Research ArticleCELL BIOLOGY

Superresolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth

See allHide authors and affiliations

Science Advances  13 Nov 2015:
Vol. 1, no. 10, e1500947
DOI: 10.1126/sciadv.1500947
  • Fig. 1 Dynamic localization of TeaR clusters.

    (A) Existing model of cell polarity establishment. Left, from the membrane transport point of view; right, from the protein point of view. (B) Wide-field images of the movement of the mEosFPthermo-TeaR signal along the growth axis (top) and along the apical membrane (bottom) of growing hyphae. Representative images from two time points marked by triangles are shown (t = 46 and 146 s). Scale bar, 1 μm. The fluorescence intensity changes at the apex (marked by yellow lines in the kymographs) are plotted below each kymograph. The intensity scale of the kymographs has been inverted for clarity. (C) Colocalization of mCherry-BglA and GFP-TeaR along the plasma membrane near the apex of a growing hypha; the elapsed time is given in minutes. Scale bar, 2 μm. (D) Comparison of TeaR (red) and MT (green) localization (0 to 16 s from movie S1); elapsed time in seconds. (E) Kymograph measured from movie S1 along the apical plasma membrane. Vertical arrow, 10 s.

  • Fig. 2 Fluorescence imaging of mEosFPthermo-TeaR clusters.

    (A and B) Images of mEosFPthermo-TeaR clusters acquired by (A) wide-field microscopy (sum of raw images for PALM) and (B) PALM (500 frames). Scale bar, 1 μm. (C) Comparison of the FWHM values from the wide-field (A) and PALM (B) images, shown by plotting the intensity profiles along the dotted line in fig. S1A. (D) TeaR clusters were quantified by cluster analysis (similar to Ripley’s K) applied to PALM images (500 frames each). To define a cluster, the number of surrounding molecules within a specified radius, r = 50 nm, was quantified and filtered by setting a threshold on the minimum number of molecules, n = 10. (E) Distribution of diameters from cluster analysis (ncluster = 80). The line represents a Gaussian fit; center position and FWHM are quoted in the graph. (F) An exponential fit of the distribution of estimated numbers of molecules; the error was given by the fit.

  • Fig. 3 TeaR clusters are regulated by TeaA and MT.

    (A) Fluorescence images of TeaR (left; PALM image, 200 frames) and MTs (middle; wide-field microscopy image, sum of raw images from the GFP channel) and the merged image (right). MTs were manually traced from the wide-field image (middle) and are shown as dashed lines over the TeaR image (left) to guide the eye. The signal in the green channel predominantly originates from GFP-MTs because of their overwhelmingly higher concentration (in diffusing dimers and filament forms) and not from the green fluorescent species of mEosFPthermo (see fig. S2A). Scale bar, 2 μm. (B) TeaR clusters were observed by wide-field epifluorescence microscopy in wild type (WT; top), ΔteaA (middle), or ΔalpA (bottom). Kymograph of the movement of the mEosFPthermo-TeaR signal along the apical membrane. (C) Fluorescence intensity along the apex of the growing hypha in (B).

  • Fig. 4 TeaR clusters define the growth zone.

    (A) Left column: Series of PALM images of a mEosFPthermo-TeaR–expressing hypha (5-min time interval, 500 frames each). Cell profiles are shown in different line styles. Right column: Overlays of PALM images from two time points (top, 0 + 5 min; middle, 5 + 10 min) and overlay of outlines reveal growth regions coinciding with TeaR cluster locations. (B) Overlay of cell profiles from a series of five PALM images of a mEosFPthermo-TeaR–expressing hypha (75-s time interval, 1500 frames each; see also fig. S3). (C) A plot of the maximum Y value from cell profiles shown in (B) shows overall cell extension. (D) Top: Comparison of cell profiles of the first and the second image in the time series. Bottom: The difference of the profiles represented in a line plot and a color map shows the subregion where the growth has taken place. Hot (red, yellow) and cold (blue, green) colors indicate regions of large and small cell extension, respectively. (E) Overlay of two successive PALM images (color and frame number indicated at the top corner). Colocalized regions will appear in white with this combination of colors. The difference of the cell profiles is shown as a color map below each set of overlaid images. (F) Reconstructed PALM image of a mEosFPthermo-TeaR–expressing hypha (1500 frames). (G) Sequence of close-ups of the region marked by a square in (F), reconstructed by moving-window binning (250 frames each with a shift increment of 50 frames or 2.5 s). They show the appearance of a new cluster (white triangle, t = 5.0 s), a translational movement (t = 10.0 s), and a spreading of the signal along with a slight shift of the pattern. (H) Overlay of the first (red) and the last (green) frame shown in (G) shows a small membrane growth. (I and J) The total lifetimes of the TeaR cluster (I) and the docking time (J) were quantified from the images. Scale bars, 1 μm (A and F); 300 nm (E, G, and H).

  • Fig. 5 Recruitment of a downstream polarity complex triggered by the MT arrival.

    (A) Top left: Wide-field fluorescence images of actin cables (green) and MTs (red). Scale bar, 5 μm. Top right: Kymograph along the apex of the growing hypha. Scale bar, 10 s. Bottom: Fluorescence intensity of actin cables (green) and MTs (red) along the apex of the growing hypha between dotted lines in (top right). (B) Kymograph of TeaR concentration at the hyphal tip generated by a simulation with part (top) or all (bottom) of the secretory vesicle population containing TeaR. (C) Five-second snapshots of the results in (B) viewed from above (top) and from the side of a hyphal tip (bottom; compare with Fig. 4). (D) Cartoon representation of the transient polarity model.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/10/e1500947/DC1

    Materials and Methods

    Fig. S1. The size quantifications and three-dimensional PALM imaging of TeaR clusters.

    Fig. S2. Simultaneous epifluorescence and PALM imaging.

    Fig. S3. Distribution of TeaR cluster at different time scales.

    Fig. S4. Modeling of TeaR movement on the plasma membrane.

    Fig. S5. Modeling of the actin and MT dynamics.

    Fig. S6. Systematic evaluations of modeling parameters.

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

    Table S2. Parameters used in the hyphal tip simulations.

    Movie S1. Dual-color wide-field fluorescence movie of mEosFPthermo-TeaR and GFP-MT in a growing filamentous fungus (Fig. 1D).

    Movie S2. PALM movie of mEosFPthermo-TeaR prepared from the moving-window binning images shown in Fig. 4, F to H.

    References (4851)

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. The size quantifications and three-dimensional PALM imaging of TeaR clusters.
    • Fig. S2. Simultaneous epifluorescence and PALM imaging.
    • Fig. S3. Distribution of TeaR cluster at different time scales.
    • Fig. S4. Modeling of TeaR movement on the plasma membrane.
    • Fig. S5. Modeling of the actin and MT dynamics.
    • Fig. S6. Systematic evaluations of modeling parameters.
    • Table S1. A. nidulans strains used in this study.
    • Table S2. Parameters used in the hyphal tip simulations.
    • Legends for movies S1 and S2
    • References (48–51)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Dual-color wide-field fluorescence movie of mEosFPthermo-TeaR and GFP-MT in a growing filamentous fungus (Fig. 1D).
    • Movie S2 (.avi format). PALM movie of mEosFPthermo-TeaR prepared from the moving-window binning images shown in Fig. 4, F to H.

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article