Fig 1.
A subresolution imaging method to monitor CW thickness dynamics in live fungal hyphae.
(A) Mid-slice confocal image of a live Aspergillus nidulans hyphal cell expressing a GFP-PH domain (plasma membrane, CW inner surface) and labeled with the lectin ConA-Alexafluor 647 (CW outer surface). (B) Scheme of CW organization showing different polysaccharides and positions of the two fluorescent signals. (C) Gaussian fits of the signal distribution of each fluorophores across the CW. The distance between the two Gaussians peaks allows to compute a local value of CW thickness, h. (D) Resulting CW thickness color map around the live cell presented in panel A. (E) Top: Mid-slice confocal image of a germling tube, with the spore body visible on the left. Bottom: corresponding CW thickness color map. Right: Measured CW thickness profile along the cell plotted as a function of the arclength distance, s, with s = 0 being the center of the cell tip (marked with an arrowhead). (F) Measurement of CW thickness, marked with a double arrow, in electron microscopy, using chemical fixation (left) or HPF (right). (G) CW thicknesses measured using our live-microscopy method (n = 81 cells) and electron microscopy from chemically fixed (n = 8) or high pressure–frozen cells (n = 8). (H) Time lapse of CW thickness maps, in relatively slow elongating germling tube and a faster mature hypha. Scale bars: (A, E, H): 2 μm. (F) top: 2 μm, bottom: 100 nm. Error bars correspond to +/− SD. Results were compared by using a two-tailed Mann–Whitney test. n.s, P > 0.05. The data underlying the graphs can be found in S1 Data. CW, cell wall; EM, electron microscopy; HPF, high pressure freezing; PM, plasma membrane.
Fig 2.
Spatial gradients of CW elasticity along fungal hyphae.
(A) Left: Distribution of the ratio of the CW thickness at cell tips to that on cell sides, with two exemplary thickness color maps of cells with different thickness polarity. Right: CW thickness gradient along the cell contour, using a symmetrized arclength distance, s’, as coordinate (s’ = 0 being the tip) (n = 58 cells). (B) Method used to compute local CW Young’s modulus around hyphal cells. Left: Bright-field images of the same cell, before (top) and after (bottom) photoablation, with measured CW thickness map before ablation. The asterisk marks the site of photoablation, and the arrow points at cytoplasmic material leaking out of the cell. Right: Segmented CW boundaries of the same cell before and after ablation used to compute the local elastic strain and deduce local values of CW Young’s elastic modulus divided by pressure, from values of thickness and elastic strains. (C) Elastic strain of the lateral CW measured as the relative radial shrinkage for osmotic shocks of different magnitudes, and compared with the value obtained from CW photoablation assays (blue dotted lines) (n > 13 cells for each osmolyte concentration). The intersection of the two curves provides an estimate of the external molarity needed to reduce turgor to zero, and thus an estimate of turgor pressure. (D-F) Distribution of CW thickness, h, Young’s modulus, Y, and surface modulus, σ = hY along the hyphae, as defined in the scheme (n > 38 cells for the CW thickness, n > 7 cells for Y and σ). Scale bar, 2 μm. Error bars correspond to +/− SD. Results were compared by using a two-tailed Mann–Whitney test. n.s, P > 0.05; **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. The data underlying the graphs can be found in S1 Data. CW, cell wall.
Fig 3.
Spatial distribution of downstream regulators of CW assembly and CW thickness profiles.
(A-C) Distribution of different tagged polar regulators of CW assembly, together with CW thickness profiles, for Myosin type V: MyoV-mCherry (A), the chitin synthase, mCherry-ChsB (B), and the post-Golgi vesicle labeling GTPase mCherry-RAB11 (C). In B, the profile of CW thickness and signal of mCherry-ChsB are plotted as a function of the arclength (s) and fitted with Gaussians to compute the FWMH, for both distributions (double arrows). (D) FWMH for each protein fluorescent signal and corresponding FWMH of the CW thickness profile, with individual cells connected by black lines. (E) FWMH distribution of different polar factors and CW thickness (n = 16, 13, 29, and 25 cells). (F) Images of the EVs marker mCherry-RAB11 in a WT and in a myoVΔ mutant cell. (G) Hyphal radius, FWMH of mCherry-RAB11 and CW thickness profiles and mean values of tip CW thickness for WT and myoVΔ mutant (n = 49, 30 cells). Scale bars, 2 μm. Error bars correspond to +/− SD. Results were compared by using a two-tailed Mann–Whitney test. ****, P < 0.0001. The data underlying the graphs can be found in S1 Data. CW, cell wall; FWMH, full width at mid height; WT, wild-type.
Fig 4.
Dynamic tracking of post-Golgi EVs together with growth and CW thickness.
(A) Time lapse of a growing WT hypha, with CW thickness maps overlaid on mCherry-RAB11 signal. (B) Quantification of the time evolution of the concentration of post-Golgi EVs at cell tips, tip elongation speed, and CW thickness. (C) Relative Stds computed over multiple time lapses for growth speed, CW thickness on cell sides and cell tips, and EVs intensity (n = 10 time lapses in different cells). Scale bar, 2 μm. Error bars correspond to +/− SD. The data underlying the graphs can be found in S1 Data. CW, cell wall; EV, exocytic vesicle; Std, standard deviation; WT, wild-type.
Fig 5.
Dynamic cotracking of EVs, CW thickness, and deformation during abrupt changes in cell growth or secretion.
(A) Scheme of a hyphal fungal tip, highlighting key assumptions of the mathematical model for tip growth: EVs cluster in the Spitzenkörper and radiate to promote the secretion of CW polysaccharides as well as remodeling enzymes. This allows the CW to thicken as well as deform under stresses created by turgor, at a strain rate, G. The pool of EVs is fed by polarized trafficking and other sources of recycling in proportion to the strain rate of the CW at cell tips (mechanical feedback). (B) Time lapse of EV accumulation (mCherry-RAB11) and CW thickness during a lateral branching event. (C) Dynamics of EVs concentration, CW strain rate, and thickness in branching cells (n = 7) and corresponding model outputs. The origin of time is defined as the first visible emergence of the new branch. (D) Time lapse of a growing cell rinsed at time t = 0 with medium supplemented with 0.2 M sorbitol to reduce turgor pressure. (E) Dynamics of EVs concentration, CW strain rate, and thickness before and after the osmotic shock (n = 10) and corresponding model outputs. The inset in the top curve represents how turgor was modulated in the model to simulate the initial rapid drop and subsequent adaption. (F) Time lapse of a cell treated with benomyl to depolymerize microtubules, displaying CW thickness profiles overlaid with EVs. (G) Dynamics of EVs concentration, CW strain rate, and thickness before and after benomyl addition (n = 5) and corresponding model outputs. (H) Time lapse of a temperature-sensitive sarA6 ts mutant cell switched to restrictive temperature at time 0, showing the CW thickness profiles overlaid with EVs. (I) Dynamics of EVs concentration, CW strain rate, and thickness in sarA6 ts cells at restrictive temperature (n = 8) and corresponding model outputs. Scale bars, 2 μm. In experimental plots presented in 5C, 5E, 5G, and 5I, the black line represents the mean of the data and the blue shade the standard deviation. The data underlying the graphs can be found in S1 Data. CW, cell wall; EV, exocytic vesicle; PM, plasma membrane.
Fig 6.
CW mechanics and secretion during steady-state hyphal growth at different elongation speeds.
(A) Time lapses of EVs (mCherry-RAB11) and CW thickness map of mature hyphae, germling tubes, and myoVΔ cells, with close-up views on EVs distribution at cell tips. (B-G) Measured geometrical, mechanical, and biochemical parameters for mature hyphae, germling tubes, and myoVΔ cells (n > 10 cells for each conditions): Tip radius (B); Tip CW strain rate (C); Turgor pressure (D); Tip CW thickness (E); Tip CW young’s modulus (F); and EVs tip concentration (G). (H-J) Model outputs in term of strain rates, CW thicknesses, and EVs tip concentration for the three steady-state hyphal growth at different elongation speeds. Scale bars, 2 μm. Error bars correspond to +/− SD for all panels, expect for turgor values in 6D in which they correspond to estimated errors from computing turgor by intersecting elastic strains obtained from laser ablation of the CW to that from ranges of osmotic shocks. The data underlying the graphs can be found in S1 Data. CW, cell wall; EV, exocytic vesicle.