Mobility, microtubule nucleation and structure of microtubule-organizing centers in multinucleated hyphae of Ashbya gossypii

Abstract

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 microm/min), rotations (up to 180 degrees in 30 s), and long-range nuclear bypassing (up to 9 microm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis-oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

Figure 1.

Localization of MTOCs in A. gossypii. (A) Images of a tip compartment expressing AgTUB4-YFP to localize MTOCs and stained with Hoechst to visualize nuclei. (B) Images of a hyphal compartment with a calcofluor stained septum and YFP-marked MTOCs. No YFP fluorescence can be seen at the septum. (C) Images of A. gossypii hyphae before and after incubation with 15 μg/ml nocodazole for 1 h and at different times after washout of the drug. Nuclei are stained with Hoechst and microtubules are labeled with anti-α-tubulin antibodies. The untreated hypha shows cMTs, one metaphase spindle before orientation along the growth axis and fluorescent foci representing SPBs. After nocodazole treatment only SPB foci are detectable with anti-α-tubulin antibodies. Twenty minutes after nocodazole washout, faintly stained short cMTs reemerged at the nuclear SPBs (arrows). By 30 min, the microtubules seemed similar to untreated cells. Bars, 5 μm (A–C).

Figure 2.

Oscillation, rotation and bypassing of nuclei in an A. gossypii hypha. Hyphae expressing AgH4-GFP to mark nuclei (green) and AgTub4-RedStar2 to mark SPBs (red) were pregrown and mounted for fluorescence videomicroscopy at room temperature as described in Materials and Methods. GFP and red fluorescent protein (RFP) fluorescence was imaged for 500 ms each in five Z-planes, 0.75 μm apart at 1-min intervals and processed (Supplemental Movie S1). (A) Maximal projections at 11 time points showing frequent changes in the position and SPB orientation of the 11 nuclei monitored in Supplemental Movie S1. During the 110 analyzed 1-min time intervals, nuclei moved forward 34 times and backward 30 times, all of which were led by the SPB. During the remaining 46 intervals, nuclei moved only marginally. Nonmoving as well as moving nuclei rotated during 53 intervals by up to 180° as seen by changes of the SPB positions. The relative position of SPBs in all fast-moving nuclei was “head-first” (at the leading edge of nuclear movement), and inversions in the direction of nuclear movement were preceded by inversion of the SPB position. Bar, 5 μm. (B). Diagram of the position of nuclei (circles) and orientation of SPBs (red dots) at 0 and 10 min. Identical nuclei are connected by dotted lines highlighting the four bypassing events. The arrow marks the 1.5-μm elongation of the hypha during 10 min, which was measured using the overilluminated montage of A to visualize the background staining of the hyphal cytoplasm. (C). Detailed diagrammatic presentation of backward and forward as well as rotational movements of two nuclei (6, white asterisk and 7, yellow asterisk) within 10 min (movie frames 3–13 of Supplemental Movie S1). Nucleus 6 bypasses nucleus 7 by migrating 5 μm backward, inverts the position of the SPB by rotation at 4 and 5 min, and migrates 3 μm forward. Nucleus 7 represents a typical tumbling nucleus showing minor movements and small angle rotations until 8 min, at which time it undergoes a rotation and then a forward movement of 2 μm. Fast-moving nuclei in A and C seem to be stretched and sometimes display two SPBs. This does not reflect the real shape of fast nuclei or duplicated SPBs however; rather, it is due to the high migration speed and the fact that these images show projections of five focal planes taken within 6 s.

Figure 3.

Migration of nuclei in the absence of cMTs. The strain expressing AgH4-GFP (green) and AgTub4-RedStar2 (red) was preincubated in the presence of 15 μg/ml nocodazole for 20 min and mounted for fluorescence videomicroscopy on agar slices containing the same concentration of nocodazole. GFP and RFP fluorescence was imaged for 500 ms each in three Z-planes, 0.75 μm apart, at 30-s intervals and processed (Supplemental Movie S2). (A) Nine maximal projections of Supplemental Movie S2 at 1-min intervals showing 11 evenly moving nuclei with SPBs only rarely oriented in the direction of migration. Bar, 5 μm. (B) Diagram of positions of nuclei (circles) and orientation of SPBs (red dots) at 0 and 8 min. Identical nuclei are connected by dotted lines. The arrow marks the elongation of the hypha within 8 min.

Figure 4.

Motility of SPBs and visualization of cMT arrays. Hyphae expressing GFP-tagged and nontagged AgTub1 were pregrown and mounted for fluorescence microscopy on thin agar slices. Images were taken from a single Z-plane every 6 s with 1.5-s exposure time for 7 min (Supplemental Movie S3) or as Z-stack with 18 planes each 0.3 μm apart and 1.5-s exposure time (Supplemental Movie S4). (A) 11 representative frames of Movie S3 showing three SPBs as distinct dots and attached cMTs as thin, weakly fluorescent filaments. The apparent connection of SPBs 1 and 2 (frames 162–180 s) represents two cMTs superimposed by chance because both are visible as two independent cMTs in frame 186 s. Bar, 5 μm. (B) Images of selected focal planes of the Z-stack (Supplemental Movie S4) of a hypha expressing GFP-AgTub1. Four SPBs and attached cMTs are seen in the different focal planes. Short and long cytoplasmic microtubules emanate from both sides of bright foci representing SPBs (1–3) and from SPBs of a metaphase spindle (4). Short cytoplasmic microtubules frequently point toward the cell cortex; long cytoplasmic microtubules run along the longitudinal axis of the hyphae and can often be followed over several focal planes. They either terminate in the cytoplasm or seem to glide along the cell cortex. The SPB from nucleus 1 is connected to the cell cortex of the tip region by three short cytoplasmic microtubules, whereas at least three longer cytoplasmic microtubules run toward the distal part of the hypha. Bar, 5 μm.

Figure 5.

EM analysis of nuclei in multinucleated hyphae. (A) Overlay of a DIC and a fluorescence image of a young A. gossypii mycelium that was stained with Hoechst to visualize nuclei. Such mycelia with five to 10 tips and 50–100 nuclei were prepared for thin section EM analysis as described in Materials and Methods. Bar, 5 μm. (B–E) EM of nuclei in different nuclear cycle stages. The continuous nuclear membrane and nuclear pore complexes within the nuclear envelope can be seen in all images. Bars, 200 nm. (B) A single SPB (asterisk) is embedded in the nuclear envelope. A higher magnification is shown in the top right corner. (C) Duplicated SPBs (asterisks) connected by a bridge are embedded in the nuclear envelope. A higher magnification is presented in the top right corner. (D) A nucleus with spindle microtubules (arrow) and continuous nuclear membrane. The two SPBs were observed in the adjacent sections at positions marked by the asterisks. Magnifications are shown in the top and bottom right corners. (E) Montage of three EM images showing a nucleus in anaphase. The continuous nuclear envelope and spindle microtubules (arrow) are visible. The SPBs are in other sections.

Figure 6.

High-resolution EM analysis of A. gossypii SPBs and microtubules. (A) Electron micrograph showing five discrete SPB layers: the inner plaque (IP), central plaque (CP), intermediate layer 2 (IL2), intermediate layer 1 (IL1) and outer plaque (OP). Hook-like appendages (asterisk) extending from the CP anchor the SPB in the nuclear envelope (NE). Nuclear microtubules (nMTs) emanate from the IP and cMTs (cMTs) from the OP. (B) Two types of cMTs, one extending from the OP in a perpendicular direction and the other in a tangential direction. (C) Three serial sections of a duplicated SPB (SPB1 and SPB2) connected by a bridge (BR). Again, perpendicular and tangential cMTs nucleate at OPs. (D) SPB with capped nuclear and cMTs (arrows). The rounded caps at the microtubule ends are associated with electron dense material. (E) Low- and (F) high-magnification image of a cMT that ends in a distinct flare at its plus end (arrow). Bars, 100 nm (A–F).

Figure 7.

Comparison of A. gossypii and S. cerevisiae SPB structure based on EM analysis. (A) Schematic depicting the S. cerevisiae and A. gossypii SPB layers and distances between SPB layers. Although the CP and IL2 are similar in size and structure in A. gossypii and S. cerevisiae, the IL1 and OP of A. gossypii are considerably smaller and the spacing between those layers is increased compared with budding yeast. The A. gossypii OP appears amorphous rather than electron dense like in S. cerevisiae. (B) Quantitation of distances between A. gossypii SPB layers with SD and number of plaques used for the measurements. The data for S. cerevisiae SPB plaques were compiled from published work (Byers and Goetsch, 1974; Bullitt et al., 1997; O'Toole et al., 1999; Schaerer et al., 2001).

Figure 8.

Two types of cMTs in A. gossypii. (A) EM micrograph of a hyphal thin section showing a nucleus with its SPB near the cell cortex. (B) Magnified view of the cortex region with a perpendicular cMT (arrows) ending at the cell cortex and two cMTs (asterisks) tangential to the nucleus and parallel to the hyphal axis (center). (C) Graphic presentation of the SPB and the emanating cMTs shown in B. Bars, 500 nm.

Figure 9.

Model of A. gossypii SPB and cMT arrays emanating at SPBs. (A) Model of three nuclei with independent cMT arrays in a hypha. The most apical nucleus has close contact to the growing tip via its short cMTs. Loss of cMTs will increase the distance between the first nucleus and the tip as shown in Figure 3. The two other nuclei are connected with the hyphal cortex via short cMTs and most likely also long cMTs. Growth and shrinkage of these cMTs provides pushing and pulling forces for short-range nuclear oscillations. The most likely pulling force in the direction of a single short cortex-connected cMT was discussed in Figure 4A. Nuclear bypassing is a long-range movement and is very likely achieved by pulling forces of a long cMT when the cortex connection of short cMTs is reduced or absent. Nuclei cannot oscillate or bypass in hyphae lacking short and long cMTs as shown in Figure 3. Nuclei with mutant SPBs able to emanate mainly/only long cMTs cannot oscillate but are still able to bypass other nuclei (unpublished observations). (B) Schematic representation of an A. gossypii SPB and associated microtubules.