Endocytic recycling via the TGN underlies the polarized hyphal mode of life

Abstract

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogenCandida albicans

“…In the SAR, we continued to observe the presence of membrane sheets, though these were found individually, were in a range of orientations (i.e., not in a parallel organisation) and were connected to many tubular extensions ( Figure 5C3 Our work provides the first indication for an on-site SV delivery mechanism in C. albicans. Indeed, endocytic recycling by way of the trans-Golgi network has recently been shown to be crucial for hyphal tip growth (Hernández- González et al, 2018). From this network, SVs bud either directly into the Spk, or in the SAR from which they are transported the short distance to the Spk, most likely along a network of actin cables that extended into the SAR (Sudbery, 2011).…”

Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase

“…An important finding of our work is that secretion of inulinase requires functional endosomes, specifically EEs, because ablation of the master RAB of EEs, RabB RAB5 , prevented InuA exocytosis without affecting ER or Golgi glycosylation, whereas ablation of its equivalent in late endosomes/vacuoles, RabS RAB7 , had no effect. In contrast, we have recently reported that ChsB, a chitin synthase that continuously recycles to the PM from the TGN does so by way of a sorting endosome that must be functionally located upstream of EEs, such that ChsB exocytosis is completely independent on RabB RAB5 (Hernández‐González et al, ). Both InuA (this work) and ChsB (Hernández‐González et al, ) require the TGN regulator HypB Sec7 for their exocytosis, but unlike the ChsB pathway, the InuA pathway does not depend on GARP, showing that this tethering complex does not participate in a retrograde step transporting inulinase between the endosomes and the TGN that is necessary for its exocytosis.…”

“…A previous study established that after its exocytosis the chitin synthase ChsB recycles from the PM to the TGN by way of a sorting endosome located upstream of EEs. Therefore, ChsB recycling is RabB RAB5 ‐independent, but depends on the GARP complex to arrive at the TGN (Hernández‐González et al, ). Fig.…”

“…D shows that, in marked contrast, InuA exocytosis is largely unaffected by ablation of Vps52 removing a key component of GARP. That vps52∆ shows only a minor effect is notable given that the mutation results in fragmentation and depolarization of the TGN and markedly reduces colony growth (Hernández‐González et al, ). Therefore, exocytosis of InuA does not require traffic between endosomes and the TGN using the ChsB/GARP pathway, but follows instead a pathway dependent on the normal function of EEs.…”

“…Following this recruitment, kinesin-1 and myosin-5 motors cooperate to transport SVs to the SPK (Zhang et al, 2011;Peñalva et al, 2017). Integral membrane proteins that undergo endocytic cycling follow this direct pathway, which can be tracked by labeling these cargoes with GFP in their cytosolic extensions (Taheri-Talesh et al, 2008;Schultzhaus et al, 2015;Hernández-González et al, 2018).…”