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

Miguel Hernández-González,Mario Pinar,Herbert N Arst,Ignacio Bravo-Plaza,Vivian De Los Ríos,Miguel A Peñalva,Amy S Gladfelter

doi:10.1371/journal.pgen.1007291

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.

Highlights

Fungal pathogenicity of plants and animals constitutes an enormous burden on human welfare, bequeathing a compelling case for understanding basic fungal biology
To sustain the strikingly rapid rates of growth [3] the secretory pathway must efficiently deliver to the apex the enzymes that synthesize the cell wall in the hyphal tip dome and the lipids required for the increase in plasma membrane (PM) surface
Vesicles at the SPK are loaded with cell wall-modifying enzymes (CWMEs) [10,11,12,13,14], demonstrating that these cargoes are exocytosed in a polarized fashion

Summary

Introduction

Fungal pathogenicity of plants and animals constitutes an enormous burden on human welfare (reviewed by [1]), bequeathing a compelling case for understanding basic fungal biology. To sustain the strikingly rapid rates of growth (circa 1 μm/min at 30 ̊C in A. nidulans) [3] the secretory pathway must efficiently deliver to the apex the enzymes that synthesize the cell wall in the hyphal tip dome and the lipids required for the increase in plasma membrane (PM) surface. Many fungi streamline this delivery by gathering a stock of secretory vesicles (SVs) at a structure denoted the Spitzenkorper (SPK), adjacent to the apical PM. Considering that key CWMEs such as β(1–3) glucan synthase [12] and chitin synthases [15] are integral membrane proteins, apical delivery alone cannot account for their polarization, which requires a mechanism(s) that counteracts retrograde diffusion across the PM

Methods

Results

Discussion

Conclusion