.

Fig. 3. Pulse-chase analysis of mEosFPthermo-ChsB in the hyphal tip regions of cells from motor mutants.

(A to C) Left: Image sequence upon photoconversion at t = 0 s for 1 s in a spot ~5 μm behind the hyphal tip (dashed circles in panels “–1”) of (A) ΔkinA, (B) ΔuncA, and (C) ΔmyoV. The elapsed time is given in seconds. Scale bars, 2 μm. Right: Corresponding kymographs; arrows indicate anterograde (blue) and retrograde (red) transport. Blue dashed lines and asterisks mark the positions of hyphal tips and photoconversion loci, respectively; red squares indicate the photoconversion intervals. Vertical scale bars, 2 μm. (D) Fluorescence intensities at (top) hyphal tips and (bottom) photoconversion loci from wild-type (WT) (black), ΔkinA (green), ΔuncA (red), and myoV (green) cells, averaged and plotted as mean ± SD (n = 3 to 5). (E) Number of anterograde (blue) and retrograde (red) transport events in six kymographs of WT, ΔuncA, and ΔmyoV. (F) Speed of anterograde (blue) and retrograde (red) transport in ΔuncA and ΔmyoV (mean ± SD). (G) Distribution of speeds in anterograde transport from WT (black), ΔuncA (red), and ΔmyoV (green) samples. a.u., arbitrary units.

Fig. 4. Pulse-chase analysis after mEosFPthermo-ChsB photoconversion at the hyphal tip.

(A to D) Left: Image sequences upon photoconversion at t = 0 s for 1 s at the hyphal tips (dashed circles in panels “–1”) of (A) a WT cell without drugs (as a control) and in the presence of (B) benomyl (Benm), (C) cytochalasin A (CytoA), and (D) a cell from the ΔuncA strain. The elapsed time is given in seconds. Right: Corresponding kymographs; blue dashed lines and asterisks mark the positions of hyphal tips and photoconversion loci, respectively; red squares indicate the photoconversion intervals. (E) Enlarged kymographs of WT (left) and ΔuncA (right) cells; vertical scale bar, 2 μm; horizontal scale bar, 1 s. Arrows show anterograde (blue) and retrograde (red) transport events; blue dashed lines indicate the hyphal tip positions. (F) Number of anterograde (blue) or retrograde (red) transport events in 20 kymographs of the control, with benomyl, and with cytochalasin A. (G) Speeds of anterograde (blue) and retrograde (red) transport in WT and ΔuncA (mean ± SD). (H) Time courses of signal intensity ratio between hyphal tip area and subapical area from a WT cell without drugs (control) and in the presence of benomyl or cytochalasin A, and in the ΔuncA strain. Further details are given in fig. S3. (I) Number of anterograde (blue) or retrograde (red) transport events from five kymographs of WT and ΔuncA cells. MT, microtubule.

Fig. 5. Images of fungal colonies and schematic depictions of ChsB transport in WT, ΔkinA, ΔmyoV, and ΔuncA strains.

(A) Images of the colonies; scale bar (for all images), 5 mm. Strains were grown on the same minimal medium glucose agar plate for 2 days. (B) Depictions of ChsB transport processes in the four strains (for details, see the text); the symbol legend is included below. SPB, spindle pole body.