Research ArticleCELL BIOLOGY

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

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Science Advances  24 Jan 2018:
Vol. 4, no. 1, e1701798
DOI: 10.1126/sciadv.1701798
  • 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.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/1/e1701798/DC1

    Supplementary Materials and Methods

    fig. S1. Transport of EEs and SVs.

    fig. S2. Pulse-chase analysis of mEosFPthermo-ChsB near the hyphal tip in the presence of microtubule or actin-depolymerizing drugs.

    fig. S3. Quantitative analysis of mEosFPthermo-ChsB numbers in the hyphal tip and expression levels.

    fig. S4. Pulse-chase analysis upon mEosFPthermo-ChsB photoconversion at the hyphal tip.

    movie S1. Superresolution movie of mEosFPthermo-ChsB clusters generated with the moving-window binning technique (500 frames binning with 50 frames shift).

    movie S2. Dynamics of ChsB clusters identified by cluster analysis.

    movie S3. Pulse-chase imaging of mEosFPthermo-ChsB in the wild-type hyphal tip region with local photoconversion ~5 μm behind the hyphal tip.

    movie S4. Transport of EEs.

    movie S5. Pulse-chase imaging of mEosFPthermo-TeaR in the wild-type hyphal tip region with a local photoconversion ~5 μm behind the hyphal tip.

    movie S6. Partial comigration of GFP-ChsB and mCherry-RabA.

    movie S7. Pulse-chase imaging of mEosFPthermo-ChsB near the hyphal tip in the presence of microtubule drugs (benomyl, 2 μg/ml).

    movie S8. Pulse-chase imaging of mEosFPthermo-ChsB near the hyphal tip in the presence of actin-depolymerizing drugs (cytochalasin A, 2 μg/ml).

    movie S9. Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the kinA deletion strains.

    movie S10. Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the uncA deletion strains.

    movie S11. Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the myoV deletion strains.

    movie S12. Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip of a wild-type cell.

    movie S13. Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip in the presence of microtubule drugs (benomyl, 2 μg/ml).

    movie S14. Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip in the presence of actin-depolymerizing drugs (cytochalasin A, 2 μg/ml).

    movie S15. Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip of a cell from the uncA deletion strain.

    table S1. A. nidulans strains used in this study.

    Reference (51)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Transport of EEs and SVs.
    • fig. S2. Pulse-chase analysis of mEosFPthermo-ChsB near the hyphal tip in the presence of microtubule or actin-depolymerizing drugs.
    • fig. S3. Quantitative analysis of mEosFPthermo-ChsB numbers in the hyphal tip and expression levels.
    • fig. S4. Pulse-chase analysis upon mEosFPthermo-ChsB photoconversion at the hyphal tip.
    • Legends for movies S1 to S15
    • table S1. A. nidulans strains used in this study.
    • Reference (51)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi file). Superresolution movie of mEosFPthermo-ChsB clusters generated with the moving-window binning technique (500 frames binning with 50 frames shift).
    • movie S2 (.avi file). Dynamics of ChsB clusters identified by cluster analysis.
    • movie S3 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB in the wild-type hyphal tip region with local photoconversion ~5 μm behind the hyphal tip.
    • movie S4 (.avi file). Transport of EEs.
    • movie S5 (.avi file). Pulse-chase imaging of mEosFPthermo-TeaR in the wild-type hyphal tip region with a local photoconversion ~5 μm behind the hyphal tip.
    • movie S6 (.mov file). Partial comigration of GFP-ChsB and mCherry-RabA.
    • movie S7 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB near the hyphal tip in the presence of microtubule drugs (benomyl, 2 μg/ml).
    • movie S8 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB near the hyphal tip in the presence of actin-depolymerizing drugs (cytochalasin A, 2 μg/ml).
    • movie S9 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the kinA deletion strains.
    • movie S10 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the uncA deletion strains.
    • movie S11 (.avi file). Pulse-chase imaging of mEosFPthermo-ChsB in the hyphal tip region of the cell from the myoV deletion strains.
    • movie S12 (.avi file). Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip of a wild-type cell.
    • movie S13 (.avi file). Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip in the presence of microtubule drugs (benomyl, 2 μg/ml).
    • movie S14 (.avi file). Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip in the presence of actin-depolymerizing drugs (cytochalasin A, 2 μg/ml).
    • movie S15 (.avi file). Pulse-chase imaging after mEosFPthermo-ChsB photoconversion at the hyphal tip of a cell from the uncA deletion strain.

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