Cytoplasmic Bulk Flow Propels Nuclei in Mature Hyphae of Neurospora crassa

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1 EUKARYOTIC CELL, Dec. 2009, p Vol. 8, No /09/$12.00 doi: /ec Copyright 2009, American Society for Microbiology. All Rights Reserved. Cytoplasmic Bulk Flow Propels Nuclei in Mature Hyphae of Neurospora crassa Silvia L. Ramos-García, 1 Robert W. Roberson, 2 Michael Freitag, 3 Salomón Bartnicki-García, 1 and Rosa R. Mouriño-Pérez 1 * Departamento de Microbiología, División de Biología Experimental y Aplicada, Centro de Investigación Científica y Educación Superior de Ensenada, Ensenada, Mexico 1 ; School of Life Sciences, Arizona State University, Tempe, Arizona 2 ; and Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon 3 Received 25 February 2009/Accepted 10 August 2009 We used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassa whose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1, and a ro-1 kin-1 double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT strain and that the ro-1 kin-1 double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants ro-1, ro-3,orkin-1 did not prevent the anterograde and retrograde migration of nuclei; however, in the ro-1 kin-1 double mutant retrograde migration was absent. In the WT strain, almost all nuclei were elongated, whereas in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The ro-1 mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Motor proteins probably play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules. Organelle movement and positioning are important aspects of cell growth and differentiation (19, 20, 27, 35). Movement and positioning of nuclei are especially important because of their implications in mitotic divisions during hyphal growth and asexual sporulation (conidiation), as well as fertilization events leading to meiosis and ascospore formation during sexual development (1, 3, 33). In yeast, nuclei move comparatively short distances (20, 32), whereas in filamentous fungi nuclei are typically transported over long distances within hyphae (1, 34, 35). Movement of nuclei in fungal cells may be either an active or a passive process. Early studies of filamentous fungi showed nuclei uniformly distributed along the entire hypha; they appeared to move with the growing hyphal apex, keeping a more or less constant distance from the cell tip. Such evidence pointed to passive displacement of nuclei by cytoplasmic bulk flow (10 12, 24), a role confirmed in our recent study on the * Corresponding author. Mailing address: Departamento de Microbiología, División de Biología Experimental y Aplicada, Centro de Investigación Científica y Educación Superior de Ensenada, Km 107, Carretera Tijuana Ensenada, Ensenada 22860, Mexico. Phone: Fax: , ext rmourino@cicese.mx. Supplemental material for this article may be found at Published ahead of print on 14 August dynamics of the microtubular cytoskeleton (28) and supported by studies with injected lipid droplets (17). Upon the discovery of motor proteins and their role in nuclear migration and positioning in filamentous fungi, attention was primarily focused on the participation of motors in nuclear events, including the movement of nuclei during hyphal extension (15, 25, 26, 29, 37), while the role of cytoplasmic bulk flow was largely discounted or disregarded. Whereas much effort has been directed toward the characterization of the components involved in motor-driven nuclear transport, the relative importance of passive nuclear propulsion has remained an open question. For the purpose of distinguishing clearly between active migration and passive displacement, we will consider migration to mean an active, motor-dependent process, while displacement will refer to passive transport of nuclei within the hypha. Movement refers either to active or passive transport of nuclei through hyphae. Here, we used strains of Neurospora crassa whose nuclei were tagged with green fluorescent protein (GFP) to examine the dynamics and distribution of nuclei in growing hyphae. In addition to evaluating nuclear movement in a wildtype (WT) strain, we examined the dynamics of nuclear movement in mutants defective in microtubule-related motor proteins: a ro-1 mutant for its deficiency in the heavy chain of dynein, a ro-3 mutant deficient in the dynactin p150 glued subunit, a kin-1 mutant deficient in conventional kinesin, and a 1880

2 VOL. 8, 2009 NUCLEAR DISPLACEMENT IN N. CRASSA 1881 Strain TABLE 1. Strains and genotypes Genotype ro-1 kin-1 a dynein-kinesin double mutant. We also tested the effect of drugs that inhibit specifically microtubules and actin microfilaments. Our study demonstrates that passive displacement plays a major role in nuclear dynamics in growing hyphae of N. crassa. MATERIALS AND METHODS Source or reference N22813A mat A his-3 ::Pccg-1-hH1 -sgfp 8 NRM104B mat a kin-1 31 FGSC4351 mat a ro-1 FGSC FGSC43 mat a ro-3 FGSC FGSC4352 mat A ro-1 FGSC FGSC3 mat A ro-3 FGSC FRM01AR mat A his-3 ::Pccg-1-drfp -hh1 This study heterokaryon mat A his-3 ::Pccg-1-Bml -sgfp XRM1752 mat A his-3 ::Pccg-1-hH1 -sgfp ro-1 M. Plamann XRM1779 his-3 ::Pccg-1-Bml -sgfp ro-1 This study XRM1764 mat a his-3 ::Pccg-1-hH1 -sgfp ro-3 This study XRM1781 his-3 ::Pccg-1-Bml -sgfp ro-3 This study XMF10258 mat a his-3 ::Pccg-1-hH1 -sgfp kin-1 This study XMF11343 his-3 ::Pccg-1-Bml -sgfp kin-1 This study XRMC0414 mat A his-3 ::Pccg-1-hH1 -sgfp ro-1 kin-1 This study Strains and culture conditions. Strains used in the present study are listed in Table 1. Strains were maintained on Vogel s minimal medium (VMM) with 2% sucrose. All manipulations were performed according to standard techniques (5). Crosses to obtain strains with motor protein mutations and GFP-labeled nuclei. The homokaryotic strain mat A his-3 ::Pccg-1-hH1 -sgfp (N2281-3A [7, 9]) was crossed to strains that carry dynein and/or kinesin mutations (ro-1, FGSC4352; ro-3, FGSC3; kin-1, NRM104B [31]; and ro-1 kin-1, XRMC0414) to obtain homokaryotic hh1 -sgfp strains with the respective mutation. Strains were crossed routinely in petri plates on synthetic crossing medium (SCM) supplemented with 1% sucrose and 2% agar (5). For each cross, the protoperithecial parent was first grown on medium for 5 days at 25 C and then fertilized by adding conidia from the second parent. After 14 days of incubation at 25 C, ascospores from the developed perithecia were collected from the petri dish cover with distilled water. Ascospores were spread onto VMM, heat shocked at 60 to 67 C for 40 to 60 min, and incubated for 12 h at 28 C. Colonies were transferred to 1- or 5-ml culture tubes with VMM and incubated for 24 h at 28 C. Strains were screened for GFP expression and the expected growth phenotype of the respective motor mutant. To construct the ro-1 kin-1 double mutant, we crossed mat A; ro-1 to mat a; kin-1. To validate the double mutant, it was crossed with the N. crassa WT strain, and both mutants were recovered from the progeny. Double labeling with -tubulin-gfp and drfp-histone1. In order to observe the relationship between microtubules and nuclei, we constructed a heterokaryon from two strains of N. crassa, one with -tubulin-gfp (9) and the other with drfp-histone1 ( drfp is shorthand for the tdimer2 [12] variant of RFP [8]). A petri plate with VMM was inoculated with spores of both strains and incubated for 10 h at 28 C. Hyphae from each strain fused forming an interconnected colony. The margin of the colony was screened to detect hyphae displaying double fluorescence. These hyphae were imaged according to the procedure described below for laser scanning confocal microscopy. Microtubule and actin depolymerization assays. Stock solutions of benomyl (BML; methyl 1-[butyl-carbamoyl]-2-benz-imidazolecarbamate; Sigma) (14) and cytochalasin A (CA; Sigma) at 10 mg ml 1 were prepared in 100% ethanol. To examine BML and CA sensitivity, serial dilutions of the drug were tested on VMM plates at 10-fold increments (0.01 to 10 g ml 1 ). The concentration that inhibited hyphal growth rate by 50% was selected for further studies. To study the effect of the anti-microtubule (Mt) drug BML (2.5 g ml 1 ) or the antimicrofilament drug CA (1.0 g ml 1 ) on nuclear distribution and movement in N. crassa, we inoculated VMM plates with either drug and incubated them at 28 C until the cells reached a young mycelium stage ( 16 h). Mycelia were observed according to the procedure described below for laser scanning confocal microscopy. Hyphae or nuclei TABLE 2. Number of measurements performed in hyphae and nuclei WT ro-1 mutant No. of measurements in N. crassa strain ro-3 mutant kin-1 mutant ro-1 kin-1 mutant BMLtreated WT CAtreated WT Hyphae 718 1, Nuclei 6,844 4,800 4,068 1,574 9,300 4,506 1,772 Laser scanning confocal microscopy of living cells. Neurospora crassa H1::GFP (WT), ro-1 H1::GFP, ro-3 H1::GFP, kin-1 H1::GFP, and ro-1 kin-1 H1::GFP strains were grown on water agar with no added nutrients and the H1::GFP WT in the presence of BML and CA. Water agar was used to minimize background fluorescence. After inoculation, the plates were incubated until the diameter of the colony was around four cm (16 to 24 h). The inverted agar block method (13) was used for live-cell imaging with an inverted laser scanning microscope (LSM-510 Meta; Carl Zeiss, Göttingen, Germany) equipped with an argon ion laser for excitation at a 488-nm wavelength and GFP filters for emission at 515 to 530 nm and with an He-Ne laser for excitation at 543 nm and RFP filters for emission at 580 to 700 nm. Two oil immersion objectives were used: 63 (differential interference contrast), 1.4 NA planapochromatic and 100 (PH3), and 1.3 NA planneofluar. Laser intensity was kept to a minimum (1.5%) to reduce photobleaching and phototoxic effects. The imaging parameters used produced no detectable background. Time-lapse imaging was performed at scan intervals of 0.5 to 4.5 s for periods up to 40 min. Image resolution was pixels and 300 dpi. Confocal images were captured by using LSM-510 software (version 3.2; Carl Zeiss) and evaluated with an LSM 510 image examiner. Time-lapse confocal and phase contrast images of hyphae were recorded simultaneously. Some of the time-lapse series were converted into AVI movies by using the same software. Phase-contrast images were captured with a photomultiplier for transmitted light using the same laser illumination for fluorescence (28). For fluorescence recovery after photobleaching (FRAP), a selected rectangular area of a hypha was overexposed at 20% intensity for 30 s (argon ion laser, 488-nm wavelength). After the photobleaching, the images were scanned at intervals of 3.5 to 4.5 s for up to 3 min (28). Nuclear kinetics and hyphal growth kinetics. Nuclear movement was measured in a hyphal length of 150 m from the apex. Only nuclei contained in these 150 m were included. Measurements were made on recorded time-lapse sequences by following, frame by frame, the advance of the leading edge of each nucleus. The movement direction was noted and speed calculated. At the same time, hyphal elongation rates were calculated by measuring frame by frame the advance of the hyphal apex. The number of frames measured for both nuclear movement and hyphal elongation is shown in Table 2. To measure growth rates in the sequences where hypha changed direction while growing, we traced the total distance covered by the hyphal apex. Cell profiles were traced with LSM 510 Examiner Software version 3.2, calibrated for the images recorded with an LSM-510 Meta microscope. Distances traveled were computed in Microsoft FIG. 1. Model illustrating cytoplasmic regions defined in the present study (modified from McDaniel and Roberson [21]). Region I contains the Spitzenkörper (Spk), region II extends from the posterior side of the Spitzenkörper to the anterior side of the first nucleus, and region III extends over a variable distance from the anterior side of the first nucleus to a zone characterized by the presence of large vacuoles. The nucleus exclusion zone is comprised of regions I and II.

3 1882 RAMOS-GARCÍA ETAL. EUKARYOT. CELL Downloaded from FIG. 2. Comparison of nuclear distribution and morphology in the apical and subapical regions of N. crassa H1::GFP: WT, ro-1, ro-3, kin-1, ro-1 kin-1, BML-treated WT, and CA-treated WT. (a to u) The panels of the left and the middle show single-focal-plane images at three different times (0:00, 2:51, and 5:47 min:s). (A to G) The panels of the right represent a three-dimensional reconstruction with ten confocal layers in subapical region III for each strain. Arrows show elongated nuclei; arrowheads point to spindle pole bodies. The scale bar in the time series is 10 m and in the three-dimensional reconstructions is 2 m. Excel spreadsheets. Table 2 shows the number of frames observed for hyphae and nuclei in each strain and treatment. The distance between the hyphal tip and the leading nucleus (n 100) was measured with the same software for each strain and treatment. We call this region the exclusion zone (Fig. 1); other pertinent regions are defined in the same figure. Distances were computed on Excel spreadsheets for statistical analysis. on July 10, 2018 by guest RESULTS Distribution and morphology of nuclei. First, we examined in greater detail the distribution of nuclei in a N. crassa strain (N22813A) that carries an hh1-sgfp fusion gene and thus expresses H1-GFP as a nuclear marker but is otherwise a WT strain (9). Nuclei were uniformly distributed throughout the subapical region of growing hyphae of the WT strain, with the leading nucleus maintaining a more or less constant distance from the apex ( 12- m constituted the exclusion zone ) (Fig. 2a to c; see also Video S1 in the supplemental material). The majority of the nuclei were elongated and oriented parallel to FIG. 3. Association of the nucleus-bound microtubule organizing center (MTOC) with microtubules. A confocal image of a germling nucleus of N. crassa strain FRM-01-AR incubated for 6 h is shown. The figure is a merged image from two channels: nucleus labeled with drfp-histone1 (red) and microtubules labeled with -tubulin-gfp (green). Arrowhead points to the predicted location of the MTOC. Scale bar, 1 m.

4 VOL. 8, 2009 NUCLEAR DISPLACEMENT IN N. CRASSA 1883 the growth axis; in some nuclei, a pyriform shape was evident (Fig. 2c and A). A brighter fluorescent spot was often observed at the leading end of the nucleus (Fig. 2A), corresponding to the expected location of the spindle pole body; the spindle pole body appeared in contact with microtubule bundles (Fig. 3). Compared to the WT, the dynein mutants showed altered nuclear morphology and distribution. In the ro-1 mutant, there were fewer nuclei in the subapical region, they were not uniformly distributed and the exclusion zone was longer (Fig. 2d to f; see Video S2 in the supplemental material). The vast majority of nuclei in this strain were spherical, although there were some examples of slightly oval or pear-shaped nuclei (Fig. 2B). The ro-3 mutant appeared similar to ro-1, but the nuclei were more clustered (Fig. 2g to i and C; see Video S3 in the supplemental material). In the conventional kinesin (kin-1) mutant, the nuclear population in the subapical region was different from that of both WT and the dynein mutants (Fig. 2j to l; see Video S4 in the supplemental material): nuclei were not clustered, were mainly spherical in shape, and were positioned closer to the apex (Fig. 2D). When both dynein and kinesin were absent (in the ro-1 kin-1 double mutant), we found a conglomeration of nuclei in the subapical region. The number of nuclei per hypha in this region appears to be much larger than in all of the other strains (Fig. 2m to o; see Video S5 in the supplemental material). All nuclei were spherical (Fig. 2E). When WT hyphae were treated with Mt or actin inhibitors opposite phenotypes were observed. In BML-treated hyphae (Fig. 2p to r; see Video S6 in the supplemental material) nuclei were elongated; the exclusion zone was longer and the distance between nuclei increased (Fig. 2F). In CA-treated hyphae, the nuclear exclusion zone essentially disappeared with some nuclei reaching the very tip of the hypha. There were fewer nuclei than in the WT (Fig. 2s to u; see Video S7 in the supplemental material), all were spherical (Fig. 2G) and uniformly distributed throughout the apical and subapical regions. Hyphal elongation rate and rate of nuclear movement. The relationship between the rates of hyphal elongation and nuclear movement is shown in Fig. 4. Rate of movement was measured every 3 s for a period of 60 s for the 10 nuclei closest to the tip of each hypha. Ten hyphae were selected at random for each different strain or inhibitor treatment (Fig. 4). The rate of hyphal elongation in the WT strain, as well as in the other strains, appeared to fluctuate, perhaps because of the pulsating nature of hyphal extension growth (18). The average extension rate in the WT strain was m s 1.A similar fluctuation was noted for the rate of nuclear movement. In the WT strain, the average rate of nuclear movement was nearly identical to the elongation rate, i.e., ms 1. To further illustrate this behavior, the movement of individual nuclei was plotted (Fig. 5). Certain nuclei exhibited acceler- FIG. 4. Average rate of nuclear movement in the apical and subapical regions of N. crassa WT, motor protein mutants, and WT treated with BML or CA. For each strain, the movement of 10 nuclei was monitored for 60 s. The data points are shown for every 3 s. The bold line represents the hyphal elongation rate, and the dotted line represents the mean of the rate of nuclear movement of the 10 leading nuclei.

5 1884 RAMOS-GARCÍA ETAL. EUKARYOT. CELL FIG. 5. Variability in the rate of nuclear movement in the apical and subapical regions of N. crassa WT, kin-1 mutant, and CA-treated WT. For each strain the movement of 10 nuclei was monitored for 60 s. The data points are shown for every 3 s. The bold line represents the hyphal elongation rate, and the gray thin lines are the rate of movement of the 10 leading nuclei. ated migrations in both anterograde and retrograde direction (Fig. 5). A positive correlation (P 0.05) between hyphal elongation rate and nuclear movement was also found in the motor protein-deficient strains and in the WT strain when it was subjected to treatment with cytoskeleton inhibitors (Fig. 6). Nevertheless, the synchrony between hyphal elongation and average rate of nuclear movement was only partial and more variable than in the WT strain (Fig. 6). Both the rate of hyphal extension and nuclear movement were much lower in the ro-1 mutant ( m s 1 and m s 1, respectively). In the ro-1 mutant, as in the WT strain, nuclear movement was mainly toward the apex and only in rare cases in the opposite direction (Fig. 6). The ro-3 mutant showed intermediate rates (and variation in the rates) of hyphal elongation and nuclear movement ( m s 1 and m min 1, respectively) (Fig. 6). Anterograde and retrograde movements were observed with higher frequency than in the ro-1 mutant. To confirm that nuclei traveled toward the hyphal apex at essentially the same speed as the hyphae elongated, we did a linear regression analysis of paired values of hyphal elongation and nuclear movement (Fig. 7 and 8). We found a strong correlation between hyphal elongation and nuclear displacement in the WT strain (Fig. 7). The same positive correlation was found for the ro-3 mutant and the ro-1 kin-1 double mutant. The ro-1 mutant and the BML-treated hyphae (Fig. 8) showed larger scattering of data. For all mutants, the slope was 1.0, indicating slightly slower nuclear movement compared to hyphal extension. Surprisingly, the ro-1 kin-1 double mutant (Fig. 8) revealed a pattern remarkably similar to the WT pattern (Fig. 8). The means of the 10 correlation values from the linear regression analysis, as well as the standard deviation and minimal and maximal values, are shown in Table 3. All of the strains and treatments had correlation coefficients of 0.8, except for the kin-1 mutant and CA-treated hyphae, where this value was 0.7 (P 0.05). Nuclear exclusion zone. Despite continuous forward movement, the nuclei of all strains examined never reach the hyphal apex, even though some of them showed an average movement faster than the rate of elongation. They were kept at a certain distance from the tip, forming a nucleus exclusion zone (Fig. 9). In the WT strain, the distance between the hyphal apex and the leading nucleus was m. The ro-1 and kin-1 mutants had the largest exclusion zones ( m and m, respectively). The ro-1 kin-1 double mutant

6 VOL. 8, 2009 NUCLEAR DISPLACEMENT IN N. CRASSA 1885 FIG. 6. Comparison of hyphal elongation and nuclear movement rates in N. crassa (these are the same strains and treatments as in Fig. 4). See Table 2 for the total number of measurements in each case. Error bars indicate the 95% confidence interval. showed an intermediate value ( m), closer to that of the WT strain, whereas the ro-3 mutant had the shortest exclusion zone ( m). Disruption of microtubules with BML did not seem to affect the exclusion zone ( m), whereas disruption of actin filaments with CA eliminated the exclusion zone ( m) (Fig. 9). Directionality and speed of nuclear movement during hyphal growth. Occasionally, in all strains the velocity and the FIG. 7. Linear regression analysis of nuclear movement and hyphal elongation in the N. crassa WT. Each point plots the distance traveled by a nucleus versus the length increase of the hypha. The movement of the first 10 nuclei (from the tip) was monitored every 3 s. For the sake of clarity, not all data points are shown; however, the regression line was calculated for the entire set of data. direction of nuclear movement became significantly different (Fig. 10). We calculated the relative proportion of passive nuclear displacement (anterograde) and active nuclear migration (retrograde or accelerated anterograde) (Fig. 10). Clearly, more than 80% of all movements are in unison with hyphal elongation. A small but variable percentage of retrograde motion was detected in all cases except in the ro-1 kin-1 double mutant. Accelerated anterograde motion occurred rarely and with variable frequency in all cases except for the CA-treated hyphae. In WT hyphae, 4.1% of the movements were retrograde while in ro-1 mutant, ro-3 mutant, and the BML-treated cells this frequency decreased to 2.4, 3.8, and 2.5%, respectively. In the kin-1 mutant we found 9.3% of all movement to be retrograde. The CA-treated cells exhibited the highest percentage of retrograde motion (22.6%). The increase in the frequency of retrograde nuclear movements in both the kin-1 mutant and the CA-treated cells was significantly different from the other strains and treatments (P 0.05). The rapid anterograde movement was most common in WT (13.5%), followed by ro-3 (13.3%), WT cells treated with BML (5.9%), the ro-1 kin-1 double mutant (3.4%), the kin-1 mutant (2.2%), the ro-1 mutant (1.1%), and CA-treated hyphae (0.1%), respectively (Fig. 10). We calculated the maximum and minimum speed for anterograde and retrograde movement for each strain and treatment (Table 4) and found that the maximum rate of retrograde motion was achieved by the ro-1 mutant, while the maximum anterograde motion was attained in the kin-1 mutant. FRAP of fluorescent nuclei. As an additional test of the dynamics of nuclear movement, we performed a FRAP experiment on hyphae of the WT strain. As seen in Fig. 11, the

7 1886 RAMOS-GARCÍA ETAL. EUKARYOT. CELL Downloaded from FIG. 8. Linear regression analysis of nuclear movement and hyphal elongation in N. crassa defective in microtubule motors or treated with cytoskeleton inhibitors (these are the same strains and treatments as in Fig. 4). The movement of the first 10 nuclei (from the tip) was monitored every 3 s. For the sake of clarity, not all data points are shown; however, the regression line was calculated for the entire set of data. on July 10, 2018 by guest TABLE 3. Correlation between hyphal elongation rate and nuclear displacement N. crassa strain r value Mean SD Minimum Maximum WT ro-1 mutant ro-3 mutant kin-1 mutant ro-1 kin-1 mutant BML-treated WT CA-treated WT rectangular area containing the bleached nuclei moved in unison with hyphal elongation. No fluorescent nuclei either in front or behind of the FRAP rectangle moved into the bleached area during a 5-min observation. Nuclear movement upon disintegration of the cytoskeleton. For most of our studies we used a concentration of BML or CA that reduced growth by ca. 50%. We also tested the effect of higher concentrations of BML (67% growth reduction at 2.5 g/ml) or CA (70% growth reduction at 1.0 g/ml). When exposed to BML, microtubules became short and disorganized, losing the preferential longitudinal alignment seen in untreated hyphae (Fig. 12a and b). Surprisingly, CA, an inhibitor of actin polymerization, also caused a severe disruption of the microtubular cytoskeleton with a substantial decrease in

8 VOL. 8, 2009 NUCLEAR DISPLACEMENT IN N. CRASSA 1887 TABLE 4. Speed of retrograde and anterograde nuclear movements Speed ( m s 1 ) a N. crassa strain Mean Minimum Maximum A R A R A R WT ro-1 mutant ro-3 mutant kin-1 mutant ro-1 kin-1 mutant BML-treated WT CA-treated WT a A, anterograde; R, retrograde. FIG. 9. Size of the nuclear exclusion zone in N. crassa (these are the same strains and treatments as in Fig. 4). Error bars indicate the 95% confidence interval. the number of Mts, and a high degree of disorganization in the alignment of the few remaining Mts. Similarly unexpected was the presence of mitotic spindles in the apical zone of CAtreated hyphae (e.g., see Fig. 12c). The disruption of the actin cytoskeleton with regard to CA was corroborated in an N. crassa strain tagged with drfp-tropomyosin (data not shown). DISCUSSION Relative importance of forces responsible for nuclear movement. In recent years, much evidence has been gathered to show that movement of nuclei in filamentous fungi can result from forces derived from Mt-related motor proteins (6, 27, 29, 30, 35, 38, 39). We present here evidence gleaned from live cell imaging of actively growing peripheral hyphae in established colonies of N. crassa that suggests that the major contributor to nuclear movement during hyphal growth is passive displacement of nuclei by cytoplasmic bulk flow. This passive flow of nuclei toward the growing tip had been observed in hyphae of Basidiobolus haptosporus (22, 23), Aspergillus nidulans (35), and N. crassa (28) but its importance relative to active migration, e.g., that powered by molecular motors, was not assessed. Although passive displacement appears to dominate nuclear movement in growing hyphae of N. crassa, the distinct instances of saltatory movements we recorded, in both retrograde or anterograde direction, suggest the participation of molecular motors, albeit in a minor proportion, in overcoming any drag exerted by cytoplasmic bulk flow. This saltatory motility has been previously described for nuclei of Ashbya gossypii and A. nidulans (35, 1) and ascribed to motor-driven processes. Our comparison of nuclear movements in motor-deficient mutants of N. crassa indicated that dynein, but not dynactin, plays a predominant role in the saltatory movements, as it does in A. nidulans (37). Even though motor-driven transport may not be the main mechanism for nuclear movement in growing hyphae, molecular motors may play a role in passive displacement, since they may serve to attach nuclei to the Mt meshwork in which nuclei seem entrapped (Fig. 13; 28). The prevalence of bulk flow in the movement of nuclei in growing hyphae of N. crassa may also apply to other developmental stages, namely, aerial hyphae, conidiophores and conidia. Two observations support this idea: (i) the reverse flow of the cytoplasm toward the conidiating region of a colony (4) and (ii) mutants devoid of molecular motors (e.g., dynein or kinesin) are capable of producing nucleated conidia (29). Barring the involvement of alternative motors, nuclei likely enter conidiophores and conidia propelled mainly by passive cytoplasmic bulk flow. We found no evidence of oscillatory nuclear motion as detected in hyphae of Ashbya gossypii (1). There was no indication that nuclei stopped advancing during growth except for FIG. 10. Frequency of different nuclear motions: retrograde (f), passive anterograde ( ), and accelerated anterograde (u) in the strains and treatments listed in Fig. 4. See Table 2 for the total number of measurements in each case.

9 1888 RAMOS-GARCÍA ETAL. EUKARYOT. CELL FIG. 11. FRAP experiment on the N. crassa WT strain. On the left, fluorescence images (optical slice of 2 m) shows that bleached nuclei are passively transported forward with the growing hypha. On the right, phase-contrast images serve to show the outline of the hypha and the nucleus exclusion zone. The rectangle in each panel shows the movement of the bleached area. very brief periods. The entire subapical set of nuclei moves as a unit, with each nucleus maintaining a more or less constant distance from one another. The concept of individual nuclear positioning described for other fungi (e.g., 35, 39) does not apply to the growing hyphae of N. crassa WT or dynein mutants. Nuclear movement in N. crassa germlings. Our present studies showing that the nuclei of a dynein mutant are propelled largely by cytoplasmic bulk flow appear to contradict earlier findings by different authors (29, 37), who assigned dynein a major role in nuclear transport. They observed that absence of dynein caused nuclei to cluster leaving long hyphal segments nucleus-free. We did not observe such clustering or nucleusfree zones (except for the apical exclusion zone) in mature FIG. 13. Model for the inferred relationships between interphase nuclei, microtubules, and microtubule motor proteins in the growing region of a mature hypha of N. crassa. The nucleus (yellow) is attached at one pole to Mts by the spindle pole body MTOC (red circle), and it is also entrapped in the microtubular network (green). Bulk flow and extension growth carry the microtubular network forward, together with the entrapped nuclei. Retrograde or accelerated anterograde movements are likely driven by the microtubular motors dynein (pink) or kinesin (blue). The latter may also serve to bind the entrapped nucleus to the microtubular network. hyphae. To test whether the discrepancy was caused by an intrinsic difference between germlings and vigorous growing hyphae, we examined nuclear dynamics in germlings of ro-1 and found that nuclei did remain clustered in the germinating conidium and moved only gradually into the germ tube (R. R. Mouriño-Perez, unpublished observations). Presumably, bulk flow is not a strong enough force to propel nuclei in the germlings until after hyphal growth is well established. What drives cytoplasmic bulk flow? It has been proposed that pressure gradients are the force driving cytoplasmic bulk flow (16, 17). However, the primary factor is apical wall expansion since no forward movement of cytoplasm will occur unless new volume is generated at the tip of the hypha or its branches. (Fig. 14). It is worth noting that pressure does play an indirect role in moving the cytoplasm forward. This is because turgor is after all the physical force that expands the plastic wall at the growing tip, a conclusion confirmed by experimental determination of the pattern of cell wall expansion in an elongating hypha (2). FIG. 12. Microtubule abundance and organization in a -tubulin- GFP strain of N. crassa. (a) Untreated; (b) BML-treated; (c) CAtreated. White arrows indicate mitotic spindles. Scale bar, 10 m. FIG. 14. Factors propelling the passive forward flow of cytoplasm. Two factors operate in unison to cause the forward movement of the cytoplasm: (i) the expansion of the apical wall continuously creates new cell volume (A3C) at the tip and (ii) water entering the hypha over its entire length to fill the increased cell volume. The net result is the forward movement of the cytosol and organelles suspended therein (a3c).

10 VOL. 8, 2009 NUCLEAR DISPLACEMENT IN N. CRASSA 1889 Cytoskeleton and nuclear movement. In a previous study, we visualized the in vivo dynamics of the cytoskeleton of N. crassa with GFP-labeled -tubulin (28). The unstained yet discernible nuclei in the growing region of a hypha could be seen trapped in the mesh of Mts moving in unison with the cytoplasmic flow. This finding suggested a tight relationship between nuclear displacement and the advance of the microtubular cytoskeleton. However, the closeness of this relationship is questionable since cytoskeleton disruption with benomyl did not stop the passive displacement of nuclei but only reduced their average speed of movement relative to that of the hyphal apex. Although the absence of specific motor proteins did not prevent the forward movement of nuclei, it did have an effect on nuclear clustering, nuclear morphology, and nuclear proximity to the apex. The changes in nuclear movement and distribution, such as reduction or enlargement of the exclusion zone, in cells treated with the Mt-destabilizing drug BML and the actin-destabilizing drug CA at subinhibitory concentrations (i.e., colony growth at 50% of the normal rate), indicates that the integrity and the dynamics of the microtubular network had an effect independent of Mt-related motor proteins. Saltatory movement decreased but nuclei were still able to move toward the apex, likely carried by cytoplasmic bulk flow. In addition to microtubules, actin plays important roles in organelle movement. The use of the actin-destabilizing drug CA caused a misdistribution of nuclei similar to what had been observed with actin (act-1) mutants of Neurospora (36). In the present study, treatment with subinhibitory concentrations of CA reduced elongation rate and nuclear displacement rate drastically but did not cause a major change in the relationship between nuclear displacement and hyphal elongation. CA did liberate nuclei from whatever cytoskeletal restraints prevented them from moving into the hyphal apex. Conclusion. Our studies demonstrate that passive displacement, rather than molecular motors, plays the dominant role in nuclear dynamics in mature growing hyphae of N. crassa. ACKNOWLEDGMENTS This study was supported by grants from the Consejo Nacional de Ciencia y Tecnología (SEP-2003-CO and SEP-2007-CB-82753) to R.R.M.-P., the American Cancer Society (RSG CCG) to M.F., and Arizona State University (LM5100R1) to R.W.R. We thank the Fungal Genetics Stock Center for strains. M.F. gratefully acknowledges the support of Eric Selker (University of Oregon); some of the early experiments leading to this publication were done in his lab. 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