Journal of Science 111, 2607-2614 (1998) 2607 Printed in Great Britain © The Company of Biologists Limited 1998 JCS7284

A cytoplasmic required for mitotic aster formation in vivo

Satoshi Inoue, O. C. Yoder, B. Gillian Turgeon and James R. Aist Department of Pathology, Cornell University, Ithaca, New York 14853, USA Αuthor for correspondence (e-mail: [email protected])

Accepted 2 July; published on WWW 13 August 1998

SUMMARY

An astral pulling force helps to elongate the mitotic spindle rate and extent of spindle elongation during B in the filamentous ascomycete, Nectria haematococca. were reduced; and spindle pole body separation almost Evidence is mounting that dynein is required for the stopped when the anaphase B spindle in the mutant was cut formation of mitotic spindles and asters. Obviously, this by a laser microbeam, demonstrating unequivocally that no would be an important mitotic function of dynein, since it astral pulling force was present. These unique results not would be a prerequisite for astral force to be applied to a only provide a demonstration that cytoplasmic dynein is spindle pole. Missing from the evidence for such a role of required for the formation of mitotic asters in N. dynein in aster formation, however, has been a dynein haematococca; they also represent the first report of mitotic mutant lacking mitotic asters. To determine whether or not phenotypes in a dynein mutant of any filamentous fungus cytoplasmic dynein is involved in mitotic aster formation in and the first cytoplasmic dynein mutant of any organism N. haematococca, a dynein-deficient mutant was made. whose mitotic phenotypes demonstrate the requirement of Immunocytochemistry visualized few or no mitotic astral cytoplasmic dynein for aster formation in vivo. in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral Key words: Anaphase, Aster, Dynein, , , Spindle, motility of membranous organelles was not apparent; the Spindle pole body

INTRODUCTION animal cell systems (Compton, 1998; Echeverri et al., 1996; Gaglio et al., 1996, 1997; Heald et al., 1996; Merdes and Cytoplasmic dynein is a microtubule (MT)-associated motor Cleveland, 1997; Merdes et al., 1996; Rodionov and Borisy, protein involved in mitotic movements (Barton and Goldstien, 1997; Vaisberg et al., 1993; Verde et al., 1991) has indicated 1996; Pfarr et al., 1990; Schroer, 1994; Steuer et al., 1990), that dynein is required for the formation and maintenance of nuclear migration in the filamentous fungi Aspergillus nidulans mitotic spindle poles and asters, a prerequisite for astral force (Xiang et al., 1994, 1995) and Neurospora crassa (Bruno et al., to be applied to a spindle pole. However, one key element of 1996; Plamann et al., 1994), and mitotic spindle orientation in vivo evidence for such a role of dynein in aster formation and astral MT dynamics in the yeast Saccharomyces cerevisiae that has, until now, been missing (cf. Compton, 1998; Merdes (Carminati and Stearns, 1997; Eshel et al., 1993; Li et al., 1993; and Cleveland, 1997), is a dynein-deficient mutant that fails Shaw et al., 1997). However, the various roles of cytoplasmic to form mitotic asters. dynein in mitosis have not yet been fully elucidated and/or To determine if cytoplasmic dynein heavy chain (CDHC) demonstrated (Holzbaur and Vallee, 1994). is required for the formation of mitotic asters in N. The spindle pole body (SPB) is a nucleus-associated haematococca, we created a CDHC-deficient mutant, Cu1, microtubule-organizing center involved in mitosis and from wild-type strain T213 by inserting a selectable marker nuclear motility in fungi (Aist, 1995; Hagan and Yanagida, into the central motor domain of the CDHC gene, DHC1 1997). Mitotic anaphase in the filamentous ascomycete, (Inoue et al., 1998). In this paper we report the first mitotic Nectria haematococca MP VI, occurs as two sequential phenotypes of a dynein mutant of a filamentous fungus (cf. stages, anaphase A and anaphase B (Aist and Bayles, 1988), Morris et al., 1995) and attribute them to the documented as defined by Inoué and Ritter (1975). Laser microbeam absence of mitotic asters in the mutant. This is the first dynein experiments and treatment with MT-depolymerizing drugs mutant of any organism whose mitotic apparatuses lack showed that astral MTs of N. haematococca are involved in mitotic asters. Thus, this report provides the missing evidence a pulling force that is transmitted to the SPB during anaphase for the essential participation of cytoplasmic dynein in B and helps to elongate the mitotic spindle (Aist and Berns, mitotic aster formation in vivo. A brief summary of these 1981; Aist et al., 1991). Recent experimental research with results has been published (Inoue et al., 1997). 2608 S. Inoue and others MATERIALS AND METHODS RESULTS Molecular manipulations Immunocytochemistry of MTs (Fig. 1) revealed prominent Detailed methods and additional molecular results have been published spindles and extensive asters of all mitotic nuclei in the (Inoue et al., 1998). DNA gel blot analysis indicated that there is only controls, wild-type T213 and isolate S01-2 (Fig. 1C,E,G). one CDHC gene in N. haematococca. To create the CDHC-deficient Although clear staining of spindles (Fig. 1I,K,M,O), and often mutant (Cu1), the CDHC gene, DHC1, in wild-type isolate T213 was of non-astral cytoplasmic MTs in the same mitotic cells (not disrupted by transformation with a vector carrying a portion of DHC1 illustrated), was seen in the mutant, no prominent asters were interrupted by a gene, hygB, for resistance to hygromycin B. The observed in the 31 anaphase B nuclei of mutant cells we control transformant, S01-2, was created by integration of the same transformation vector at an ectopic site, leaving DHC1 intact. videotaped (Fig. 1M and O). In a small minority (7 of 31) of these mitoses in the mutant, one or two thin, 2-6 µm long MTs Microscopy and microscopic analyses or MT bundles were observed projecting from one of the two Procedures for microscopic analyses were similar to those previously SPBs at anaphase B. From these unique results we inferred that described (Aist and Bayles, 1988; Aist et al., 1991; Wu et al., 1998). little or no mitotic aster was formed in the CDHC-deficient Briefly, cultures were grown on glass microscope slides coated with mutant and that CDHC has as essential role in the formation yeast extract-glucose (YEG)-Gelrite medium and videotaped at of asters in N. haematococca. ×10,000 magnification using phase-contrast optics and real-time This lack of prominent asters in fixed and stained cells of image processing. Videotaped images were captured and processed the mutant was confirmed by several unique observations and with the computer program Image-Pro Plus (Media Cybernetics, analyses of aster function performed with living cells. First, Silver Spring, MD) and printed with a Codonics NP-1600 Photographic Network Printer (Codonics, Middleburg Heights, OH). rapid migrations of small organelles toward the SPB and back A customized software program designed by W. Schubarg (Empire again within the astral regions (intra-astral motility; Aist and Imaging Systems, Cicero, NY) was used to acquire mitotic data from Bayles, 1991a) were numerous and easily observed during videotapes. From the mitotic data spreadsheets, plots of spindle anaphase B in control cells, but none was observed in the elongation and movements of the SPBs and mitotic apparatus of each mutant, despite extensive efforts to detect them by carefully analyzed mitosis were created by Sigma Plot for Windows™ (SPSS reviewing videotaped sequences of 26 mitoses. Instead, small Inc., Chicago, IL). vesicles and lipid bodies moved rapidly and freely, by Brownian motion, in the presumed astral regions of the mutant, Laser microsurgery apparently due to the absence of astral MTs. Second, the rate Cultures were grown for 5-6 days on glass microscope slides coated and extent of spindle elongation during anaphase B of the with YEG-Gelrite medium. We conducted the laser microsurgery experiments in the Biological Microscopy and Image Reconstruction mutant were reduced, with the elongation rate being only about Resource at the Wadsworth Center in Albany, NY. Development of half that of the controls (Table 1). This slower rate of spindle the differential interference contrast-based light microscopic system elongation in the mutant is consistent with previous we used for laser microsurgery was described elsewhere (Cole et al., observations (Aist and Bayles, 1991b) in which a similarly 1995). A Q-switched, pulsed (10 Hz), neodymium-yttrium- slow spindle elongation rate was inferred to be caused by a aluminum-garnet laser was used for microsurgery. After preliminary momentary absence of the astral pulling force during spindle experiments, we decided to use a laser power between 4.5 and 6.0 µJ, bending episodes in wild type. Third, the rate of migration which consistently broke the early anaphase B spindle without (Table 1) and the frequency of oscillations (not illustrated) of apparent, short-term, deleterious effects on the cells. One to three the mitotic apparatus during anaphase B in the mutant were pulse trains of 0.5-1.0 seconds each were required to cut the spindles, reduced, reflecting absence of the astral pulling force (Aist and and comparable irradiations were given to the nucleoplasm (beside the spindle) in control cells. Each experiment was videotaped at Bayles, 1988). And fourth, we performed laser microsurgery ×12,000 magnification with a Nikon ×100 PlanApo objective lens and on the anaphase B spindle of the mutant, using the ectopic real-time image processing using the computer program Image 1 transformant (S01-2) as a control. When the spindle of the (Universal Imaging Corp., West Chester, PA). Images were captured control was cut by laser irradiation, the SPBs began to move from videotapes, and selected linear measurements were calculated as described above under Microscopy and microscopic analyses. Table 1. In vivo characteristics of anaphase B spindles of MT immunocytochemistry wild-type (T213), ectopic transformant-control (S01-2) Tubulin immunocytochemistry has been shown to be an effective and dynein mutant (Cu1) strains of N. haematococca* approach to visualization of MTs of the mitotic apparatus (asters and spindle) in yeast when compared to either electron microscopy or Rate of forward Final spindle Spindle migration‡ of the GFP-tubulin fluorescence (Carminati and Stearns, 1997). Moreover, length elongation rate mitotic apparatus when applied to the mitotic apparatus of N. haematococca (Aist et (µm) (µm/minute) (µm/minute) al., 1991), immunocytochemistry gave results corresponding closely Strain n=7 n=10 n=10 to those obtained using freeze-substitution fixation followed by electron microscopy of serial thin-sections and computerized three- T213 (>20.00)§ 7.05 (±1.26) a 4.00 (±2.11) a Cu1 15.00 (±2.42) 3.58 (±0.85) b 0.80 (±0.58) b dimensional reconstruction methodologies (Jensen et al., 1991). In S01-2 (>20.00)§ 7.83 (±1.16) a 2.65 (±1.33) a addition to astral MTs, non-astral cytoplasmic MTs in N. haematococca are readily visualized by this procedure (Inoue et al., *Data are means (±1 s.d.). Values in each column not followed by the 1998; Wu et al., 1998). same letter were significantly different (P≤0.01) by the two-sample t-test. In the present study, two day-old germlings on YEG-Gelrite-coated ‡The mitotic apparatus in hyphal tip cells usually migrates toward the tip. glass slides were used. Immunofluorescent MT staining was §Since the final spindle length of T213 and S01-2 was usually more than performed following the protocol of Aist et al. (1991). The optics were 20 µm, a complete data set could not be collected because most SPBs were a Zeiss ×63 Neofluar objective (NA 1.25) and a ×16 projection lens. off the video screen at the end of anaphase B. Dynein in aster formation and function 2609 apart immediately after irradiation, separating three times as of asters in the mutant. The rate of SPB separation (a measure rapidly as those of the nucleoplasm-irradiated controls (Figs 2 of the spindle elongation rate) in the Cu1 nucleoplasm- and 3). Presumably, this was the rate of movement that the irradiated controls (Table 2) was almost the same as the spindle asters generated on their own, without limitation by the elongation rate we obtained in unirradiated cells (Table 1), spindle. This result confirmed that a strong astral pulling force showing that the laser irradiation itself did not have a exists in control cells, counterbalanced by the intact spindle significant affect on the spindle elongation/SPB separation which limits the rate of SPB separation, as first reported by rate. Aist and Berns (1981). In contrast, SPB separation almost Laser microsurgery on the anaphase B spindle of the CDHC- stopped in the CDHC-deficient mutant when the spindle was deficient mutant made it possible to observe a unique behavior cut with the laser microbeam (Figs 2 and 3), showing of the severed spindle segments, because, in contrast to wild conclusively that without cytoplasmic dynein there was no type, the SPBs in the mutant became almost stationary after astral pulling force; because the asters were absent, only the the spindle was cut, rather than migrating rapidly apart and out spindle pushing force was available to separate the SPBs when of view (Fig. 2). The cut spindle segments first rotated slightly the spindle was left intact. Thus, cytoplasmic dynein is in opposite directions (Fig. 2C), and then each elongated required for the astral pulling force to be manifested during toward the opposite SPB until their free ends had passed each anaphase B in vivo, a result that is consistent with the absence other (Fig. 2D). The average elongation rate of the cut spindle

Fig. 1. Mitotic nuclei of the ectopic transformant control (A,B,E-H), the wild- type control (C,D) and the dynein mutant (I-P) stained with FITC-labeled antibody (for MTs) (A,C,E,G,I,K,M,O) and with 4′,6-diamidino-2-phenylindole (for chromatin) (B,D,F,H,J,L,N,P). As, aster; IP, interphase nucleus. Triangles indicate the approximate positions of SPBs. Note that no aster is visualized in these typical anaphase A (K) and anaphase B (M and O) nuclei in the mutant, whereas developing asters were often visualized at anaphase A (C) and prominent asters were consistently visualized at anaphase B (E and G) in the controls. The left end of the spindle in M is slightly out of the plane of focus, accounting for the diminishing fluorescence signal from the spindle near the left SPB. Bar, 5 µm. 2610 S. Inoue and others

Table 2. In vivo SPB separation rates following control (SO1-2) strains was similar, indicating that comparable laser microbeam irradiation of spindles transformation itself had no appreciable affect on mitosis. (spindle cut) or nucleoplasm (spindle intact) in ectopic Moreover, the mitotic phenotypes caused by CDHC deficiency transformant-control (S01-2) and dynein mutant (Cu1) in Cu1 were restricted to anaphase B: mitosis occurred strains of N. haematococca normally from to anaphase A (Fig. 4), except for a Mean (±1 s.d.)* marked diminution of oscillatory movements and rotations of Strain/treatment n (µm/minute) the mitotic apparatus which was obvious during review of the ± video tapes. The duration (23 to 29 seconds), net spindle S01-2/spindle intact 9 5.69( 0.90) a µ S01-2/spindle cut 8 16.35(±7.66) b elongation (approx. 2.0 m) and rate of spindle elongation (3.3 Cu1/spindle intact 8 3.29(±1.15) c to 4.2 µm/minute) during anaphase A in the mutant were Cu1/spindle cut 11 0.88(±0.71) d similar to those in the controls. The separation distance of incipient daughter nuclei during anaphase B was diminished in ∗Values in this column not followed by the same letter were significantly different (P≤0.01) by the two sample t-test. the mutant (Fig. 4C and F; Table 1). segments was 4.32 (± 2.04 s.d.) µm/minute (n=6). Eventually, DISCUSSION the cut spindle segments rotated back until they were parallel to each other, and then they appeared to re-join, forming what Role of dynein in aster formation we are calling apparently intact spindles (Fig. 2E). As revealed We have not ruled out the possibility that in the absence of by careful still-frame reviews of the videotapes, these functional cytoplasmic dynein, the astral MTs in our dynein apparently intact spindles were formed when the overlapping, mutant are preferentially unstable and therefore selectively free ends of the elongated, parallel cut spindle segments sensitive to depolymerization during fixation. Nevertheless, became associated laterally, giving the initial impression of a our tubulin immunocytochemistry protocol has been shown to regenerated spindle. Apparently intact spindles are be reliable, our MT staining results were clear and consistent, nonfunctional, as they do not achieve further SPB separation. and the pattern of organelle movements in all astral regions of Mitosis in the wild-type (T213) and ectopic transformant- the mutant cells was indicative of the absence of astral MTs.

Fig. 2. Differential interference- contrast, time-lapse videomicrographs showing laser microbeam irradiation and cutting of the central spindle in the CDHC-deficient mutant Cu1 (A-E) and the ectopic transformant control S01-2 (F-J). Elapsed time (minutes and seconds) is shown in the lower left corner of each panel. NE, nuclear envelope; Sp, spindle; AiSp, apparently intact spindle. Triangles indicate the positions of SPBs. Arrows indicate where and when laser irradiation was applied. Circles indicate the free ends of cut spindle segments. Immediately after the second irradiation, the cut spindle segments in Cu1 rotated out of parallel with each other (C). Then they each elongated toward the opposite SPB until the free ends had passed each other (D). Next, the cut spindle segments rotated back until they were parallel to each other, forming what we are calling an apparently intact spindle (E). An apparently intact spindle is the intranuclear structure formed when the overlapping, free ends of the now parallel, elongated cut spindle segments associate laterally, giving the initial impression of a regenerated spindle. After the spindle in Cu1 was cut (B and C), the SPBs remained at nearly the same positions (C-E), whereas the SPBs moved apart rapidly after cutting of the spindle in S01-2 (H-J), migrating almost out of view in only 8 seconds. Bar, 5 µm. Dynein in aster formation and function 2611

30 in which possible mechanisms of this dynein function in N. S01-2 Control haematococca may be envisaged. Astral MTs polymerize from S01-2 CUT 25 Cu1 Control the SPBs during anaphase A and B and elongate into the

m) Cu1 CUT cytoplasm to form roughly cone-shaped arrays (Aist and µ Bayles, 1991c; Aist et al., 1991). These asters are composed 20 Laser Irradiation of numerous MTs of variable length, some of which apparently are attached to the SPBs (polar MTs), but most of which are 15 not attached directly to the SPBs (free MTs). Thus, we inferred that the astral MTs have a rapid turn over rate and exhibit 10 dynamic instability (Aist and Bayles, 1991c). Many of the astral MTs are bundled, being cross-linked by numerous Distance between SPBs ( Distance between 5 bridges (Jensen et al., 1991). A few of the astral MTs terminate in the cell cortex near the 0 plasma membrane in a tuft of fine, fibrillo-granular material, 0 60 120 180 240 300 360 suggestive of a functional interaction of the astral MT plus ends Time (sec) with some as yet unidentified component of the cortex (Aist and Bayles, 1991c; Aist and Berns, 1981). Dynactin has been Fig. 3. Plots of SPB separation of the dynein mutant, Cu1, and the found not only in animal cells, but also in the filamentous ectopic transformant-control, S01-2, following laser microbeam cutting of the spindle (CUT) or comparable control irradiation of the ascomycete N. crassa (Plamann et al., 1994; Tinsley et al., nucleoplasm (Control) at early anaphase B in N. haematococca. Note 1996). It can bind to both dynein and MTs and apparently that SPB separation was accelerated two- to threefold in S01-2 when functions to anchor dynein to subcellular binding sites on the spindle was cut. In contrast, SPB separation almost stopped when various organelles (Vallee and Sheetz, 1996). the spindle was cut in Cu1, demonstrating the absence of the astral Based on the preceding information, we suggest four pulling force in the dynein mutant. possible mechanisms for the participation of a cytoplasmic dynein-dynactin complex in aster formation in wild-type N. haematococca (Fig. 5). Such a complex may bridge polar and Therefore, the best interpretation of our results is that mitotic free MTs to each other, thus holding the free MTs in the asters cells of our dynein mutant are unable to form mitotic asters, (Fig. 5A). Cross bridging may involve additional free MTs as which points to a role for dynein in aster formation. well, to form small MT bundles that are held similarly in the Our previous studies and those of others provide a context asters (Fig. 5B). As a minus end-directed motor (Vallee, 1993),

Fig. 4. Phase-contrast, time-lapse videomicrographs of representative mitoses in living cells of wild-type T213 (A,B,C) and the CDHC-deficient mutant Cu1 (D,E,F). Elapsed time (minutes and seconds) is shown in the upper right corner of each panel. Ch, ; NE, nuclear envelope; Sp, spindle; V, vacuole. Triangles indicate the positions of SPBs. Note that chromosomes were clustered on the spindle during in both isolates (A and D). The right end of the spindle in D was momentarily out of the plane of focus. Chromatids were then segregated to the poles during anaphase A in both isolates, where they formed dark clusters near the SPBs at the beginning of anaphase B (B and E). These chromatid clusters momentarily obscured the SPBs from view. There was no discernible difference between wild type and mutant up to the beginning of anaphase B, except for a marked diminution of oscillatory and rotatory movements of the mitotic apparatus in the mutant. During anaphase B (Aist and Bayles, 1988) in both isolates, the SPBs were further separated from each other as the spindle elongated, and the nuclear envelope constricted behind each incipient daughter nucleus, which appeared as small (2-3 µm wide), pear-shaped organelles, each attached to its SPB (triangles, C and F). The spindle eventually disappeared, at the end of anaphase B (C and F). Compared to wild type (C), separation of the SPBs during anaphase B was less extensive in the mutant (F). Bar, 5 µm. 2612 S. Inoue and others

yeast retain the ability to form mitotic asters (Eshel et al., 1993; Li et al., 1993; Yeh et al., 1995) and the astral MTs are longer than in wild type (Carminati and Stearns, 1997; Cottingham and Hoyt, 1997). Aster-less mitotic apparatuses were found also in S. cerevisiae kip2∆, a kinesin-related protein mutant (Cottingham and Hoyt, 1997) and tub2-401, a β-tubulin mutant (Sullivan and Huffaker, 1992). It is now clear that several different motor proteins are involved in aster formation and the dynamics of astral MTs (Compton, 1998). The finding of aster-less mitotic apparatuses in our dynein mutant is consistent with results obtained by others using Xenopus and mammalian cells and cell-free preparations (Gaglio et al., 1996; Heald et al., 1996; Merdes et al., 1996; Vaisberg et al., 1993; Verde et al., 1991) in which astral and spindle MTs were not focused properly in the absence of functional CDHC, as well as with those from animal cells overexpressing a subunit of dynactin, in which ‘...few or no astral microtubules were visible...’ (Echeverri et al., 1996). A dynein-like motor was shown to be necessary also for the formation of radial arrays of cytoplasmic MTs in fragments of fish melanophore cells (Rodionov and Borisy, 1997). As the Fig. 5. Some possible mechanisms of cytoplasmic dynein first reported case of a CDHC-deficient mutant, in any participation in mitotic aster formation in N. haematococca, based on organism, that is unable to form mitotic asters, our work our previous and present results with this fungus and the work of supplies a key element that has been missing from the others on dynein and dynactin in other organisms. This preliminary, mounting evidence that cytoplasmic dynein has an essential hypothetical, working model embraces components of models for role in aster formation (cf. Compton, 1998; Merdes and spindle pole formation proposed by Compton (1998), Gaglio et al. Cleveland, 1997). However, the spindle poles in our dynein- (1997), Heald et al. (1997) and Merdes and Cleveland (1997), deficient mutant appeared to be normal, in contrast to the models for nuclear migration proposed by Plamann et al. (1994) and experimental results obtained with animal cells, but consistent Morris et al. (1995), and a model for astral MT dynamics derived with the results obtained with dynein mutants of the yeast, S. from the work of Carminati and Stearns (1997) and Shaw et al. (1997). fMT, free microtubule; pMT, polar microtubule; SPB, spindle cerevisiae (Eshel et al., 1993; Li et al., 1993). In view of the pole body; TC, hypothetical tethering component. A cytoplasmic disparate results mentioned above, we infer that cytoplasmic dynein-dynactin complex may (A) hold fMTs to pMTs, (B) bundle dynein may be more important in the formation of mitotic fMTs and pMTS, (C) tether pMTs to a hypothetical tethering asters in the cells of filamentous fungi and higher eukaryotes component of SPBs via dynactin, and/or (D) stabilize the plus ends (e.g. Nectria, Xenopus and mammalian cells) than in the cells of astral MTs. of lower eukaryotes, such as Dictyostelium and Saccharomyces, in which its function may be limited more specifically to anchoring the plus ends of astral MTs in the cell cytoplasmic dynein associated with polar MTs would be cortex and/or to generating a pulling force that is manifested expected to travel to the SPB. After arriving at the SPB, the via astral MTs (Carminati and Stearns, 1997; Eshel et al., 1993; dynein-dynactin complex could then tether polar MTs to the Koonce, 1996; Koonce and Samsó, 1996). Whether or not SPB by binding to the polar MTs via dynein and to a cytoplasmic dynein in N. haematococca and/or animal cells hypothetical tethering component of the SPB via dynactin (Fig. also generates the astral pulling force is an important matter 5C). In yeast, cytoplasmic dynein is localized at the cell cortex for future investigations. (Yeh et al., 1995), and disruption of the dynein gene altered the dynamics of astral MTs (Carminati and Stearns, 1997). Mechanics of anaphase B Furthermore, overexpression of a dynein-green fluorescent Several important conclusions can now be drawn about the protein fusion stabilized astral MTs, producing longer MTs and mechanics of anaphase B in N. haematococca, because of our making the asters persist longer (Shaw et al., 1997). Thus, it present in vivo observations and experiments on mitosis in seems reasonable to speculate that a cytoplasmic dynein- dynein mutant cells that are unable to form asters. First, the dynactin complex could stabilize astral MTs in N. fact that the spindle elongates without the aid of the astral haematococca via interactions with their plus ends (Fig. 5D), pulling force demonstrates, unequivocally, the presence of the thereby contributing to aster formation. Of course, further spindle pushing force, which was only inferred previously research will be required to determine if dynein is located in from the dynamics of spindle bending episodes in wild-type the asters, at the SPBs and/or in the cell cortex and to then cells (Aist and Bayles, 1991b). Second, our present laser distinguish among the four possible mechanisms depicted in microbeam results confirmed the absence of the astral pulling this preliminary, hypothetical, working model. force and the presence of the spindle pushing force in the dynein mutant. Thus, these two forces are now firmly Counterparts of the aster-less mitotic apparatus established as redundant mechanisms for separating the This is the first report of any CDHC-deficient transformant that mitotic SPBs in wild-type N. haematococca. Third, our lacks mitotic asters. By contrast, CDHC-deficient mutants of results are consistent with those obtained with other fungi Dynein in aster formation and function 2613

(Hoyt and Geiser, 1996) in demonstrating that dynein is not during mitosis in Fusarium (fungi imperfecti): New evidence from required for spindle elongation. Research from other ultrastructural and laser microbeam experiments. J. Cell Biol. 91, 446-458. laboratories has suggested that MT-associated motor proteins Aist, J. R., Liang, H. and Berns, M. W. (1992). Astral and spindle forces in PtK2 cells during anaphase B: a laser microbeam study. J. Cell Sci. 104, other than cytoplasmic dynein, perhaps a bimC kinesin- 1207-1216. related protein, may be involved in generating the spindle Barton, N. R. and Goldstein, L. S. B. (1996). Going mobile: Microtubule pushing force (Barton and Goldstein, 1996; Bloom and motors and segregation. Proc. Nat. Acad. Sci. USA 93, 1735- Endow, 1995; Saunders, 1993), and we have recently cloned 1742. Bloom, G. S. and Endow, S. A. (1995). Motor proteins 1: kinesins. Protein and sequenced a gene for such a motor protein in N. Profile 2, 1109-1171. haematococca (T. M. Sandrock et al., unpublished results). Bruno, K. S., Tinsley, J. H., Minkle, P. F. and Plamann, M. (1996). Genetic And fourth, we found that the spindle in the dynein mutant interactions among cytoplasmic dynein, dynactin and nuclear distribution can elongate under its own power (i.e., in the absence of mutants of Neurospora crassa. Proc. Nat. Acad. Sci. USA 93, 4775-4780. asters) at about 4 µm/minute. Although it has been possible Carminati, J. L. and Stearns, T. (1997). Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex. to deduce that the astral force accounts for about 50% of the J. Cell Biol. 138, 629-641. rate of the slow phase of spindle elongation in S. cerevisiae Cole, R. W., Khodjakov, A., Wright, W. H. and Rieder, C. L. (1995). A (Saunders et al., 1995; Yeh et al., 1995), this is the first time differential interference contrast-based light microscopic system for laser that the relative contribution of the astral pulling force to the microsurgery and optical trapping of selected chromosomes during mitosis in vivo. J. Microsc. Soc. Am. 1, 203-215. rate of spindle elongation in a filamentous fungus has been Compton, D. A. (1998). Focusing on spindle poles. J. Cell Sci. 111, 1477- determined. Moreover, this rate is similar to that observed 1481. during anaphase A in control cells, when asters are just Cottingham, F. R. and Hoyt, M. A. (1997). Mitotic spindle positioning in beginning to develop (Fig. 1c), and is in agreement with our Saccharomyces cerevisiae is accomplished by antagonistically acting previous inference, based on the results of cutting only one microtubule motor proteins. J. Cell Biol. 138, 1041-1053. Echeverri, C. J., Paschal, B. M., Vaughan, K. T. and Vallee, R. B. (1996). of several bundles of spindle MTs at early anaphase B with Molecular characterization of the 50-kD subunit of dynactin reveals function a laser microbeam, that the astral pulling force aids in spindle for the complex in chromosome alignment and spindle organization during elongation (Aist et al., 1991). Thus, it now seems abundantly mitosis. J. Cell Biol. 132, 617-633. clear that the substantial increase (from about 4 to about 7 Eshel, D., Urrestarazu, L. A., Vissers, S., Jauniaux, J.-C., van Vliet- µm/minute) in the spindle elongation rate occurring from Reedijk, J. C., Planta, R. J. and Gibbons, I. R. (1993). Cytoplasmic dynein is required for normal nuclear segregation in yeast. Proc. Nat. Acad. anaphase A to anaphase B in N. haematococca represents Sci. USA 90, 11172-11176. primarily the contribution of the astral pulling force to spindle Gaglio, T., Saredi, A., Bingham, J. B., Hasbani, M. J., Gill, S.R., Schroer, elongation. Although the presence of the astral pulling force T. A. and Compton, D. A. (1996). Opposing motor activities are required in animal mitosis has been known for some time now (Aist for the organization of the mammalian mitotic spindle pole. J. Cell Biol. 135, 399-414. et al., 1992), its relative contribution to spindle elongation in Gaglio, T., Dionne, M. A. and Compton, D. A. (1997). Mitotic spindle poles any animal cell is yet to be determined. are organized by structural and motor proteins in addition to . J. Cell Biol. 138, 1055-1066. We thank Dr T. C. Huffaker for his useful advice during the project. Hagan, I. and Yanagida, M. (1997). Evidence for -specific, spindle We appreciate the access provided to his laser microbeam facility by pole body-mediated, nuclear positioning in the fission yeast Dr Conly L. Rieder and the expert technical assistance of Mr Richard Schizosaccharomyces pombe. J. Cell Sci. 110, 1851-1866. Cole during the laser microbeam experiments. 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