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Fungal Genetics and Biology 37 (2002) 171–179 www.academicpress.com

The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa Meritxell Riquelme,a,1 Robert W. Roberson,b,* Dennis P. McDaniel,c n Bartnicki-Garcıad and Salomo b

a Department of Plant Pathology, University of California, Riverside, CA 92521-0122, USA Department of Plant Biology, Molecular and Cellular Biology Program, Arizona State University, Tempe, AZ 85287-1601, USA c W.M. Keck Bioimaging Laboratory, Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA d Centro de Investigaci on Cientıfica y de Educaci on Superior de Ensenada (CICESE), 22830, Baja California, Mexico

Received 19 February 2002; accepted 11 June 2002

Abstract We have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa and to better understand the role(s) of dynein during hyphal tip growth. Based on video-enhanced DIC light microscopy, the mature, growing hyphae of N. crassa wild type could be divided into four regions according to cytoplasmic organization and behavior: the apical region (I) and three subapical regions (II, III, and IV). A well-defined Spitzenk€ orper dominated the cytoplasm of region I. In region II, vesicles (0.48 lm diameter) and mitochondria maintained primarily a constant location within the advancing cytoplasm. This region was typically void of nuclei. Vesicles exhibited anterograde and retrograde motility in regions III and IV and followed generally parallel paths along the longitudinal axis of the cell. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position in the advancing cytoplasm in regions III and IV. Many nuclei occupied the cytoplasm of regions III and IV. In ro-1 hyphae, discrete cytoplasmic regions were not recognized and the motility and/or positioning of vesicles, mitochondria, and nuclei were altered to varying degrees, relative to the wild type cells. Immunofluorescence microscopy revealed that the microtubule cytoskeleton was severely disrupted in ro-1 cells. Transmission electron microscopy of cryofixed cells confirmed that region I of wild-type hyphae contained a Spitzenk€ orper composed of an aggregation of small apical vesicles that surrounded entirely or partially a central core composed, in part, of microvesicles embedded in a dense granular to fibrillar matrix. The apex of ro-1 the hypha contained a Spitzenk€ orper with reduced numbers of apical vesicles but maintained a defined central core. Clearly, dynein deficiency in the mutant caused profound perturbation in microtubule organization and function and, consequently, organelle dynamics and positioning. These perturbations impact negatively on the organization and stability of the Spitzenk€ orper, which, in turn, led to severe reduction in growth rate and altered hyphal morphology. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Neurospora crassa; Ropy; Dynein; Laser scanning confocal microscopy; Microtubules; Spitzenk€ orper; Ultrastructure; Video-enhanced light microscopy

1. Introduction The cytoskeleton is the platform on which cytoplasmic organization is maintained and organelle movement and positioning are controlled. Our understanding of the mechanistic aspects of intracellular motility in fungal *

Corresponding author. Fax: +480-965-6899. E-mail address: [email protected] (R.W. Roberson). 1 Present Address: Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB, England.

hyphae has been expanded by the identification and subsequent characterization of mutants defective in microtubule-associated proteins and molecular motors (Lee and Plamann, 2001). For example, sequence analysis of the nudA and nudG genes in nuclear distribution mutants of Aspergillus nidulans revealed that they encode, respectively, the heavy and the 8-kDa light chains of cytoplasmic dynein (Xiang et al., 1994; Beckwith et al., 1998). Mutant strains of Nectria haematococca (Inoue et al., 1998) and Neurospora crassa (Plamann et al., 1994; Bruno et al., 1996; Tinsley et al., 1996; Minke

1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 0 6 - 6

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et al., 1999a; Minke et al., 1999b) that are defective in components of the dynein complex display similar nuclear distribution phenotypes as the Aspergillus nud mutants. Cytoplasmic dynein is a multisubunit protein that functions as a minus-end-directed microtubule molecular motor in eukaryotic cells. Dynein and dynactin are involved in the transport and/or positioning of membranous organelles, e.g., vesicles, mitochondria, nuclei, Golgi complex, and endoplasmic reticulum (Yamashita and May, 1998). Molecular characterization of the ropy-1 (ro-1)2 mutation in N. crassa revealed that it is deficient in one of the heavy chains of cytoplasmic dynein (Plamann et al., 1994). This genetic lesion results in a variety of phenotypes including: distortion of hyphal morphology (Garnjobst and Tatum, 1967), disruption of nuclear movement and positioning (Plamann et al., 1994), reduced cell growth rates, and altered Spitzenk€ orper organization and behavior (Riquelme et al., 1998; Riquelme et al., 2000). Studies of germlings and mature hyphae of several ro mutants describe additional phenotypes such as disruption of conidia germination, displacement of septa position, and atypical mitochondrial morphology (Minke et al., 1999a,b). Contrary to the conclusions reached by Seiler et al. (1999), we found by video-enhanced light microscopy (VELM) that ro-1 mutants have a seriously impaired Spitzenk€ orper. In ro-1 and ro-3 mutants, the Spitzenk€ orper is not only much smaller than the wild type Spitzenk€ orper but its size and trajectory are more variable (Riquelme et al., 2000). From our observations of ropy mutants and previous findings on wild-type N. crassa (Riquelme et al., 1998), we proposed that the functional and structural integrity of the microtubular cytoskeleton was essential for regular hyphal morphogenesis. To test the validity of this hypothesis, and to explore the impact of dynein deficiency in hyphal tip growth, we have used VELM and laser scanning confocal microscopy (LSCM) plus transmission electron microscopy (TEM), to compare cytoplasmic organization, organelle motility, microtubule distribution, and Spitzenk€ orper organization in a wild-type strain vs. ro-1 mutant of N. crassa.

Stock Center (Department of Microbiology, University of Kansas Medical Center, Kansas City, KS). The strains were grown at ca. 21 °C in 8.5 cm plastic petri dishes containing 20 ml of VogelÕs complete medium (VCM) (Vogel, 1956) with 1.5% (w/v) sucrose as the carbon source. 2.2. Video-enhanced light microscopy Hyphal growth and cytoplasmic behavior were documented and analyzed using VELM. Hyphae were grown on sterile glass slides pre-coated with a thin layer (1–2 mm) of VCM. An Axioskop microscope (Carl Zeiss, Thornwood, NY) with differential interference contrast (DIC) optics (Plan-Neofluar 100x/1.3 N.A. oil immersion objective lens and 1.4 N.A. oil immersion condenser lens) was used to visualize selected hyphae. The microscope was also equipped with infrared and green (550 nm) filters to reduce heat and chromatic aberration (C. Bracker, personal communication), respectively, placed in between the light source and condenser. Images were captured with a Hamamatsu C2400-07 (tube type) video camera coupled to an analog camera control unit (Hamamatsu Photonic Systems Corp., Bridgewater, NJ). Real time digital contrast enhancement was done with an Argus 10 image processor (Hamamatsu Photonic Systems Corp.). Motion sequences were recorded at 30 frames/s with a Panasonic AG-6370 S-VHS videocassette recorder (Panasonic Broadcast and Television Systems Company, Seacaucus, NJ) on S-VHS videotape. For tracking intracellular motion, video sequences were digitized (30 frames/s) with Adobe Premiere 4.2 (Adobe Systems, Mountain View, CA). Selected frames were exported to Adobe Photoshop 6.0 (Adobe Systems) where organelle position was tracked over time. The velocity of organelles was determined by plotting their positions at time intervals of 0.5 to 1 s. Vesicle size was measured with Argus 10 software. All measurements of organelle velocities and sizes represent the mean +/) standard deviation (SD). For image presentation, single frames from videotaped sequences were digitized and further processed in Photoshop 6.0 (Adobe Systems) and printed using an NP-1600M Medical Color Printer (Codonics, Inc., Middleburg Heights, OH).

2. Methods and materials 2.3. Laser scanning confocal microscopy 2.1. Organism and media N. crassa wild-type (FGSC 988) and ro-1 (FGSC 4351) strains were obtained from the Fungal Genetics 2 Abbreviations: DAPI 40 , 6-diamidino-2-phenylindole; DIC, differential interference contrast; LSCM, laser scanning confocal microscopy; ro-1, Ropy-1; TEM, transmission electron microscopy; VELM, video-enhanced light microscopy; VCM, VogelÕs Complete Medium.

Reliable preservation and labeling of microtubules in hyphae of N. crassa were obtained using the cryofixation/freeze substitution and methacrylate de-embedment protocol reported by Bourett et al. (1998), with only few modifications. In brief, small portions of actively growing mycelia were aseptically transferred onto 5  7 mm pieces of sterile dialysis membrane on VCM. After 6 to 8 h, the membranes with the hyphae were

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cryofixed in cold ()20 °C) anhydrous acetone. Cryofixed cells were freeze-substituted for 72 h at )85 °C in anhydrous acetone (20 ml), rinsed in several changes of cold, anhydrous ethanol, and then warmed to room temperature in ethanol containing 2% formaldehyde. After sample rinsing in ethanol, they were infiltrated with butyl-methyl methacrylate (methacrylate; 20 ml butyl methacrylate, 5 ml methyl methacrylate, 0.25 g benzoin methyl ether, and 0.4 mg DTT; T. Bourett, personal communication), flat embedded between sheets of Teflon (FEP 3000 L, Dupont, Wilmington, DE), and UV polymerized for 24 h under nitrogen gas at 0 °C in an FSU 010 Freeze-Substitution Unit (Bal-Tec Products, Inc., Middlebury, CT). The resin was removed from the cells, i.e., de-embedded, at 4 °C in 3 changes of acetone over a period of 6 to 12 h and then rehydrated in phosphate buffer (0.1 M, 6.8 pH) amended with saline (0.15 M; PBS). The cryofixation/freeze substitution and methacrylate de-embedment protocol did not require the use of enzymatic treatments to partially digest the cell wall, which may compromise cytoplasmic integrity. Cells were blocked with 10% goat serum and 3% bovine serum albumin (BSA) in PBS for 1 h and incubated overnight at 4 °C in an a-tubulin monoclonal antibody (Accurate Chemical and Scientific Corporation, Westbury, NY) diluted 500 fold in PBS/BSA (PBS, 0.1% BSA, pH 6.8, and 0.02% sodium azide). After rinsing in PBS/BSA, the cells were incubated overnight at 4 °C in an anti-mouse secondary antibody (goat anti-mouse conjugated to Alexa 488 [Molecular Probes, Eugene, OR]) diluted 100 fold in PBS/BSA. Following the removal of secondary antibody by rinsing with PBS, the DNA fluorophore 4,6-diamidino-2-phenylindole (DAPI) was applied to cells at a concentration of 0.1 lg/ ml in H2 O for 5 to 10 min. Rinsed cells and dialysis membrane were mounted cell side up on a clean glass slide in a drop of 90% glycerol:10% PBS (pH 8.6) containing 0.1% n-propyl gallate to retard photobleaching and overlaid with a clean coverslip. Specimens were examined and images were collected by LSCM using a Leica TCS NT (Leica Imaging System, Exton, PA). Lasers used for excitation of fluorochromes included an argon/UV laser for DAPI and an argon laser for Alexa 488. Hyphae were optically scanned longitudinally from top to bottom as a series of 16, 0.5 lm-thick sections and illustrated as composites of all sections (Figs. 31–42) or as composites of four sections through the upper (Fig. 27) median (Figs. 28, 29) and lower (Fig. 30) focal planes. Digitized images were processed in Scion 3.0 (http://www.scioncorp.com) or Photoshop 6.0 (Adobe Systems) and printed as described above. 2.4. Transmission electron microscopy Fixation and preparation of cells for TEM were carried out using cryofixation and freeze substitution

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methods described in McDaniel and Roberson (2000). Thin sections were examined and images were recorded using a Philips CM12S TEM (Philips Electronic Instruments, Co., Mahwah, NJ) coupled to a Gatan 689 CCD digital camera (1024  1024 pixel area; Gatan Inc., Pleasanton, CA). Digitized images were processed in Photoshop 6.0 (Adobe Systems) and printed as described above.

3. Results 3.1. Cytoplasmic organization and organelle motility In the wild-type strain, mature leading hyphae that made up the advancing edge of the mycelium and exhibited optimal growth characteristics, i.e., hyphoidshaped tip, growth rates of ca. 0.37 lm/sec (0:05) (N ¼ 25), and the presence of a well-defined, apically positioned Spitzenk€ orper (Figs. 1–6), were chosen for light microscope observations. Based on video-enhanced DIC optical microscopy of 45 mature hyphae, four cytoplasmic regions (I, II, III, and IV) were recognized (Figs. 6–8). Smaller secondary hyphae were located behind the leading edge of the mycelium and were not selected for analysis in this study. Region I extended back approximately 2.0 to 3.0 lm from the apical pole (Fig. 6) and corresponded to the hyphal apex, as defined by Bartnicki-Garcıa (1990) and L opez-Franco and Bracker (1996). In this region, a well-defined Spitzenk€ orper (1.25 lm [ 0.23] diameter, N ¼ 10) dominated the cytoplasm. One or two large spherical organelles ranging from 1.0 to 1.5 lm in diameter were observed occasionally near the Spitzenk€ orper (Fig. 6). Region II extended 10 to 20 lm behind region I (Fig. 6). Vesicles [ca. 0.48 lm (0:11, N ¼ 45) diameter] and mitochondria were the dominant structures in this region and moved forward with the advancing cytoplasm. This region was typically void of nuclei. Region III extended subapically 30 to 40 lm behind region II. Nuclei were present in the cytoplasm of region III (Fig. 7). Rapid movements of vesicles characterized by frequent starts and stops, i.e., saltatory motility (Rebhun, 1972), that were independent of the overall cytoplasmic migration and movements of other adjacent organelles, typified both the central and cortical cytoplasm of regions III and IV. These vesicles exhibited anterograde or retrograde saltatory motility (Figs. 9–14) at approximate rates of 3.1 (0:36) lm/sec (N ¼ 30) and 3.2 (0:56) lm/ sec (N ¼ 30), respectively. Region IV was distinguished from region III by an increased density of nuclei, mitochondria, and vesicles (Fig. 8). Mitochondria and nuclei migrated in an anterograde manner in regions III and IV via bulk cytoplasmic flow. A small population of mitochondria displayed rapid anterograde and retrograde saltatory movements in regions III and IV.

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Figs. 1–26. Video-enhanced DIC images in mature hyphae of wild-type (1–14) and ro-1 (15–26) strains of N. crassa. Numbers in upper right corner of images indicate time (min:sec) elapsed. 1–5. Time-lapsed view of wild-type hyphal elongation. The Spitzenk€ orper (arrows) is located within the hyphal apex during this 1:55 min growth sequence. Little changes in tip morphology, growth rate, or growth direction were observed. Bar ¼ 5 lm. 6– 8. Four cytoplasmic regions of N. crassa wild-type hyphae were recognized in this study (I, II, III, and IV) and illustrated in this individual hypha. The Spitzenk€ orper (Spk), large spherical organelles (arrowhead), small vesicles (arrows), long mitochondria (M), and nuclei (N) are noted. Bar ¼ 5 lm. 9–14. Region III of N. crassa illustrating the motility of a small vesicle in the cortical cytoplasm. In (14), a single frame from the sequence is brightened and overlaid with circles and lines tracing the position and movement of the vesicle during this sequence. Bar ¼ 0.7 lm. 15–19. Timelapsed view of ro-1 hyphal elongation and cytoplasmic organization. Discrete cytoplasmic regions in ro-1 were not detectable and, when present, the Spitzenk€ orper was small and difficult to discern. Small vesicles (arrows) maintain a close position to the Spitzenk€ orper during this 4:33 min sequence. A change in the Spitzenk€ orper position (19) leads to a change in tip morphology and possibly a shift in the direction of hyphal growth. Large irregularly shaped inclusions (asterisk) and vacuoles were common in subapical regions. Bar ¼ 1.5 lm. 20. Subapical region of ro-1 illustrating large irregularly shaped inclusion (asterisk). Bar ¼ 1.5 lm. 21–26. Anterograde and retrograde saltatory motility of small vesicles (arrowheads) were greatly reduced in ro-1 cells. Bar ¼ 4.8 lm.

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The hyphae of ro-1 showed no discrete cytoplasmic regions recognizable by DIC optics and the entire cytoplasm appeared less dense. When present, the Spitzenk€ orper was small (0.85 lm diameter [0:22, N ¼ 10]) and difficult to discern (Figs. 15–19). Subapical regions contained large irregularly shaped inclusions (Figs. 19, 20) composed of a granular matrix that exhibited a constant churning or mixing motion when viewed by VELM. Mitochondria, nuclei and, for the most part, vesicles (Figs. 21–26) displayed primarily bulk cytoplasmic flow. Anterograde or retrograde saltatory vesicle motility was rarely observed in ro-1 cells. 3.2. The microtubule cytoskeleton and nuclear distribution Wild-type mature hyphae contained abundant microtubules (Figs. 27–42). Microtubule organization followed an alignment that was mostly parallel to the growing axis of the hypha. Nuclei were rarely seen in region II but were distributed throughout the cytoplasm of regions III and IV (Figs. 31, 38, 39). Associations between microtubules and nuclei were common in region IV (Figs. 37–39) and likely reflected direct microtubule association with spindle pole bodies. In ro-1 mutants, microtubules were less abundant and more often obliquely oriented and variously curved (Figs. 32–36, 40–42) relative to wild-type hyphae. Associations between microtubules and nuclei occurred throughout the cytoplasm (Figs. 32, 40–42). In distal hyphal regions, aggregations of nuclei were common (Figs. 41, 42). Little evidence of cellular damage due to processing for immunofluorescence labeling was observed when labeled cells were viewed with DIC optics (not shown). 3.3. Transmission electron microscopy In wild-type hyphae of N. crassa, the Spitzenk€ orper was composed of an aggregation of apical vesicles (ca. 85 nm diameter [7:5], N ¼ 55) surrounding entirely or partially a differentiated core composed of microvesicles (ca. 35 nm diameter [7:2], N ¼ 35) embedded in a dense granular to fibrillar matrix (Figs. 43, 44). Apical vesicles contained a finely granular, electron-opaque matrix and were also widely distributed throughout all cytoplasmic regions (Figs. 43, 46–48). Vesicles with an electrontransparent lumen (ca. 0.15 lm diameter [47], N ¼ 75) and multivesicular bodies (ca. 0.2 lm diameter [46], N ¼ 46) were abundant throughout hyphal cytoplasmic regions II, III, and IV but were rarely seen in region I (Figs. 45, 46–49). Spherical to hexagonal shaped organelles, containing an electron-opaque matrix, were observed in close proximity to the Spitzenk€ orper (Fig. 45). Solitary and bundled microtubules were located throughout the cytoplasm, including the Spitzenk€ orper (Figs. 43, 44, 46, 47). Close associations (ca. 14 to 22 nm,

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N ¼ 20) between microtubules and other cellular components, e.g., apical vesicles, electron-transparent vesicles (Figs. 46, 47), multivesicular bodies, mitochondria, nuclei (not shown), were often observed. Typical organelles were present throughout the subapical cytoplasmic regions (Figs. 48, 49). Golgi body equivalents were often in close proximity to mitochondria (Fig. 48). Due to the poor cryopreservation of region IV, fewer details of the cytoplasmic organization were available. In ro-1 hyphae that contained a recognizable Spitzenk€ orper, the numbers of associated apical vesicles and microvesicles were greatly reduced relative to wild-type Spitzenk€ orper (Figs. 50–55). Golgi body equivalents and mitochondria were often present in apical regions of hyphae (Figs. 50–53). Though not abundant, microtubule profiles were observed throughout the cytoplasm (e.g., Fig. 50). In subapical regions of ro-1 hyphae, large aggregations of vesicles with an electron translucent lumen and multivesicular bodies were present (Figs. 54, 55). Aggregates of apical and microvesicles were not observed.

4. Discussion To better understand the contributions of dynein towards sustaining hyphal tip growth and to complement previous studies, we have compared the cytoplasmic organization and dynamics of mature leader hyphae in wild-type and ro-1 mutant strains of N. crassa using light and electron microscopy. Our current findings reinforce previous conclusions that the microtubule cytoskeleton of N. crassa plays a major role in apical growth (Riquelme et al., 1998, 2000) and also that dynein and dynactin deficiencies of two ropy mutants distorted hyphal morphogenesis by destabilizing the Spitzenk€ orper and causing it to deviate widely from an axial trajectory (Riquelme et al., 2000). Although Seiler et al. (1999) reported a prominent Spitzenk€ orper in N. crassa ro-1 hyphae, TEM of cryofixed cells confirmed previous phase contrast microscopy findings (Riquelme et al., 2000). The EM images show unmistakably that the Spitzenk€ orper of ro-1 was greatly diminished, having fewer apical vesicles associated with its core. Reduced Spitzenk€ orper size has also been observed in dynein-deficient mutants of Nectria haematococcum (Inoue et al., 1998) and kinesin-deficient mutants of N. haematococcum and N. crassa. (Wu et al., 1998; Seiler et al., 1997, 1999), suggesting that dynein and kinesin are both involved in the formation of the Spitzenk€ orper in these cells. Interestingly, the Spitzenk€ orper core of ro-1 cells maintained a discrete organization, suggesting a degree of independence with the surrounding cloud of apical vesicles. This has been suggested in previous cell growth and inhibitor studies (Grove and Sweigard, 1981, 1996; Roberson unpublished observations).

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Figs. 27–42. LSCM showing the microtubule cytoskeleton and nuclei in mature hyphae of wild-type (27–31, 37–39) and ro-1 (32–36, 40–42) strains of N. crassa. 27–31. LSCM demonstrates the abundance of microtubules and nuclei in a wild-type hypha. Microtubules were generally aligned parallel along the growing axis of the hypha. Some were obliquely oriented and organized into bundles. Bar ¼ 5 lm. 32. Microtubules (arrows) and nuclei (asterisks) in ro-1 hypha. Microtubules were less abundant and their organization was altered in ro-1 hyphae. Associations between microtubules and nuclear positions were observed (arrowheads). Bar ¼ 5.8 lm. 33–36. LSCM through ro-1 hyphae illustrating microtubules (arrows) and nuclei (arrowheads). Bars ¼ 4.2 lm (33, 35) and 2.8 lm (34, 36). 37–39. LSCM of region IV of wild-type hypha illustrating microtubule (37) and nuclear distribution (38). Fig. 39 represents an overlay of Figures 37 and 38. Associations between microtubules and nuclear positions were common (arrowheads). Bar ¼ 2.5 lm. 40–42. Microtubule (40) and nuclear (41) distributions in the subapical region of ro-1 hypha. Fig. 42 represents an overlay of figures 40 and 41. Associations (arrowheads) between microtubules and nuclei were observed. Bar ¼ 12.5 lm.

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Figs. 43–49. Transmission electron microscopy of cryofixed and freeze-substituted wild-type hyphae of N. crassa. 43. Near-median longitudinal section through hypha illustrating portions of regions I and II. The Spitzenk€ orper was composed of a cloud of apical vesicles (arrows) surrounding a core (asterisk). Microtubules (arrowheads) and mitochondria (M) are noted. The lack of a true hyphoid apical profile reflects a slight disruption of growth just prior to cryofixation. Bar ¼ 3.3 lm. 44. High magnification median view through the Spitzenk€ orper core (asterisk). This specialized cytoplasmic area consisted of a granular to fibrillar matrix in which microvesicles (arrowheads) were embedded. Note apical vesicles (white arrows) and oblique view of a microtubule (black arrow). Bar ¼ 0.25 lm. 45. Section through region I showing electron-opaque hexagonal organelles (arrows) in close proximity to the Spitzenk€ orper. Apical vesicles and vesicle with electron transparent matrix (arrowhead) are present. Bar ¼ 28 lm. 46, 47. High magnification of cortical cytoplasm in region III showing close associations of apical vesicles (white arrows) and electron-transparent vesicles (asterisk) with microtubules (black arrows). Bars ¼ 0.16 lm. 48. Longitudinal section through region III illustrating profiles of nuclei (N), nucleoli (asterisk), mitochondria (M), endoplasmic reticulum (white arrows), and numerous electron transparent vesicles (arrowheads). Golgi body equivalents (black arrows) were often in close proximity to mitochondria. Bar ¼ 1.4 lm. 49. Enlarged region of central cytoplasm of region III. Nuclei (N), nucleoli (asterisk), Golgi body equivalent (G), vacuoles (V), and vesicles with electron transparent matrix (arrowheads) are pointed out. Bar ¼ 0.47 lm.

The reduction of Spitzenk€ orper size in N. crassa was accompanied, and probably caused, by the perturbation of the ropy-1 microtubule cytoskeleton. Alterations in microtubule distribution have been reported in related studies of Ashbya gossypii (Alberti-Segui et al., 2001) and A. nidulans (Han et al., 2001). However, in previous studies of ropy strains of N. crassa (Minke et al.,

1999a,b) and dynein-deficient stains of N. haematococcum (Inoue et al., 1998; Wu et al., 1998), little or no discernable microtubule perturbations were noted. In our view, it is clear that dynein deficiency causes profound disruption in the microtubule distribution and function. Such perturbations resulted in cytoplasmic disorganization and altered intracellular motility that

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Figs. 50–55. Transmission electron microscopy of cryofixed and freeze-substituted hyphae of the ro-1 mutant of N. crassa. 50. Near-median longitudinal section of the hyphal tip. The number of apical vesicles (arrows) associated with the Spitzenk€ orper was greatly reduced. Interestingly, the presence and size of the Spitzenk€ orper core (asterisk) were less susceptible to the effects of the mutation. Cisternae of Golgi body equivalents (G), mitochondria (M), microtubules (arrowheads), and vesicles with electron transparent matrix (V) are noted. Bar ¼ 0.36 lm. 51–53. Three consecutive sections of hyphal apex of figure 50 showing distributions of apical vesicles (arrows), Golgi body equivalents (G), and mitochondria (M). Bar ¼ 0.3 lm. 54, 55. In subapical regions of ro-1 hyphae large aggregations of vesicles with an electron transparent matrix were common (asterisk). Multivesicular bodies (arrowheads) are also associated with these vesicle clusters. A mitochondrion is noted (M). Subapical regions in ro-1 hyphae, were very susceptible to freeze damage noted by reticulate appearance of membrane. Bar ¼ 0.5 lm (54) and 0.18 lm (55).

impacted negatively on the organization and stability of the Spitzenk€ orper. In the end, this led to severe reduction in growth rate and altered hyphal morphology. Saltatory motility of vesicles was abundant and easily observed in wild-type hyphal regions III and IV using VELM. The diameter of these vesicles (median crosssectional area) was ca. 0.48 lm, based on VELM measurements. Similar sized vesicles were observed in wild-type hyphae and cells of wall-less mutants of Neurospora (Steinberg and Schliwa, 1993; Seiler et al., 1999). It is difficult to identify the ultrastructural equivalent of these vesicles. However, based on cytoplasmic distribution and association with microtubules,

we conclude that their ultrastructural counterparts were electron-transparent vesicles (ca. 0.14 lm diameter) and/ or multivesicular bodies (ca. 0.2 lm diameter). The discrepancies in the diameters measured by VELM and TEM were due to the fact that structures smaller than the limit of resolution for light microscopy (0.25 lm) may be detected but their measurable diameter will be no smaller than the diffraction limit of the optical system (Inoue and Spring, 1997). The intracellular dynamics of these vesicles are useful indicators for cytoskeletal function; however, their role(s) in hyphal growth is not clear. Transmission EM data revealed that electrontransparent vesicles and multivesicular bodies aggregate

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into large, irregularly shaped clusters in the subapical hyphal regions in ro-1 mutants and represent the large inclusions observed using VELM. These data indicate a direct role for microtubules and dynein in maintaining a correct cytoplasmic distribution and motility of these vesicles. Similar roles for dynein/dynactin have been suggested for nuclear distribution (Plamann et al., 1994; Xiang et al., 1994; Inoue et al., 1998). Aggregates of other vesicle types, i.e., apical and microvesicles, were not observed in ro-1 hyphae. In conclusion, our results support the notion that the dynein motor is required for the proper distribution and function of microtubules. In the absence of dynein, the cytoplasm becomes partially disorganized and the function and organization of the Spitzenk€ orper are significantly compromised.

Acknowledgments Research at UC Riverside was supported in part by University resources and resources accumulated during the many years when the NIH and the NSF provided SBG with generous grant support, the most recent being GM-48257 and IBN-9204541 respectively. This work was initially supported by a Graduate DeanÕs Dissertation Research Grant from the University of California, Riverside to MR. Research at ASU was supported by the Plant Biology Department. Standard epifluorescence and LSCM were performed in the W.M. Keck Bioimaging Laboratory (ASU). Electron microscopy was performed in the Life Sciences Electron Microscopy Facility (ASU).

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