The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

Abstract

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.

Figure 1.

Growth and microtubule distribution in an early germling. (A) Time-lapse images (Fig 1.mov) of a binucleate GFP-α-tubulin–expressing germling growing and going through mitosis. Images are projections of Z-axis stacks acquired using a wide-field microscope. Note that microtubules are less brightly fluorescent in young germlings than in hyphal tip cells, presumably because the GFP has had longer to mature in the older hyphal tip cells. The time, in seconds, after the start of observation is indicated in the lower left corner of each panel. At the 4322-s time point, the germling is entering mitosis. Two small spindles have formed and only a few very faint cytoplasmic microtubule fragments remain (arrows). At the 4502-s time point, all cytoplasmic microtubules have disassembled. Cytoplasmic microtubules reassemble after mitosis (5582-s time point). (B) Profile of tip growth. Growth of the germling tip (in micrometers) is plotted with respect to time (in seconds). Each open diamond represents a single time point. The slope (growth rate) over two intervals was determined by regression analysis and is shown with black lines. Thus, for the interval from 1801 to 4502 s, the growth rate was 0.015 μm/min, and for the interval from 8180 to 14147 s, the growth rate was 0.194 μm/min. This germling went through mitosis during the observation period (gray region). The spindle length is shown with filled circles. Note that for this germling tip growth rates varied greatly over time. However, growth rates were generally faster after nuclear division when there were four nuclei in the germling instead of two.

Figure 2.

Effects of benomyl on hyphal tip growth. (A) Time-lapse images of a GFP-α-tubulin–expressing strain from Fig 2.mov. Images are projections of Z-stacks obtained with a wide-field microscope and the time (in seconds) after the start of observation is indicated in the upper right corner of each panel. Benomyl was added 900 s after the start of observations. The hypha at the left was in interphase and the hypha at the right was in anaphase when benomyl was added. A mitotic spindle is visible in the hypha at the right at the 840-s time point. The growth of both hyphae dramatically slowed after the addition of benomyl. Bulging of tips after benomyl addition is indicated by arrows, and bulging of a medial region of the hypha at the left is indicated by an arrowhead. (B) Profile of the growth of the left hypha in A. The time of benomyl addition is designated with the filled arrow and the completion of cytoplasmic microtubule disassembly is designated by the open arrow. The rates of tip growth before and after the addition of benomyl were determined by regression analysis (gray lines) and are indicated.

Figure 3.

Growth recovery after benomyl washout. A rapidly growing hypha was treated with benomyl for 15 min. The benomyl was then washed out and tip growth was monitored. (A) The hyphal tip just before benomyl addition. (B) The same hyphal tip after 15 min in benomyl and immediately before benomyl washout. The microtubules have disassembled. (C) Time course images (Fig 3.mov) of the same hypha after benomyl washout. Imaging was restarted as soon as possible after benomyl washout (time 0). Microtubules are clearly visible at 540 s, and there is a visible bulge in the hyphal tip (arrowhead). The bulge is larger than before the benomyl washout. Normal tip growth has resumed by the 1440-s time point, and the hyphal bulge is still visible (arrowhead). (D) The growth of the hyphal tip was plotted against time after the completion of benomyl washout. Tip growth accelerated after the benomyl washout and reached a consistent rate of 0.458 μm (gray line, determined by regression analysis).

Figure 5.

Cytoplasmic microtubules persist during mitosis near the tips of rapidly growing hyphae. (A and C) Time-lapse images of the hyphal tip regions of a GFP-α-tubulin–expressing hyphae. The images in A are from Fig 5A.mov, and the images in C are from Fig 5C.mov. All images are maximum intensity projections of Z-stacks acquired using a spinning-disk confocal microscope. The time (in seconds) after the start of observation is indicated in the upper right corner of each panel. The series begins in mitosis and ends in interphase. Schematic drawings of microtubule distributions at the zero time points are shown at the top of each panel. Cytoplasmic microtubules are present near the hyphal tip throughout mitosis. (B) Profile of tip growth. Elongation of the hyphal tip (filled squares) and the length of the spindle closest to the hyphal tip in A (open circles) were plotted. The growth rate of the hypha (gray lines, determined by regression analysis) was the same in mitosis and the period afterward.

Figure 4.

Time-lapse images of GFP-α-tubulin in the medial region of a hyphal tip cell undergoing mitosis from Fig 4.mov. Images are maximum intensity projections of Z-stacks acquired with a spinning-disk confocal microscope. The time (in seconds) after the start of observation is indicated in the upper right corner of each panel. Three spindles go into anaphase successively. Note that the cytoplasmic microtubules disassemble completely in the early stages of spindle formation (100 s).