A Myosin Light Chain Is Critical for Fungal Growth Robustness in Candida albicans

Abstract

Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In Candida albicans , as in many filamentous fungi, a cluster of vesicles, called a Spitzenkörper, is observed at the tip of growing hyphae that is thought to function as a vesicle supply center.

Two distinct lipid transporters together regulate invasive filamentous growth in the human fungal pathogen Candida albicans

“…These data are consistent with a defect in polarized growth in the drs2 mutant after the first septin ring forms. In agreement with this, we observed that, upon filament extension, active Cdc42, visualized with the CRIB (Cdc42 Rac1 Interactive Binding domain) reporter [48], became depolarized in drs2 cells and the SPK (visualized with the myosin light chain Mlc1, [49,50]), was not maintained at the filament tip following cell division, compared to the control cells (Figures 3D and 3E). Furthermore, comparison by time-lapse microscopy of the wild-type and drs2 cells expressing both reporters for active Cdc42 and the Spitzenkörper, indicates that depolarization of Cdc42 occurred prior to the SPK delocalization from the filament tip.…”

“…To visualize the distribution of phosphatidylserine, a fusion of GFP with the discoidin-like C2 domain of lactadherin (GFP-LactC2) was used, as previously described [36]. Strains expressing Mlc1-iRFP-GNB were generated by homologous recombination, using pFA-iRFP670-GNB-URA3, as in [50]. To visualize the distribution of ergosterol, we used filipin staining, as described [58], as well as the genetically encoded biosensor D4H [60].…”

“…Hence, we sought to determine whether a specific localization of Drs2 is critical for distinct functions. To address this question, we used a synthetic physical interaction (SPI) approach to stabilize Drs2 at the SPK [50]. To generate a mutant with Drs2 restricted/stabilized at the SPK, we used a strain expressing a GFP nanobody (GNB) fused to one copy of Mlc1-iRFP, together with Drs2GFP as the Drs2 sole copy.…”

Two distinct lipid transporters together regulate invasive filamentous growth in the human fungal pathogen Candida albicans

“…These data are consistent with a defect in polarized growth in the drs2 mutant after the first septin ring forms. In agreement with this, we observed that, upon filament extension, active Cdc42, visualized with the CRIB (Cdc42 Rac1 Interactive Binding domain) reporter [48], became depolarized in drs2 cells and the SPK (visualized with the myosin light chain Mlc1, [49,50]), was not maintained at the filament tip following cell division, cells) of the indicated strains and the survival was assessed. C) Hyphal specific gene are induced in drs2 cells upon serum exposure.…”

The type V myosin-containing complex HUM is a RAB11 effector powering movement of secretory vesicles

“…However, contrasting to myoE Δ mutants, sec4 Δ mutants are still capable of gathering RAB11 SVs at the tip ( Figure 1 D), indicating that Sec4 is not essential for transport. Instead, we observed that the cluster of SVs accumulating in sec4 Δ tips was twice as broad as in the wild type (1.68 ± 0.5 SD versus 0.74 ± 0.2 μm 2 , p = 0.0001 in a Mann-Whitney t test), while still displaying a similar average intensity per pixel, which suggests that the number of vesicles accumulating in the mutant was consistently higher than the wild type ( Puerner et al., 2021 ). This cluster of RAB11 SVs at the tip, less tightly packed in the sec4 Δ mutant than in the wild type, appears to be submitted to the opposing forces exerted by anterograde and retrograde motor teams, often causing the detachment of RAB11 SV 'lumps' away from the apex ( Video S1 ).…”