The <i>Aspergillus nidulans</i> Peripheral ER: Disorganization by ER Stress and Persistence during Mitosis

<div><p>The genetically amenable fungus <i>Aspergillus nidulans</i> is well suited for cell biology studies involving the secretory pathway and its relationship with hyphal tip growth by apical extension. We exploited live-cell epifluorescence microscopy of the ER labeled with the translocon component Sec63, endogenously tagged with GFP, to study the organization of ‘secretory’ ER domains. The Sec63 <i>A. nidulans</i> ER network includes brightly fluorescent peripheral strands and more faintly labeled nuclear envelopes. In hyphae, the most abundant peripheral ER structures correspond to plasma membrane-associated strands that are polarized, but do not invade the hyphal tip dome, at least in part because the subapical collar of endocytic actin patches constrict the cortical strands in this region. Thus the subapical endocytic ring might provide an attachment for ER strands, thereby ensuring that the growing tip remains ‘loaded’ with secretory ER. Acute disruption of secretory ER function by reductive stress-mediated induction of the unfolded protein response results in the reversible aggregation of ER strands, cessation of exocytosis and swelling of the hyphal tips. The secretory ER is insensitive to brefeldin A treatment and does not undergo changes during mitosis, in agreement with the reports that apical extension continues at normal rates during this period.</p></div

Changes in the ER in swollen conidia, germlings and mature hyphae.

<p><b>A.</b> Swollen conidium after the first mitotic division. Sec63-labelled ER is shown on the left in inverted grey contrast. The right image is a merge of the Sec63-GFP (green) and HhoA-mCherry (that labels nuclei/chromatin) channels<b>.</b> <b>B.</b> A germling, with fluorescent markers displayed as in <b>A. C.</b> Sec63-GFP ER (inverted contrast) in germlings imaged at different stages after polarity establishment (all images displayed at the same magnification). Peripheral ER strands concentrated near the tip are visible at the stages shown in <b>C3</b> through <b>C6</b>. The prominence of the tip pm-associated ER strands increases with the length of the germtube. <b>D.</b> Long hypha. A linescan of the Sec63-GFP signal across the indicated line is shown on the right (FAU, fluorescence arbitrary units). <b>E.</b> Cortical ER strands do not overlap with the plasma membrane, stained with FM4-64.</p

Reductive stress leads to aggregation of peripheral ER strands.

<p><b>A.</b> Sec63-GFP images (inverted greyscale) of cells treated with DTT for the indicated time periods. Images correspond to maximal intensity projections of z-stacks processed with an ‘unsharp’ filter. In the 45 min GFP image, green arrowheads indicate aggregates of ER strands and magenta arrows indicate nuclei, whose NEs, unlike peripheral ER strands, appear unaltered. All images are shown at the same magnification. Bars, 5 µm. <b>B.</b> Examples of ring-shaped ER aggregates in cells treated with DTT for 130 min.</p

Role of F-actin and of the subapical collar of actin patches in determining peripheral ER morphology.

<p><b>A.</b> Individual images of a z-stack of Sec63-GFP in an untreated hyphal tip cell control. <b>B.</b> Individual images of a z-stack of Sec63-GFP in a hyphal tip cell treated with latrunculin B. <b>A2</b> and <b>B2</b> correspond to the rectangular regions indicated in <b>A</b> and <b>B</b>, respectively, shown at double magnification. <b>C.</b> Dual-channel images of Sec63-GFP and mRFP-AbpA. Two examples of hyphal tip cells are shown. Images represent maximal intensity projections of z-stacks treated with an ‘unsharp’ filter.</p

The ER does not undergo major changes following microtubule depolymerization.

<p><b>A.</b> Control experiment demonstrating the depolymerization of MTs (green) upon treatment with benomyl. Unpolymerized TubA-GFP accumulates in the cytosol, leaving the nuclei empty (nuclei are labeled with HhoA-mCherry). The picture was taken after after 17 min of incubation in the presence of the drug. <b>B.</b> Effect of benomyl treatment on the Sec63-GFP-labeled ER. The representative example shown on the right corresponds to a cell incubated for 30 min in the presence of benomyl. The positions of the nuclei are revealed by the HhoA-mCherry fluorescence.</p

The ER visualized with Sec63-GFP.

<p>Different planes (z coordinates indicated in µm) of z-stack of images of a hyphal tip cell. The position of the nucleus is indicated in the middle plane. The asterisk indicates the apical border of the hypha, whereas the arrow indicates the apex-proximal ER protusion.</p

The peripheral Sec63 ER is not disorganized during mitosis.

<p><b>A.</b> Nuclear division in a tip-distant region of a cell coexpressing Sec63-GFP and HhoA-mCherry (‘green’ and ‘red’ channels displayed in inverted greyscale). Time is indicated in min:sec. Whenever possible, the position of the intact NEs of the dividing nuclei is indicated with arrows. <b>B.</b> The peripheral ER does not undergo disorganization during mitosis. The top greyscale image is a kymograph displaying growth of the hyphal tip cell shown below. The kymograph was traced as schematized on the right. The series of images displayed below the kymograph (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067154#pone.0067154.s003" target="_blank">Video S3</a> should be consulted) show that the peripheral ER does not undergo rearrangements during mitosis.</p

The ER is brefeldin A-insensitive.

<p>A strain coexpressing Sec63-GFP with the late Golgi marker mRFP-PH^OSBP was treated with brefeldin A for 25 min. Under these conditions, late Golgi cisternae form aggregates. In contrast, the ER appears to be largely resistant to the drug. Images represent maximal intensity projections of z-stacks of deconvolved images.</p

Endocytic recycling <i>via</i> the TGN underlies the polarized hyphal mode of life

<div><p>Intracellular traffic in <i>Aspergillus nidulans</i> 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 <i>geaA</i>^<i>gea1</i> 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 <i>via</i> 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.</p></div

Endocytic recycling <i>via</i> the TGN underlies the polarized hyphal mode of life - Fig 5

<p><b>Recycling of ChsB from endosomes necessitates dynein</b> (A) Left, schematics summarizing the rationale of these experiments. Middle images: localization of GFP-ChsB in hyphal tips of the wt and of strains carrying ts mutations in <i>nudA</i> encoding the dynein heavy chain, before and after shifting cells from 28°C to 37°C. Note the aggregate of ChsB in the <i>nudA</i> mutants at 37°C (see scheme), which forms apparently at the expense of the signal in the apical dome and the SPK (which is not detectable in the mutants at 37°C). Right, linescans of the ChsB channel for the hyphae displayed in the images (1 px, 0.103 μm). (B) Hyphal tip of a <i>nudA2</i> cell shifted to 42°C, stained with the endocytosed membrane tracker FM4-64.</p