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Cell wall dynamics stabilize tip growth in a filamentous fungus [1]

['Louis Chevalier', 'Université Paris Cité', 'Cnrs', 'Institut Jacques Monod', 'Paris', 'Equipe Labellisée Ligue Contre Le Cancer', 'Mario Pinar', 'Department Of Cellular And Molecular Biology', 'Centro De Investigaciones Biológicas Margarita Salas', 'Madrid']

Date: 2023-01

Hyphal tip growth allows filamentous fungi to colonize space, reproduce, or infect. It features remarkable morphogenetic plasticity including unusually fast elongation rates, tip turning, branching, or bulging. These shape changes are all driven from the expansion of a protective cell wall (CW) secreted from apical pools of exocytic vesicles. How CW secretion, remodeling, and deformation are modulated in concert to support rapid tip growth and morphogenesis while ensuring surface integrity remains poorly understood. We implemented subresolution imaging to map the dynamics of CW thickness and secretory vesicles in Aspergillus nidulans. We found that tip growth is associated with balanced rates of CW secretion and expansion, which limit temporal fluctuations in CW thickness, elongation speed, and vesicle amount, to less than 10% to 20%. Affecting this balance through modulations of growth or trafficking yield to near-immediate changes in CW thickness, mechanics, and shape. We developed a model with mechanical feedback that accounts for steady states of hyphal growth as well as rapid adaptation of CW mechanics and vesicle recruitment to different perturbations. These data provide unprecedented details on how CW dynamics emerges from material secretion and expansion, to stabilize fungal tip growth as well as promote its morphogenetic plasticity.

Funding: This work was supported by grants from the "Fondation de la Recherche Médicale" (n°13171) to L.C., the "Spain’s Ministerio de Ciencia e Innovación" (grant RTI2018-093344-B100) and the "Comunidad de Madrid and he European Regional Development and European Social Funds" (grant S2017/BMD-3691) to M.A.P, the "La Ligue Contre le Cancer" (EL2021.LNCC/ NiM) and the "European Research Council" (ERC CoG “Forcaster” no. 647073) to N.M., as well as the "Agence Nationale pour la Recherche" (ANR, “CellWallSense” no. ANR-20-CE13-0003-02) to N.M. and A.B. "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Here, we used Aspergillus nidulans, an established tractable model fungus that features rapid hyphal growth [ 42 , 43 ], to understand in quantitative terms how CWs are built and remodeled from EV pools during tip growth. We developed a super-resolution live imaging method to map CW thickness around growing hyphal cells, which we combine with the dynamic imaging of RAB11-labeled EVs, and with biochemical and genetic interventions affecting growth, turgor, trafficking, or secretion. We propose a mathematical model for tip growth that we systematically test and calibrate against dynamic perturbations. Combined with experimental findings, this model suggests the existence of a mechanical feedback from CW growth to vesicle accumulation that accounts for stable steady-state hyphal growth at various elongation speeds.

The composition and assembly of the fungal CW define its material properties that underpin its ability to protect hyphal cells and allow them to grow. CWs have thicknesses that may vary between approximately 50 to 500 nm and bulk elastic moduli of approximately 10 to 100 of MPa, akin to a material like rubber [ 28 , 29 ]. The CW is put under tension by a large cytoplasmic pressure of several atmospheres, called turgor, which is osmotically generated. Turgor serves as a core mechanical engine to deform freshly assembled CW portions at cell tips and, thus, power cell growth, but also entails risk of CW failure and cell death [ 8 ]. CW growth for tip elongation has been modeled in multiple instances. Some models primarily focused on secretory vesicle supplies disregarding contributions from turgor and CW material properties [ 30 – 32 ]. Others have been based on frameworks of visco-elasto-plastic thin shells, assuming that newly assembled CW portions at the apex undergo plastic irreversible deformation above a threshold stress but used simplified descriptions of material supply from secretory vesicles [ 33 – 38 ]. Interestingly, both modeling and experimental work have suggested the existence of mechanical feedbacks, whereby enhanced strain rates in the CW may promote the recruitment or stability of polar secretory domains [ 34 , 39 – 41 ]. Despite the potential predictive power of these models, quantitative comparison with experimental data has been limited. Accordingly, we still lack quantitative models and experiments that enable to understand how the dynamics of CW secretion, expansion, and mechanics may be regulated during fungal growth and shape changes.

Fungal CWs are composed of reticulated polysaccharides including chitin, α- and β-glucan and mannose polymers, as well as remodeling enzymes like hydrolases and transferases [ 6 ]. Post-Golgi RAB11 exocytic vesicles (EVs) are thought to secrete a subset of sugars and proteins into the CW and also to carry transmembrane enzymes to the plasma membrane that catalyze the elongation of other sets of sugars [ 3 , 7 ]. Therefore, secretory vesicles may promote both CW material assembly and extensibility needed to support mechanical stability and growth [ 4 , 8 ]. Vesicles are trafficked toward the hyphal tip along microtubules tracks and recycled via a subapical endocytic ring domain [ 3 , 9 , 10 ]. At cell tips, they are clustered by F-actin and myosin type V motors around a dense reservoir called the Spitzenkörper, thought to be adapted to rapid hyphal growth in many but not all fungal species [ 11 – 18 ]. Secretory vesicles radiate from this local reservoir, through transport and diffusion, to eventually tether and fuse with the plasma membrane and fuel CW assembly [ 17 – 21 ]. Chemical or genetic conditions that affect the polarized trafficking of EVs halt tip growth and often yield to defects in tip shape [ 22 – 24 ]. Accordingly, variations in EVs concentration, apical domain sizes and shapes have been correlated to tip expansion speeds, and diameters in multiple fungi [ 25 – 27 ]. Yet, to date, a detailed assessment of how secretory vesicle pools contribute to actual CW material assembly and expansion in live growing cells is still lacking.

Filamentous fungi are generally nonmotile but exploit fast polar tip growth for surface colonization, mating, or host infection [ 1 ]. In typical vegetative life cycles, for instance, fungal spores germinate to outgrow polarized hyphae that expand rapidly at their tips and undergo branching, turning, and sometimes fusion to generate the complex mycelium network [ 2 , 3 ]. Hyphal cell shape and growth are defined by the dynamic expansion of their cell wall (CW), which surrounds and protects the plasma membrane [ 4 , 5 ]. In general, however, how the CW undergoes such rapid and diverse shape changes while ensuring surface mechanical integrity remains poorly understood.

Results

A spatial gradient of cell wall stiffness is associated to hyphal polar growth Many models for tip growth posit that cell tips shall feature softer and/or thinner CWs to account for polarized CW deformation [51,52]. In mature hyphae, we found that CW thickness exhibited a relatively shallow gradient, with tips being on average only approximately 13% thinner than cell sides. In addition, we noted that a fraction of cells exhibited a reversed pattern, with a thicker CW at cell tips (Fig 2A). Furthermore, inspections of time-lapse sequences taken at one frame per minute suggested that this CW thickness polarity could even become inverted in a time course as short as approximately few minutes, indicative of rapid CW remodeling activity at cell tips (S1 Movie). We conclude that CW thickness gradients may not be sufficient to polarize CW mechanics for tip growth in these cells. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001981.g002 To compute local values of CW elasticity around cells, we built on the analysis of thickness, h, to compute the CW Young’s modulus, Y, which reflects bulk material properties and its surface modulus, σ, which is the product of thickness and Young’s modulus, hY, and represents the apparent CW stiffness [28]. We imaged cells to map CW thickness and rapidly photoablated the CW using a focalized UV laser [34]. This caused the pressurized cytosolic material to flow out of cells within seconds, yielding cell deflation and CW elastic relaxation. CW relaxation allowed to compute a local elastic strain, , with R 0 and R 1 the local cell radii before and after deflation. This first showed that the CW relaxed twice as much along the radial axis as compared to the longitudinal axis of the cell, suggesting relatively low anisotropies in the CW material (S2A Fig) [33]. Second, it allowed to compute local values of Y/P, with P the turgor pressure, from force balance relationships in the pressurized CW, with at cell tips, and for lateral CWs (Figs 2B and S2B) [28,53]. Therefore, in order to compute exact local values of CW elastic moduli, we measured turgor pressure. We assumed turgor to be homogenous within hyphal compartments and monitored shape changes of the CW, as above, but in response to hyperosmotic shocks of different magnitudes. This led to estimates of turgor values of P ~ 1.1 to 1.3 MPa, from the medium osmolarity needed to shrink cells as much as with CW photoablation [53,54] (Figs 2C and S2C). Together, these analyses show that the CW Young’s modulus follows a steep gradient from Y tip = 64 ± 45 MPa at cell tips, up to Y side = 210 +/− 103 MPa on cell sides approximately 10 to 14 μm away from cell tips (Fig 2E) [29]. Combining thickness and Young’s modulus, we obtained local values of CW surface moduli or apparent stiffness (hY) that evolved from 4.3 ± 2.5 N/m at cell tips up to 15.3 ± 6.2 N/m on cell sides (Fig 2F). Such gradients in CW stiffness might reflect spatial differences in the cross-linking of CW components. Therefore, hyphal polar growth in Aspergillus nidulans is accompanied by a steep gradient of surface stiffness, dominated by spatial variations in bulk material properties, with tips CWs being approximately 2 to 3× softer than lateral CWs.

Spatial patterns of secretory vesicle accumulation and cell wall mechanics To assay if these local variations of CW thickness and mechanics at cell tips reflected polarized CW synthesis, vesicle transport, or endocytosis, we used three-color imaging to coimage CW thickness with important regulators of these processes. This included mCherry-labeled type V myosin motor, (MyoV-mCherry), which functions to transport EVs and marks the Spitzenkörper; mCherry-RAB11 to directly visualize the pool of post-Golgi RAB11 EVs; the transmembrane chitin synthase, mCherry-ChsB, which serves as a proxy for CW synthesis; and Lifeact-tdTomato as reporter for F-actin [9,55,56]. As previously reported, Lifeact preferentially labeled endocytic patches along a subapical collar, while all other factors localized to cell tips [10,57] (Figs 3A–3C and S3A). To assay which of these markers may best represent local mechanical variations of tip CWs, we selected cells exhibiting a marked gradient in CW thickness being either thicker or thinner at cell tips and compared the width of the polarity zone formed by different markers with the width of the tip thickness profile. This analysis revealed that the MyoV-mCherry signal was more focused than zones of CW thickness variations, while mCherry-ChsB had a broader distribution. Similarly, the zones delimited by the F-actin endocytic ring were about twice as large as the width of the thickness gradient. Interestingly, mCherry-RAB11, provided the closest width to that of CW thickness gradients (Figs 3D, 3E, S3B and S3C). Accordingly, affecting the distribution of mCherry-RAB11, using a myoVΔ mutant, led to wider cells, with significantly wider distributions in both EV domains and CW thickness gradients at cell tips (Fig 3F and 3G) [15]. Thus, although these results do not rule out contributions from polarized transport, endocytosis, and CW synthesis to both EVs and CW thickness distribution, they suggest that the mCherry-RAB11 signal may be used as a close proxy for CW mechanical changes at cell tips. Together, these analyses directly highlight in living cells the spatial relationships between exocytic carrier distribution and local modulations in CW mechanics [33]. PPT PowerPoint slide

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TIFF original image Download: 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. https://doi.org/10.1371/journal.pbio.3001981.g003

Dynamic coevolution of CW thickness, tip growth and exocytosis, during hyphal tip shape changes We next sought to test the model against abrupt changes in growth or secretion. One first natural instance during which growth or secretion may rapidly evolve in mycelial colonies is de novo tip growth at emerging lateral branches [59,60]. We monitored branch formation by focusing on hyphal compartments bound by two division septa (Fig 5B). Branching followed stereotypical ordered processes. First, EVs spontaneously gathered in sizable patches, reflecting positive feedbacks in EVs domain formation. These patches then fluctuated in intensity and position to eventually stabilize and promote branch emergence. Upon emergence, both growth speeds and EVs concentration increased to approach stable steady-state values within approximately 10 to 20 min (Figs 5C and S6A and S4 Movie). However, in spite of these drastic changes in both growth and vesicle concentration, the thickness of the CW did not exhibit any systematic thickening or thinning (S6A Fig). In the model, we recreated the branching process by starting with a low value of EV, no growth, and the reference value for CW thickness. The model faithfully reproduced the observed rapid increase and saturation in both growth speed, and EV levels with similar timescales as in experiments, as well as the near-constant CW thickness values (Fig 5C). These analyses suggest that CW synthesis and expansion increase in a balanced manner during de novo tip growth. Conversely, we stopped growth by abruptly reducing turgor pressure. We rinsed cells with a low dose of 0.2 M sorbitol supplemented in the medium, which completely halted tip growth within a minute, for a duration of approximately 10 min. As described previously, turgor rapidly adapted and allowed growth to restart at a speed close to that before sorbitol treatment within 10 to 15 min [40,61]. Interestingly, upon sorbitol treatments, we observed a progressive delocalization of RAB11-labeled EVs domain from cell tips, which occurred slightly slower than drops in growth speeds. These observations parallel previous reports in fission yeast [40] and further support the general hypothesis of mechanical feedback in the model. Interestingly, in contrast to de novo growth at branching sites, growth arrest upon turgor reduction was also accompanied by an increase in CW thickness of approximately 30% to 50% over 3 to 5 min, reflecting a significant transient imbalance between CW synthesis and expansion. We interpret this as a result of the slower drop of EVs concentration in comparison to CW strain rates, which presumably yield to leftover synthesis with no deformation thereby thickening the CW (Fig 5D and S5 Movie). Accordingly, when tip growth restarted, due to turgor adaptation, the EVs signal recovered its initial intensity, and CW thickness progressively decreased toward original values before the sorbitol shock. To test the model against these turgor modulations, we inputted an abrupt drop in turgor values followed by a progressive adaptation. This allowed to recapitulate growth arrest followed by growth restart, the progressive decrease of EV concentration upon turgor loss and its recovery upon growth restart, as well the dynamics of CW thickening followed by progressive thinning, though this effect was less pronounced in the model than in experiments (Fig 5E). These findings demonstrate how temporal delays between CW secretion and expansion at cell tips can transiently impact CW thickness and mechanics. To more directly affect vesicle accumulation at cell tips, independently of turgor manipulations, we next used two independent assays to alter EV trafficking. We first depolymerized microtubules that serve as important tracks for EV polarized trafficking. We treated hyphal cells with low doses of benomyl, which caused microtubules to disappear within 2 to 4 min (S6B Fig). This led to a fraction of cells that kept on elongating at a slower rate and exhibited frequent turns in growth direction, and others that completely halted growth, and exhibited tip bulging concomitant with a progressive dispersion and loss of EVs [23,62]. Remarkably, as a consequence of growth arrest and EV reduction, the CW at the bulging tip exhibited significant thickening transiting from values of approximately 65 nm, up to approximately 100 nm in a timescale of approximately 20 min (Fig 5F and 5G and S6 Movie). We also affected vesicle accumulation using sarA6, a temperature-sensitive allele of sarA encoding the ARF GTPase governing ER exit, which results in Golgi disassembly, thereby blocking the production of post-Golgi EVs [22]. When cells were shifted to the restrictive temperature, the polar domain of RAB11 EVs completely dissipated from cell tips in a timescale of 10 to 20 min (Fig 5H and 5I and S7 Movie). Remarkably and in agreement with microtubule depolymerization experiments, this disappearance was concomitant with a growth arrest, marked bulging at cell tips, and significant CW thickening. Furthermore, growing sarA6 for extended periods of times at restrictive temperature, yielded to large balloon-shaped tips of up to 25 μm in diameter featuring CW thicknesses reaching up to 250 nm (S6C and S6D Fig) [22]. In the model, we simulated both benomyl and sarA6 results by reducing the source and sink terms that control the dynamic concentration of EVs. This allowed to reproduce both EV and growth reduction as well as CW thickening over similar timescales as in experiments. Therefore, in response to alterations in either growth or secretion, CW assembly appears to occur faster than expansion, ensuring that CW thicken rather than thin to safeguard cell surface integrity.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001981

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