Figure 1.
The ER visualized with Sec63-GFP.
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.
Figure 2.
Changes in the ER in swollen conidia, germlings and mature hyphae.
A. 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. A germling, with fluorescent markers displayed as in A. C. 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 C3 through C6. The prominence of the tip pm-associated ER strands increases with the length of the germtube. D. Long hypha. A linescan of the Sec63-GFP signal across the indicated line is shown on the right (FAU, fluorescence arbitrary units). E. Cortical ER strands do not overlap with the plasma membrane, stained with FM4-64.
Figure 3.
The ER does not undergo major changes following microtubule depolymerization.
A. 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. 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.
Figure 4.
Role of F-actin and of the subapical collar of actin patches in determining peripheral ER morphology.
A. Individual images of a z-stack of Sec63-GFP in an untreated hyphal tip cell control. B. Individual images of a z-stack of Sec63-GFP in a hyphal tip cell treated with latrunculin B. A2 and B2 correspond to the rectangular regions indicated in A and B, respectively, shown at double magnification. C. 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.
Figure 5.
Reductive ER stress results in reversible impairment of exocytosis.
GFP-SynA (inverted greyscale) and Nomarski (DIC) images of hyphal tips of cells incubated with DTT for the indicated times. The wash-out tip example corresponds to a DTT-treated cell further incubated in medium lacking the reducing agent.
Figure 6.
Reductive stress leads to aggregation of peripheral ER strands.
A. 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. Examples of ring-shaped ER aggregates in cells treated with DTT for 130 min.
Figure 7.
The ER is brefeldin A-insensitive.
A strain coexpressing Sec63-GFP with the late Golgi marker mRFP-PHOSBP 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.
Figure 8.
The peripheral Sec63 ER is not disorganized during mitosis.
A. 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. 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 (Video S3 should be consulted) show that the peripheral ER does not undergo rearrangements during mitosis.