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First published online August 16, 2005
doi: 10.1242/10.1242/jcs.02501

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Role of the spindle-pole-body protein ApsB and the cortex protein ApsA in microtubule organization and nuclear migration in Aspergillus nidulans

¹ Max-Planck-Institute for Terrestrial Microbiology, Department of Biochemistry, Karl-von Frisch Str., 35043 Marburg, Germany
² University of Karlsruhe, Applied Microbiology, Hertzstr. 16, 76187 Karlsruhe, Germany
³ Department of Pharmacology, 675 Hoes Lane, UMDNJ-R.W. Johnson Medical School, Piscataway, NJ 08854-5635, USA

View larger version (92K): [in a new window]   Fig. 1. Influence of apsA and apsB on cytoplasmic and mitotic MTs. (A) Compared with wild type (strain SJW02) (a), where more than three MTs can be observed at the same time, in apsB10-mutant cells (SDV16) (b) only one dominant cytoplasmic MT is apparent. By contrast, the number of cytoplasmic MTs in apsA mutant cells (SNS9) (c) is increased and MTs are more curved and appear thinner. A similar phenotype was observed in a temperature-sensitive dynein mutant strain (SDV26) at restrictive temperature (42°C) (d), while MTs showed wild-type-like organization at permissive temperature (e). (B) Astral MTs (arrowheads) of the mitotic spindle are dramatically reduced in an apsB10 strain (a,d), but not completely lost, in comparison with wild type (b,e). The difference is obvious in short and long spindles. The number of astral MTs was not reduced in apsA strains (c,f). (C,D) Oscillation and movement of the mitotic spindle is common in wild-type strains (C,a-e), but was never observed in a apsA strain (D,a-e). MTs were visualized with a GFP-TubA fusion protein. Dotted lines in (C,D) indicate spindle centers. Bar, 4 µm for (A), 2 µm for (B), 5 µm for (C and D) (see supplementary material Movies 6 and 7).

View larger version (116K): [in a new window]   Fig. 2. Subcellular localization of ApsB. (A) Both ApsB-GFP (SDM1000) (a) and GFP-ApsB (SEa3) (b) localized to the nuclear SPB (green dots at nuclei) and to the cytoplasm (arrowheads). Cytoplasmic ApsB was always bound to MTs and moved fast up and down the filaments (see text and supplementary material, Movies 8-10a). Nuclei were stained with red fluorescent protein (DsRedT4) in (a) or with DAPI in (b). (B) C-terminally tagged ApsB as well as N-terminally tagged ApsB (C) was detected at the poles of the mitotic spindle. In (B) ApsB-GFP and GFP-TubA is visible (SDM40), while a red signal was used to set apart the mRFP1-ApsB (C,b) from the green GFP-TubA (C,a) (SDV1B). In (C,c) an overlay of (Ca,b) is shown. (D) In addition, N-terminally tagged GFP-ApsB localized to septa [arrows in (D,a,b)], while C-terminally tagged ApsB-GFP (E) did not [arrowheads in (E,a,b)]. Septa were not stained completely, but GFP-ApsB is seen as dots near the septal hole, as insert in (D, a) shows. The septa in (E) were negatively stained by weak cytoplasmic GFP background [insert in (E,a)]. Bar, 3 µm.

View larger version (77K): [in a new window]   Fig. 3. Dominant-negative effect of C-terminally tagged ApsB-GFP on MT organization. (A) In wild type, MTs (GFP-TubA (a) connect adjacent nuclei [StuA(NLS)-DsRedT4 (b)] (SJW100) and several additional MTs emerge from the nuclear SPB [arrowheads in overlay (c)]. (B) In strains with N-terminally tagged ApsB, MTs were organized like in wild type (SDV1B). (b) mRFP1-ApsB dots indicate the position of the nuclear SPB of two nuclei along cytoplasmic MTs [GFP-TubA (a)] [dark areas and arrowheads in overlay (c)=nuclei]. (C) C-terminally tagged ApsB-GFP (SDM40) displayed a dominant negative phenotype reducing the number of MTs similar to the apsB10 mutation (D) (SDV16). In (C) notice the MT-bound ApsB-GFP dot (middle) and the nuclear SPB-associated ApsB-GFP [bright, white dots connecting two nuclei to MT (oval, white areas of StuA(NLS)-GFP]. Bar, 3 µm for (A and B) and 4 µm for (C and D).

View larger version (61K): [in a new window]   Fig. 4. Activities of nuclear and septal MTOCs in wild type and apsB-mutant strains. (A) Scheme of MTOC activities. Cytoplasmic MTs (white arrows) emerge from different MTOCs (small white dots), e.g. the nuclear SPB (left) and MTOCs near the hyphal septa (right) (grey ball=nucleus; vertical black line=septum). (B) KipA is a MT plus-end marker and its fluorescence signal can be followed as comets (GFP-KipA) during MT growth (Konzack et al., 2005). The white arrow follows a GFP-KipA signal emerging at a septal MTOC (asterisk) (see supplementary material, Movies 11). Time is indicated in seconds. Bar, 3 µm. (C) Quantitative analysis of GFP-KipA counts at different MTOCs. Values are the average of 100-150 MTOCs. Wild-type strain: SSK92. Mutant strains: SDV24, SDV25.

View larger version (107K): [in a new window]   Fig. 5. Time-lapse sequences of nuclear migration and MT organization. (A,B) Interphase nuclei (dark, round or oval areas) move with the SPB (arrow) at their leading edge. The pulling force is applied to cytoplasmic MTs, which are connected with their minus ends to the SPB, thus pulling the nucleus. Notice that the nucleus in (A) migrates upwards and MTs (GFP-TubA) were located only at its front side (leading SPB). In (B) the pulling force was applied on the opposite of the former leading side, thereby moving the SPB from the upper end of the nucleus to the lower end, subsequently dragging the entire nucleus downwards. (C) If connected to the same MT, adjacent nuclei move synchronously when a pulling force is applied. (D) The lower nucleus moved upwards, although MTs coming from the SPB did not interact with the cortex, but only one central MT bundle was present. In (C,D) nuclei, microtubules and ApsB (SPB, arrows) were stained with GFP. Notice that GFP is C-terminally fused to ApsB. Bar, 2 µm for (A,D), and 3 µm for (C,D). Times between each picture is 30 seconds for (A-C) and for (D) as indicated in minutes (A,B: SJW02; C,D: SDM40). See supplementary material, Movies 12-16.

View larger version (68K): [in a new window]   Fig. 6. Genetic interaction of apsA and apsB with molecular motors. (A) Strains were grown for three days at 37°C on glucose medium (a) to repress or on ethanol medium (b) to induce the alcA-promoter. A kinA strain (SNR3) grows more compact compared to wild type, but sporulation is not affected. Hyphal growth of a apsA strain (SRF30) is similar to wild type (RMS011), but spore production is reduced (brown colony). A double mutation of kinA and apsA (SDM24) results in a synthetic lethal phenotype, while strains, which contained an inducible alcA(p)::apsA::GFP construct in addition to the kinA and apsA mutation (SDM23), produces spores and shows normal growth on ethanol medium apart from the compact colony morphology due to the influence of the kinA mutation. The cortical localization of ApsA-GFP was not influenced by the kinA mutation (not shown). (B) nudA and nudF strains grow very poorly, but grow better when suppressed by the apsB14 mutation. In comparison, apsA5 has no effect on the dynein deletion strain. Strains were incubated at 43°C for 3 days. Strains from left to right are: apsB14/A-7, apsB14/A-9, apsB14/F-9, apsB14/F-11, apsA5/A-6, XX60, apsB14/F-3, apsB14/F-6, SEwild-type and apsB14. Bars, 1 cm (A,B) and 1.5 cm (B).

View larger version (77K): [in a new window]   Fig. 7. ApsB accumulates at the hyphal tip in dynein-mutant cells. Cytoplasmic, MT-associated ApsB (white spots) often localizes at the hyphal tip as one single spot. This is independent of whether apsB is tagged N- or C-terminally with GFP (A,B) (SEa3, SDM1000). The localization pattern was not changed, neither in a kinA (C) (SDM64) nor in a kipA (D) (SDV19) background. However, up to 10 ApsB-GFP dots accumulated in nudA strains (E) (SDM92). Despite their accumulation at the hyphal tip, each spot was still highly mobile (see supplementary material, Movies 17 and 18). (F) ApsB localization in apsA1 mutant stains (SDV31). Scale bar is 3 µm for (A-D,F) and 2 µm for (E).

View larger version (28K): [in a new window]   Fig. 8. Scheme of MT organization and nuclear migration in A. nidulans. (A) Cytoplasmic MTs are generated from SPBs. They elongate and eventually overlap to form anti-parallel bundles (1), thereby connecting adjacent nuclei. Some grow tipwards or make contact with the cortex (2). Nuclei can be moved by pulling forces of motor proteins, which could be located between overlapping filaments (1) or at the cortex (2), as the right nucleus indicates. (B) In wild-type cells, MTs contact ApsA or associated proteins (a). In apsB mutant cells, only a dominant, central microtubule bundle passes through the compartment, while additional MTs are absent (b). Although present, MTs cannot successfully contact the cortex in apsA mutant cells, hence elongating, which results in the appearance of curved MTs (c). Because certain MTs are not present in apsB mutants, nuclear positioning is interrupted, while MTs in apsA mutants miss the interaction with ApsA. Therefore, apsA and apsB mutants show very similar nuclear migration defects. Although cortical pulling forces (2) may be ineffective here, pulling forces between overlapping filaments (1) probably still work and are responsible for the observed remaining nuclear migration activity.