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Apical Branching in a Temperature Sensitive Mutant ofAspergillus niger
Fungal Genetics and Biology 22, 153–167 (1997) Article No. FG971003
Apical Branching in a Temperature Sensitive Mutant of Aspergillus niger1
Cristina G. Reynaga-Pen ˜ a and Salomon Bartnicki-Garcia2 Department of Plant Pathology, University of California, Riverside, California 92521
Accepted for publication June 30, 1997
Reynaga-Pen˜a, C. G., Bartnicki-Garcia, S. 1997. Apical Branching in a Temperature Sensitive Mutant of Aspergillus niger. Fungal Genetics and Biology 22, 153– 167. An apical branching, temperature-sensitive, mutant of Aspergillus niger (ramosa-1) was isolated by UV mutagenesis. Ramosa-1 has a wild type morphology at 23°C, but branches apically when shifted to 34°C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenko¨rper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210–240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenko¨rper two new Spitzenko¨rper appeared, each giving rise to an apical branch. The second branch Spitzenko¨rper appeared with a 60- to 1
A video sequence of the actual apical branching process is available through the Internet: http://math3130.ucr.edu/fungus/homepg1.html. 2 To whom correspondence should be addressed. Fax: (909)787-5113. E-mail: [email protected]. 3 Abbreviations used: ACM, Aspergillus complete medium; VCM, Vogel’s complete medium; VCMPG, VCM 1 17% gelatin and 0.36% PhytaGel.
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100-s delay. We did not observe the original Spitzenko¨rper dividing in two; instead, the new Spitzenko¨rper arose de novo from vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenko¨rper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenko¨rper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization. r 1997 Academic Press
Index Descriptors: apical branching; Aspergillus niger mutant; ramosa-1 ; cytoplasmic contraction; Spitzenko¨rper role; Spitzenko¨rper ontogeny. Mycelial fungi grow by production of long tubular cells (hyphae) which branch and extend through the nutrient medium to form colonies. Fungal hyphae elongate at their apices by a process of polarized exocytosis. The wallbuilding vesicles involved in this polarized secretion accumulate in large numbers at the apices of growing hyphae (McClure et al., 1968; Girbardt, 1969; Grove and Bracker, 1970). The aggregate of vesicles forms a visible structure called the Spitzenko¨rper, long suspected of playing a key role in fungal growth (Brunswick, 1924; Girbardt, 1955, 1957). Hyphal branching makes possible mycelium development. In most instances, branches arise laterally at some distance from the primary tip, but in certain fungi, e.g., Aspergillus nidulans or Geotrichum candidum, apical
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branching is common (Trinci, 1970). Apical branching can also be part of a stress response (Robertson, 1958, 1959; Robinson and Smith, 1980; Reissig and Kinney, 1983). Although the kinetics of branching has been extensively studied in several fungi (Trinci, 1970, 1984), the details of the cytology of branching have not been elucidated, largely because of the limitations of observing unstained live specimens by ordinary light microscopy. These can be largely overcome with the help of video-enhanced phase contrast microscopy, which allows examination of fine intracellular details (1003 phase-contrast objective lens) and tape recording of fast-occurring events in growing cells. This methodology gave us a unique opportunity to study Spitzenko¨rper physiology and ontogeny. In this paper, we report the isolation and characterization of a temperature sensitive mutant of Aspergillus niger (ramosa-1). We have used this mutant to elucidate physiological and cytological details of the apical branching process. Because apical branching takes place within the hyphal apex, it was feasible to track the future site of branch formation at the high magnification needed to resolve cytological details. At the final magnification on the video screen (75003), the field of view was restricted to only 30 µm.
Reynaga-Pen˜a and Bartnicki-Garcia
Spores of ramosa-1 mutant and wild type strains were stored in silica gel at 4°C (Perkins, 1962).
Mutagenesis Conidia of A. niger were suspended in 50 mM phosphate buffer at a concentration of 5 3 107 conidia/ml and irradiated with a short-wave (254 nm) UV lamp (Fisher Scientific, Pittsburgh, PA) with a dose necessary to attain a ,10% survival rate. Mutants affected in hyphal tip morphology were selected after a filtration enrichment procedure modified from Boschloo et al. (1990). Irradiated conidia were incubated in liquid ACM at 40°C for a total of 3 days. Every 24 h, mycelia that formed in the culture were removed by filtration through four layers of cheesecloth. After three filtrations, any ungerminated conidia were collected by centrifugation, spread on ACM agar-plates, and incubated at the permissive temperature (23°C) for 48–72 h. When the colonies were about 2–3 mm in diameter, the plates were placed in an incubator at 40°C for 2 h. Individual colonies with abnormal apical growth were selected by microscopic observation with a 103 objective lens immediately after the 40°C incubation. Selected colonies were transferred to fresh ACM–agar plates for phenotype confirmation.
MATERIALS AND METHODS Culture Conditions Organism We used A. niger wild type, T312, from the fungal collection of P. H. Tsao (Department of Plant Pathology, University of California, Riverside).
Culture Media Wild type and mutant strains were grown and maintained in Aspergillus complete medium (ACM)3 (MackIntosh and Pritchard, 1963) at 23°C. All video microscopy was done with hyphae grown on VCMPG medium: Vogel’s complete medium (VCM) (Vogel, 1956) solidified with 17% gelatin (DIFCO) and 0.36% PhytaGel (Sigma Chemical Company, St. Louis, MO, USA). Gelatin was essential to minimize phase haloes around the cells (Mason and Powelson, 1956; Girbardt, 1957; Lo´pez-Franco, 1992); since the gelatin medium melts at temperatures higher than 30°C, we added PhytaGel as a second solidifying agent. For lower-magnification observations on colony growth and morphology we used both VCMPG and VCM solidified with 1.5% agar.
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For observation of growing fungi by phase-contrast microscopy, we used the slide culture chamber described by Lo´pez-Franco (1992). This chamber was a modification of the chamber developed by Girbardt (1956). The chambers consisted of 1-mm-thick microscope slides (Carolina Biological Supply Co., Burlington, NC, catalog No. 632000) on which two strips of fingernail polish were painted near the long edges. The fingernail polish strips supported a coverslip (Fisher Scientific, Pittsburgh, PA; No. 1, 22 3 50 mm) positioned in the center of the slide. Silicone sealant was used to attach the coverslip to the microscope slide and to provide a flexible hinge. A thin layer of warm VCMPG was applied to sterile slide chambers and inoculated with a mycelial plug from the edges of colonies. The chambers were incubated inside a humid petri dish at 23°C for 48 h. During incubation, the hinged coverslip was maintained open so it would not come in contact with the medium or the fungus. Once the colony extended onto the middle of the slide chamber (i.e., it had grown 20 to 30 mm), the inoculum plug plus excess mycelia with aerial growth were removed with a razor blade. After 20 min, the
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coverslip was carefully lowered to contact the gel medium. Hyphae growing in the slide chamber were observed by phase contrast optics 10 to 20 min after the coverslip was lowered to allow the fungus to adapt to the conditions of the chamber.
Video Microscopy Microscopic observations were made with an Olympus Vanox microscope (Olympus Optical Co., LTD, Tokyo, Japan) fitted with a phase contrast 1003 oil-immersion objective lens (n.a. 1.25) and a 253 W.F. eye piece (American Optical, No. 186) in the TV port. Video images were obtained with a Hamamatsu C2400-07 camera and enhanced with an Argus-10 image processor (Hamamatsu Photonic Systems Corp., Bridgewater, NJ) and displayed on a black and white, 12-inch monitor (Sony Corporation of America, San Jose, CA; Model PVM-122). Final magnification obtained on the video screen was 75003. Images were recorded in real time on S-VHS videotapes. Apical branching was induced by increasing the temperature of the slide chamber directly on the microscope stage using a household electric hair dryer (Conair Corporation, Model 187A) with its nozzle positioned 13 cm from the specimen. Air flow and temperature were regulated with a rheostat to maintain the air temperature in the vicinity of the chamber at 40 6 1°C. Slide chamber temperature was determined with an ultrathin thermocouple (made from 0.00059 5 12 µm thick foil embedded in a polymer glass laminate; Model CO1-T, Omega Engineering Inc., Stamford, CT) placed inside the chamber over a layer of VCMPG.
Measurements Colony growth rate was measured on petri plate cultures. The plates of VCM agar were inoculated with a spore suspension of either the wild type or mutant (ramosa-1) strains. Plates were incubated at 23°C (permissive temperature) for the first 24 h, until a small (3–5 mm) colony was established. For incubation at the restrictive temperature, the plates were shifted to incubators at 34 or 40°C. Colony diameter was measured in duplicate plates approximately every 24 h until the colonies reached the edge of the plate. Growth rate was calculated by linear regression. The diameter of peripheral hyphae was determined in colonies growing in petri plates or in slide chambers, at final magnifications of 15003 or 75003, respectively. Readings were made on the monitor screen with the
Argus-10 image processor. Diameter was measured at about 15–20 µm from the tip, at which distance, and for all practical purposes, diameter had reached its maximum.
RESULTS Isolation of Morphological Mutants UV mutagenesis of A. niger conidia yielded a variety of morphological mutants, several of which were branching mutants with a small, compact, colony phenotype. From 18 mutants isolated, only 3 maintained the mutant phenotype after reisolation. Among them, we recovered two temperature-sensitive mutants capable of apical branching when shifted to 40°C, and we named them ramosa-1 and ramosa-2. Since ramosa-1 underwent apical branching consistently when shifted to the restrictive temperature, we chose it to analyze the process of apical branching by video-enhanced phase contrast microscopy. Although the restrictive temperature initially used was 40°C, we subsequently found that the apical branching phenotype was also expressed at a lower temperature (34°C).
Colony Growth and Morphology At the permissive temperature (23°C), in any of the culture media we tested, both colony morphology (Fig. 1) and radial growth rate (Fig. 2) of the ramosa-1 mutant were similar to those in the parental wild type strain. In fact, the radial growth of mutant colonies was about 5% higher than wild type. The average growth rate of wild type and ramosa-1 on VCM plates was 9 to 10 µm/min. Both strains conidiated profusely. At the restrictive temperature (34–40°C), colony morphology and growth rate of wild type and mutant differed sharply. While the wild-type strain maintained its colony appearance (Fig. 1C), the overall aspect of ramosa-1 changed dramatically. It turned button-like or ‘‘colonial’’ (as defined by Barratt and Garnjobst, 1949). At this temperature, ramosa-1 did not conidiate (Fig. 1D). At either 34 or 40°C, the radial growth rate of ramosa-1 was drastically reduced to only 11 or 7%, respectively, of that at 23°C (Fig. 2). In contrast, when the wild-type strain was grown at 34 or 40°C two different responses were observed. At 34°C, the growth rate doubled but at 40°C it was reduced to 80% of that at 23°C (Fig. 2).
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FIG. 1. Colony morphology of A. niger wild type and ramosa-1 grown at the permissive and restrictive temperatures on VCM for 5 days. (A) Wild type strain at 23°C. (B) Ramosa-1 at 23°C. (C) Wild type strain at 34°C. (D) Ramosa-1 at 34°C. Magnification 1.123.
FIG. 2. Colony growth of A. niger wild type and ramosa-1. Radial growth rate of wild type (W) and ramosa-1 (X) strains at the permissive (23°C) and restrictive (34 and 40°C) temperatures. Agar plates of VCM were inoculated in the center with a spore suspension, preincubated for 24 h at 23°C, and then transferred to 34 or 40°C or maintained at 23°C. Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Observed at low magnification, the wild-type phenotype consisted of long hyphae with widely spaced lateral branches. Typically, the first lateral branch emerged at 150–300 µm from the growing tip (Fig. 3A). The same phenotype was maintained after a change to the restrictive temperature (Fig. 3C). At the permissive temperature, ramosa-1 phenotype was similar to the wild type, with only a slight reduction (5–7%) in hyphal diameter (Fig. 3A vs Fig. 3B). After transfer to the restrictive temperature (34 to 40°C), all leading hyphae of ramosa-1 branched apically within 45–75 min (Fig. 3D). When colonies of ramosa-1 were kept at the restrictive temperature for additional 20 to 30 min after the first branching, each new tip branched again within 20 µm of the previous branch (Fig. 3E). Incubation at the restrictive temperature for 90 to 120 min also produced increased septation and formation of small lateral branches next to
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the septa (Fig. 3F). Subsequently, these lateral branches developed apical branches (not shown). Expression of the apical branching phenotype was reversible; mutant colonies that were returned to the permissive temperature ceased producing apical branches and grew long hyphae with lateral branches similar to the wild-type strain.
of about 0.4 µm, although at times this zone was not evident. Numerous mitochondria were found just outside this phase-light zone, mostly aligned with the longitudinal axis of the hypha. Mitochondria were in continuous motion; individual mitochondria were seen to dart frequently into the apical region and interact momentarily with the Spitzenko¨rper.
Hyphal Tip Organization4
Response of Ramosa-1 during Shift to the Restrictive Temperature
At 23°C, hyphae of ramosa-1, observed at high magnification by phase contrast optics, were similar to those of the wild type (Figs. 4A and 4B). The overall distribution of organelles in the apical and subapical region followed the pattern typical of Spitzenko¨rper-bearing fungi (Girbardt, 1969; Grove and Bracker, 1970). The Spitzenko¨rper appeared as a single, phase-dark, ellipsoidal structure within the apical dome. Its size, measured in 30 different hyphae, varied from 0.9 to 1.5 µm in its short axis (1.1 µm average) and 1.2 to 1.9 µm in its long axis (1.5 µm average). This phase-dark structure corresponds to the vesicle cluster described by Lo´pez-Franco and Bracker (1996). The two other components of the Spitzenko¨rper of A. niger described by Lo´pez-Franco and Bracker were less evident: we could see a diffuse vesicle cloud surrounding the vesicle cluster but, except on rare occasions, we could not detect a core inside the vesicle cluster. In single, undisturbed growing hyphae of wild type or mutant, Spitzenko¨rper shape and size remained relatively constant. The Spitzenko¨rper position varied slightly; most of the time, we could see a small but distinct separation (median value 5 0.26 µm, n 5 30) between the Spitzenko¨rper and the apical pole (Fig. 4). During hyphal elongation, the Spitzenko¨rper moved along a straight path but with minor transverse oscillations (up to 6 pixels 5 0.35 µm from the longitudinal axis). The Spitzenko¨rper lies in an exclusion zone described by Lo´pez-Franco and Bracker (1996) as ‘‘a region in the apex where other large organelles were scarce or absent, except for occasional intruding mitochondria.’’ In our observations of A. niger, both wild type and ramosa-1, we found that behind the Spitzenko¨rper there was a phase-light zone 4 The nomenclature for the different parts of a hypha is the one adopted by Lo´pez-Franco and Bracker (1996) from an earlier proposal (Bartnicki-Garcia, 1990). The different hyphal regions are subdivided on the basis of d values, d being the distance between the VSC and the apical pole in the hyphoid equation (Bartnicki-Garcia et al., 1989). The apical dome extends between 0 and 1d; the hyphal apex between 0 and 2d; the subapical region begins at 2d. For the parent hypha in Fig. 7A, d 5 1.3 µm.
During a shift from permissive (23°C; room temperature) to restrictive temperature (34°C), the temperature in the slide chamber first rose rapidly to 30°C and then climbed more gradually between 30 and 34°C (Fig. 5). Within 20 min, the temperature inside the chamber had reached 34°C and remained at 34 6 1°C as long as the heat source was on. We recorded the behavior of 120 individual hyphae of ramosa-1 during the temperature shift to 34°C. The following responses were noted: (1) In about half of all hyphae (52%) the Spitzenko¨rper displayed abnormal behavior accompanied by abnormal cell morphology. Within this group, there were two categories: (i) About 21% of all hyphae underwent cytoplasmic contractions (see description below), and these contractions were followed by a sharp reduction in elongation rate. In most cases, the Spitzenko¨rper disappeared from view. Preliminary observations indicated occasional apical branching in such hyphae but the large number of nongrowing, seemingly dead hyphae indicated that, in the confinement of the slide chamber, heating had a deleterious effect not observed in petri plate cultures. We circumvented this problem by discontinuing the heating of the slide chamber as soon as the Spitzenko¨rper disappeared. Accordingly, about half of the hyphae that underwent cytoplasmic contractions branched apically (the temperature in the slide chamber dropped an average of 4°C within the first 5 min after the heating source was turned off). We analyzed a total of 13 videotaped sequences of apical branching to follow the behavior of the Spitzenko¨rper and related cytological events (see below). (ii) Of all hyphae, 31% showed morphological alterations without manifesting cytoplasmic contractions. The shapes produced departed significantly from the normal hyphoid5 shape; the beaded morphology observed in Fig. 6A was a distinct phenotype shown by 5% of the total number of hyphae. 5
A hyphoid shape is one that equals, or approximates closely, the shape described by the hyphoid equation y 5 x cot (xV/N) from the VSC model for fungal morphogenesis (Bartnicki-Garcı´a et al., 1989). Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 3. Effect of temperature on the morphology of peripheral (leading) hyphae in colonies of A. niger wild type and ramosa-1. (A) Wild type strain at 23°C. (B) Ramosa-1 at 23°C. (C) Wild type strain at 40°C. (D–F) Ramosa-1 at 40°C.
(2) In the other half of the hyphae observed (46%) there were no gross abnormalities and growth simply ceased within 10–30 min; typically, the apical zone of exclusion increased accompanied by a gradual reduction in growth rate, cell diameter, and Spitzenko¨rper size (Fig. 6B). No cytoplasmic contractions were detected in these hyphae. (3) Only 2% of the hyphae observed continued elongating normally during the entire treatment period (60 to 90 min).
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Events during Apical Branching in Ramosa-1 The following observations on hyphal cytology and physiology pertain to the apex and subapical region 20 µm long, which was the average length of hypha within the field of view. Apical branching was accompanied by marked changes in Spitzenko¨rper behavior and internal
Apical Branching in A. niger Mutant
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organization of the hyphal apex and subapex. The following description of events is a composite picture made from 13 examples of apical branching. However, the illustrations (Figs. 7 to 10) and the description of Spitzenko¨rper ontogeny and branch development come from a single representative sequence of apical branching where all the described events were clearly visible. We recognized six stages in this morphogenetic process: (a) Cytoplasmic contraction. The first discernible event preceding apical branching was a cytoplasmic contraction; it occurred within 20 to 60 min after the heat source was turned on, by the time the chamber temperature had stabilized at 33 to 35°C (Fig. 5). The contraction included two simultaneous events: (1) a flash created by a sharp change in refractive index (darkening) in a localized area inside the cell with a corresponding lightening of the
FIG. 5. Temperature increase inside the slide chamber placed on the microscope stage during the heating procedure described under Materials and Methods.
FIG. 4. Hyphal tips of A. niger wild type (A) and ramosa-1 (B) growing at 23°C, observed by phase contrast microscopy. Arrows point to the Spitzenko¨rper.
adjacent zone outside the cell (Fig. 7B) and (2) a sudden synchronous movement of organelles toward the apex lasting about 1 s (Reynaga-Pen˜a, 1996). The overall aspect of the hyphal segment under observation was affected by the contraction. The cytoplasm darkened, and the organelles (notably mitochondria) became ‘‘compacted’’ (Fig. 7C); these changes were reversed within 5–10 s. (b) Spitzenko¨rper retraction. Within 5 s following the cytoplasmic contraction, the Spitzenko¨rper retracted from its polar position in the apical dome (Fig. 7D) and moved backward up to 0.4 µm. During retraction, the Spitzenko¨rper shape appeared more spheroidal compared to the ellipsoidal shape when it was next to the apical pole (Figs. 7A and 7B vs Fig. 7D) and measured an average of 1.4 µm in diameter. During retraction, a phase-light zone of exclusion was maintained around the Spitzenko¨rper and it was possible to see thin filaments radiating from the Spitzenko¨rper region (Fig. 7D), crossing the zone of exclusion, and extending forward to the cell wall and backward to nearby mitochondria. (c) Spitzenko¨rper disappearance. After its momentary retraction, the Spitzenko¨rper migrated back toward the apical pole (Fig. 7E), and its size decreased gradually as it meandered within a confined area at the apex, and finally vanished (Figs. 7F and 7G). The time elapsed between the contraction and the disappearance of the Spitzenko¨rper varied from 20 to 58 s. As the Spitzenko¨rper disappeared, mitochondria migrated toward the subapical region and clustered in a subapical zone between 5 and 10 µm from the apical pole (Fig. 7G); this caused the hyphal apex to acquire an ‘‘empty’’ appearance. This organelle-depleted
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FIG. 6. Abnormal morphology and behavior of some hyphae of ramosa-1 growing at the restrictive temperature of 34°C. (A) Beaded appearance. (B) Reduction in Spitzenko¨rper size and hyphal diameter and increase in exclusion zone size prior to cessation of growth.
zone extended up to 6 µm from the apical pole. Organelle clustering occurred only in a small subapical zone (within 15 µm from the apical pole). Occasionally during the period when no Spitzenko¨rper was visible, the cluster of mitochondria dispersed for a few seconds to later cluster again. (d) Transition phase. During the period when no Spitzenko¨rper was visible (60–260 s) elongation ceased (Fig. 11) but some growth continued, albeit at a much slower rate (Reynaga-Pen˜a et al., 1997). During this time, the apex lost its characteristic hyphoid shape and became enlarged and blunt (Fig. 8). From opposite sides of this deformed apex, the two branches later emerged with their corresponding Spitzenko¨rper.
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Reynaga-Pen˜a and Bartnicki-Garcia
(e) Spitzenko¨rper ontogeny. At the sites where the new apical branches originated, there was no evidence of a specific, discrete structure preceding the appearance of the new Spitzenko¨rper. Small clouds of irregular outline and medium density were commonly seen moving in the area where new Spitzenko¨rper formed, sometimes aggregating into a denser cloud that gave the appearance of an incipient Spitzenko¨rper (Fig. 7H). The Spitzenko¨rper-like body did not seem to be stable; it moved along the cell periphery and rapidly dissolved. Frequently, but momentarily, mitochondria migrated into the area where new Spitzenko¨rper formed. Details of the ontogeny of the two Spitzenko¨rper formed in the apically branching hypha are shown in Figs. 9 and 10. The first new Spitzenko¨rper became established and remained distinctly visible 263 s after the cytoplasmic contraction (Fig. 9C). This Spitzenko¨rper increased in size rapidly and within 20 to 30 s it reached a maximum size of 1.1 µm by 1.3 µm (Fig. 9G). As the Spitzenko¨rper developed, the nearby cell wall expanded and the first apical branch emerged (Figs. 7I–7L and 8). In the five apical branching sequences where we could follow the development of the two Spitzenko¨rper simultaneously, we found that they did not develop in unison. One Spitzenko¨rper appeared first, the second one sometime later (60 to 100 s after the first) (Figs. 7J, 7L). The second Spitzenko¨rper was first visible at 360 s (Fig. 10B); it was smaller than the first branch Spitzenko¨rper and measured 0.8 µm by 1.0 µm when it reached a maximum size (Fig. 10F). (f) Recovery of normal tip organization. Recovery of the typical organelle distribution at the hyphal tip occurred gradually, and was simultaneously accompanied by recovery of apical elongation. First, mitochondria moved slowly toward the apex and became gradually positioned close to the area where the first of the two new apices later appeared (Fig. 7I). As branches developed, organelle distribution in the emerging tips gradually attained that of normal growing hyphae (Figs. 7K–7N). (g) Branch development. Two branches were formed on opposite sides of the main hyphal axis at angles of 50° to 60°. In the two instances when both apical branches grew on the same focal plane, it became feasible to simultaneously compare their development. As judged by comparison of cell profiles, the two emerging branches were clearly evident at 310 s (Fig. 8). From the outset, one branch became dominant. By 270 s there was already evidence of two incipient branches, one slightly larger than the other. Because of this size discrepancy it was hard to tell if both
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branches began to grow at exactly the same time or whether the dominant branch preceded the other; in any case, we estimate the time difference to be less than 20 s. The first branch was of a larger size and by the end of the observations (Fig. 7N), it had attained a diameter of 6.0 µm at its base, while the diameter of the second branch was 4.7 µm (78% of the diameter of the first branch). Occasionally, more than two branches formed at the apex of the ramosa-1 mutant after a shift to the restrictive temperature.
Changes in Cell Wall Aspect As the Spitzenko¨rper began to disappear, the appearance of the apical cell wall changed (Fig. 7). Its outline became progressively darker and wider, suggesting thickening of the wall. The change in appearance was more perceptible in the vicinity of the area where the cytoplasmic contraction occurred. As the branches began to develop, the newly formed wall at the two apical poles had the appearance of a normal growing hypha.
enhanced phase-contrast microscopy allowed us to define more precisely the cytological events in apical branching. Although the physiology of apical branching had been studied earlier in A. nidulans (Trinci, 1970; Trinci and Morris, 1979), this seems to be the first time that cytological details in hyphal tip organization and Spitzenko¨rper behavior have been described during apical branch formation. The recording of microscopic images on videotape was essential for the subsequent qualitative and quantitative analysis of events. Videotape analysis made it possible to disclose important short-term events associated with apical branching in ramosa-1 such as cytoplasmic contractions, the retraction of the Spitzenko¨rper, its disappearance, and the appearance of two new Spitzenko¨rper. The profound changes in growth rate and hyphal morphology observed during apical branching were accompanied by drastic changes in Spitzenko¨rper behavior. These findings reinforce the idea that this structure plays a decisive role in apical growth and morphogenesis (Girbardt, 1957, 1969; McClure et al., 1968; Grove and Bracker, 1970; Bartnicki-Garcı´a, 1990).
Growth Rates Spitzenko¨rper Morphology
The apical branching process was accompanied by profound changes in elongation rate of hyphae (Fig. 11). Within 2 s after the cytoplasmic contraction, the elongation rate began to drop. By 30 s, the rate had diminished to 35% of the original value (from 5.60 to 1.98 µm/min). Between 30 and 60 s it dropped to 20% of the original rate, 1.12 µm/min. During the time when the Spitzenko¨rper was not visible (60 to 260 s) the average elongation rate dropped further to 7.5% of the original rate, 0.42 µm/min. From 150 to 180 s, no elongation was detected. As the first and second Spitzenko¨rper appeared, the cell began to recover and the elongation rate of the branches slowly started to rise. From 263 s (the time when the first Spitzenko¨rper was visible) to 330 s, the first branch elongated at 2.22 µm/min, and attained a rate of 2.76 µm/min by the end of our observations (570 s). Similarly, from 360 s (when the second Spitzenko¨rper appeared) to 420 s, the second branch elongated at 1.61 µm/min, and by 570 s it reached 2.46 µm/min (Fig. 11).
The Spitzenko¨rper of A. niger corresponds to pattern 4 described by Lo´pez-Franco and Bracker (1996). Our observations on the Spitzenko¨rper morphology of A. niger, both wild type and ramosa-1, correspond to what Lo´pezFranco and Bracker described as the vesicle cluster (the phase-dark spheroidal body in close proximity to the apical pole). We could not distinguish the central dark core from the outer vesicle cluster, and there was usually a distinct phase-light zone of separation between the Spitzenko¨rper and the apical cell membrane. Generally, the Spitzenko¨rper of our strain of A. niger maintained an ellipsoidal shape, but during retraction its shape changed from ellipsoidal to spherical. This could be another example of Spitzenko¨rper pleomorphism (Lo´pez-Franco and Bracker, 1996); on the other hand, the ellipsoidal shape could be the result of optical distortion by the high curvature of the apical dome, in which case, the spheroidal shape might be the true shape of the Spitzenko¨rper.
DISCUSSION
Spitzenko¨rper Behavior
The isolation of temperature sensitive mutants of A. niger (ramosa-1 and ramosa-2) and the use of video-
The Spitzenko¨rper is a dynamic structure that advances continuously as the hypha elongates (Girbardt, 1957; Lo´pez-Franco and Bracker, 1996). As it moves on along the
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FIG. 7. Cytological events during apical branching of ramosa-1. (A) Normal hyphal growth before the cytoplasmic contraction. (B) Cytoplasmic contraction; arrow points to sharp change in refractive index. (C) Hyphal tip after the contraction. (D) Spitzenko¨rper retraction; arrows indicate filaments radiating from the Spitzenko¨rper. (E) Spitzenko¨rper returning to the apical pole. (F–G) Spitzenko¨rper disappearance; arrow in G points to cluster of mitochondria. (H) Transient vesicle cloud (arrow) appeared to form incipient Spitzenko¨rper but later disappeared. (I) Blunt hyphal tip prior to Spitzenko¨rper appearance. (J–L) Formation of new branch Spitzenko¨rper. (M–N) Branch elongation. Bar 5 5 µm.
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FIG. 7—Continued
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FIG. 9. Ontogeny of the first Spitzenko¨rper (Fig. 7, upper branch) formed during the ramosa-1 apical branching sequence. Arrows indicate the vesicle cloud (B) that gave rise to a Spitzenko¨rper (C).
FIG. 8. Progressive changes in hyphal tip morphology preceding the appearance of apical branches. (A) The traced cell profiles at 0 and 246 s were superimposed on the image captured at 310 s. Note the transformation of the apex from a typical hyphoid-like shape (0 s) to a blunt shape tip prior to the emergence of the two incipient branches (310 s). (B) A hyphoid plot was superimposed on the image of the blunt apex (246 s) to show the departure from hyphoid morphology.
longitudinal axis of the hypha, it also shows minor oscillations in the transverse direction. The factors responsible for advancing and maintaining the position of the Spitzenko¨rper are not well understood. Our observations on cytoplasmic contractions (see below) suggest the involvement of the cytoskeleton in Spitzenko¨rper dynamics. A number of studies provide evidence for a Spitzenko¨rper–cytoskeleton connection. Examination of hyphal tips of Magnaporthe grisea (Bourett and Howard, 1991) and Allomyces macrogynus (Vargas et al., 1993) revealed actin filaments and microtubules in the Spitzenko¨rper region. Although the role of cytoskeletal elements in Spitzenko¨rper structure and function is not well established, several
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reports suggest that the cytoskeleton may propel and control the motion of the Spitzenko¨rper. Howard and Aist (1977) showed that the Spitzenko¨rper of Fusarium acuminatum vanished after treatment with MBC, an anti-tubulin agent. Similarly, Grove and Sweigard (1981), Roberson and Vargas (1994), and Srinivasan et al. (1996) showed that disruption of either microtubules or actin filaments caused the detachment and disappearance of the Spitzenko¨rper of Rhizoctonia solani or A. macrogynus. An association between cytoskeletal filaments and the
FIG. 10. Ontogeny of the second Spitzenko¨rper (Fig. 7, lower branch) formed during the ramosa-1 apical branching sequence. Arrows in B and C point to the incipient Spitzenko¨rper.
Apical Branching in A. niger Mutant
FIG. 11. Hyphal elongation during apical branching. Time zero corresponds to the cytoplasmic contraction. X, Main branch; W, first branch; M, second branch. Measurements started at 226 s for the main hypha, and at 246 s for the branches.
Spitzenko¨rper is also suggested by our observations of phase-dark filaments radiating from the Spitzenko¨rper of ramosa-1 during its detachment from the apical pole. Similar filaments were reported by Lo´pez-Franco and Bracker (1996) for the Spitzenko¨rper of R. solani.
Spitzenko¨rper Ontogeny From our findings on apical branching, we can make some tentative conclusions about Spitzenko¨rper ontogeny. Although it seems certain that the original Spitzenko¨rper did not split into two to form the Spitzenko¨rper of the apical branches, we cannot rule out that invisible remnants (core?) of the original Spitzenko¨rper may have contributed or given rise to the two new Spitzenko¨rper. We favor the view that the two branch Spitzenko¨rper were formed de novo from vesicle accumulations around an invisible nucleation site. Whether this presumed nucleation site becomes, or forms part, of the Spitzenko¨rper core remains an intriguing question, and one that is particularly difficult to address since the nature and structure of the Spitzenko¨rper core have yet to be elucidated. The first recognizable event in Spitzenko¨rper formation was the appearance of a vesicle cloud in the apical region. Although we could not identify, unequivocally, these accumulations of medium density material as being vesicle populations, such explanation is likely, at least for those which later condensed and became a Spitzenko¨rper. Note
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that some of these clouds did not necessarily produce a ‘‘new’’ Spitzenko¨rper, they dissolved or drifted away. Perhaps Spitzenko¨rper development from a vesicle cloud may require an ‘‘anchoring’’ step to stabilize it in place. Conceivably, actin and tubulin could be involved in anchoring the Spitzenko¨rper and/or in maintaining its integrity. The idea that ‘‘Spitzenko¨rper integrity might require fully functional microtubules’’ was previously suggested by Howard and Aist (1977). Although our observations and conclusions on Spitzenko¨rper ontogeny pertain exclusively to apical branching, the question arises as to whether they apply to lateral branching which is, by far, the most prevalent mode of branching in fungi. A de novo mechanism for Spitzenko¨rper formation seems likely in lateral branching, especially since the main Spitzenko¨rper lies too far away from the branching site to have any direct effect on its formation. Whether or not a localized cytoplasmic contraction also precedes lateral branching remains an intriguing possibility.
Apical Dominance and Branch Ontogeny The apical branching we observed in ramosa-1 is not a perfectly dichotomous branching in which the parental hypha would split evenly into two branches of equal standing. The apical branches of ramosa-1 were uneven in three respects: time of emergence, growth rate, and size. One branch, the first one to appear, became dominant, it grew faster and wider than the other. This raises intriguing questions about the basis of this dominance and also questions about definitions. On the latter, Trinci (1970) acknowledged the difficulty in determining whether the branching in A. nidulans was truly apical or subapical (if it occurred beyond 4 µm). With the much higher magnification we employed and with the subsequent image analysis, we were able to distinguish more precisely the origin of the two branches in A. niger. Figure 8 shows clearly that both branches arise from equivalent but opposite sites on the foremost part of the hyphal apex, which by this time had become enlarged and its shape was no longer hyphoid but blunt. Therefore, on the basis of location, the branching we observed is truly apical. As to the dominance exerted by the first branch, presumably this arises from cytoskeletal connections that give the first Spitzenko¨rper a larger share of the supply of cytoplasmic vesicles from the parental hypha. Once this is set, the second branch Spitzenko¨rper can marshal only a minor share of the flow of wall-building vesicles.
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Kinetics of Apical Branching Our observations on the kinetics of apical branching in the ramosa-1 mutant of A. niger agree with previous conclusions made for apical branching in Fusarium oxysporum (Robertson, 1958) and A. nidulans (Trinci, 1970). In both instances, there was a drastic but transient reduction in elongation rate prior to appearance of apical branches. Unlike Trinci’s (1970) findings on A. nidulans, the branches in ramosa-1 did not recover the elongation rate of the parent hypha previous to branching; this discrepancy, however, could be due to the temperature shift down of the slide chamber before the appearance of the apical branches. The presumed alteration in cytoskeleton organization during a cytoplasmic contraction may also affect vesicle travel from the subapex, where vesicles originate (Grove and Bracker, 1970), to the apex. A drastic reduction in the flow of wall-building vesicles reaching the tip would account for the momentary but sharp reduction in cell expansion following the cytoplasmic contraction.
Phenotype Expression Ramosa-1 expressed its apical branching phenotype readily and consistently on solid media in ordinary petri dishes. However, phenotype expression inside closed slide chambers, placed under a 1003 oil-immersion objective, was a more difficult challenge. On petri plates essentially all peripheral hyphae branched apically when shifted from 23 to 34°C, whereas only 11% of the hyphae examined underwent apical branching inside the chambers. To understand this low response, we have to consider that the hyphae were already under considerable stress caused by the limiting environment inside the slide chambers. For instance, Lo´pez-Franco et al. (1994) found that elongation rates of hyphae growing in closed slide chambers were only 27 to 69% of those measured in cells growing in petri plates. Presumably, the temperature increase required to express the apical branching phenotype compounded other stresses created by confinement in the slide chambers. The high incidence of cell death (46%) and abnormal morphology (31%), observed within the first few minutes of heating indicate a highly stressful environment.
Cytoplasmic Contractions Cytoplasmic contractions are not unique to the ramosa-1 mutant. We have also observed them in the wild-type strain of A. niger, though at a much lower
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Reynaga-Pen˜a and Bartnicki-Garcia
frequency. Localized cytoplasmic contractions can also be detected in hyphae of other fungi, e.g., Neurospora crassa and Trichoderma harzianum (Reynaga-Pen˜a, unpublished results). In R. solani, ‘‘cytoplasmic waves’’ with some similarity to the cytoplasmic contractions in A. niger have also been reported (Lo´pez-Franco and Bracker, 1996). Although the cause and nature of the cytoplasmic contractions is unclear, it seems safe to conclude that the observed unidirectional movement of organelles involved a contraction of a cytoskeletal network that links all these structures together. Cytoplasmic contractions of longer duration were induced in hyphae of Basidiobolus magnus (McKerracher and Heath, 1986) and Saprolegnia ferax (Jackson and Heath, 1992; Kaminskyj et al., 1992). These reports suggested that calcium and actin–myosin interactions were involved in the mechanism causing the contractions. In all instances where cytoplasmic contractions were detected in ramosa-1 (21% of total hyphae), we found a significant drop in hyphal elongation rate. In the majority of these hyphae (16% of the total hyphae analyzed), Spitzenko¨rper behavior was also affected: it either retracted from its polar position and disappeared from view, or momentarily stopped advancing. Our finding that cytoplasmic contractions were consistently followed by Spitzenko¨rper dislocation and/or disappearance suggests the existence of a dynamic connection between the cytoskeletal network and the Spitzenko¨rper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization, manifested by the cytoplasmic contraction. A logical next step in understanding the biochemical nature of the mechanism responsible for apical branching would be the characterization of the genetic locus affected in ramosa-1.
ACKNOWLEDGMENTS Supported in part by grants from the National Institutes of Health (GM-48257), the National Science Foundation (IBN-9204541), and a predoctoral fellowship from the ‘‘Consejo Nacional de Ciencia y Tecnologı´a’’ (CONACYT, Me´xico) to the senior author.
REFERENCES Barratt, R. W., and Garnjobst, L. 1949. Genetics of a colonial microconidiating mutant strain of Neurospora crassa. Genetics 34: 351–369.
Apical Branching in A. niger Mutant
Bartnicki-Garcia, S., Hergert, F., and Gierz, G. 1989. Computer simulation of hyphal morphogenesis and the mathematical basis for the hyphal (tip) growth. Protoplasma 153: 46–57. Bartnicki-Garcia, S. 1990. Role of vesicles in apical growth and a new mathematical model of hyphal morphogenesis. In Tip Growth in Plant and Fungal Cells ( I. B. Heath, Ed.), pp. 211–232. Academic Press, San Diego. Boschloo, J. G., Paffen, A., Koot, T., van den Twell, W. J. J., van Gorcom, R. F. M., Cordewener, J. H. G. and Bos, C. J. 1990. Genetic analysis of benzoate metabolism in Aspergillus niger. Appl. Microbiol. Biotechnol. 34: 225–228. Bourett, T. M., and Howard, R. J. 1991. Ultrastructural immunolocalization of actin in a fungus. Protoplasma 163: 199–202. Brunswik, H. 1924. Untersuchungen u¨ber geschlechts—un kernverha¨ltnisse bei der hymenomyzetengattung Coprinus. In Botanische Abhandlung (K. Goebel, Ed.), pp. 1–152. Gustav Fisher Verlag, Jena. Girbardt, M. 1955. Lebendbeobachtungen an Polystictus versicolor (L.) Flora 142: 540–563. Girbardt, M. 1956. Eine methode zum vergleich lebender mit fixierten strukturen bei pilzen. Z. wiss. Mikrosk. 63: 16–21. Girbardt, M. 1957. Der Spitzenko¨rper von Polystictus versicolor (L.). Planta 50: 47– 59. Girbardt, M. 1969. Die ultrastruktur del apikalregion von pilzhyphen. Protoplasma 67: 413–441. Grove, S. N., and Bracker, C. E. 1970. Protoplasmic organization of hyphal tips among fungi: Vesicles and Spitzenko¨rper. J. Bacteriol. 104: 989–1009. Grove, S. N., and Sweigard, J. A. 1981. The Spitzenko¨rper persists after tip growth is arrested by cytochalasins. Mycol. Soc. Am. Newsl. 32: 33. [Note: A full version of this work is available on the Internet at http://epress.com/w3jbio/grove] Howard, R. J., and Aist, J. R. 1977. Effects of MBC on hyphal tip organization, growth, and mitosis of Fusarium acuminatum, and their antagonism by D2O. Protoplasma 92: 195–210. Jackson, S. L., and Heath, I. B. 1992. UV microirradiations elicit Ca21 dependent apex-directed cytoplasmic contractions in hyphae. Protoplasma 170: 46–52. Kaminskyj, S. G. W., Jackson, S. L., and Heath, I. B. 1992. Fixation induces differential polarized translocation of organelles in hyphae of Saprolegnia ferax. J. Microsc. 167: 153–168. Lo´pez-Franco, R. 1992. Organization and Dynamics of the Spitzenko¨rper in Growing Hyphal Tips. Ph.D. dissertation, Purdue Univ., West Lafayette, IN. Lo´pez-Franco, R., and Bracker, C. E. 1996. Diversity and dynamics of the Spitzenko¨rper in growing hyphal tips of higher fungi. Protoplasma 195: 90– 111. Lo´pez-Franco, R., Bartnicki-Garcia, S., and Bracker, C. E. 1994. Pulsed growth of fungal hyphal tips. Proc. Natl. Acad. Sci. USA 91: 12228– 12232. MackIntosh, M. E., and Pritchard, R. H. 1963. The production and
167 replica plating of micro-colonies of Aspergillus nidulans. Genet. Res. 4: 320–322. Mason, D. J., and Powelson, D. M. 1956. Nuclear division as observed in live bacteria by a new technique. J. Bacteriol. 71: 474–479. McClure, W. K., Park, D., and Robinson, P. M. 1968. Apical organization in the somatic hyphae of fungi. J. Gen. Microbiol. 50: 177–182. McKerracher, L. J., and Heath, I. B. 1986. Polarized cytoplasmic movement and inhibition of saltations induced by calcium-mediated effects of microbeams in fungal hyphae. Cell Motil. Cytoskeleton 6: 136–145. Perkins, D. D. 1962. Preservation of Neurospora stock cultures with anhydrous silica gel. Can. J. Microbiol. 8: 591–594. Reissig, J. L., and Kinney, S. G. 1983. Calcium as a branching signal in Neurospora crassa. J. Bacteriol. 154: 1397–1402. Reynaga-Pen˜a, C. G. 1996. Morphogenetic analysis of apical branching in a temperature-sensitive mutant of Aspergillus niger. Ph.D. Dissertation. University of California, Riverside. Riverside, CA. Reynaga-Pen˜a, C. G., Gierz, G. and Bartnicki- Garcı´a, S. 1997. Analysis of the role of the Spitzenko¨rper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger. Proc. Nat. Acad. Sci. U.S.A. 94: 9096–9101. Roberson, R. W., and Vargas, M. M. 1994. The tubulin cytoskeleton and its sites of nucleation in hyphal tips of Allomyces macrogynus. Protoplasma 182: 19–31. Robertson, N. F. 1958. Observations of the effect of water on the hyphal apices of Fusarium oxysporum. Ann. Bot. 22: 159–173. Robertson, N. F. 1959. Experimental control of hyphal branching and branch form in hyphomycetous fungi. J. Linn. Soc. London Bot. 56: 207–211. Robinson, P. M., and Smith, J. M. 1980. Apical branch formation and cyclic development in Geotrichum Candidum. Trans. Br. Mycol. Soc. 75: 233–238. Srinivasan, S., Vargas, M. M., and Roberson, R. W. 1996. Functional, organizational, and biochemical analysis of actin in hyphal tip cells of Allomyces macrogynus. Mycologia 88: 57–70. Trinci, A. P. J. 1970. Kinetics of apical and lateral branching in Aspergillus nidulans and Geotrichum lactis. Trans. Br. Mycol. Soc. 55: 17– 28. Trinci, A. P. J. 1984. Regulation of hyphal branching and hyphal orientation. Symposium of British Mycological Society: The ecology and Physiology of the Fungal Mycelium (D. H. Jennings and A. D. M. Rayner, Eds.). Cambridge Univ. Press, Cambridge, UK. pp. 23–52. Trinci, A. P. J., and Morris, N. R. 1979. Morphology and growth of a temperature-sensitive mutant of Aspergillus nidulans which forms aseptate mycelia at non-permissive temperatures. J. Gen. Microbiol. 114: 53–59. Vargas, M. M., Aronson, J. M., and Roberson, R. W. 1993. The cytoplasmic organization of hyphal tip cells in the fungus Allomyces macrogynus. Protoplasma 176: 43–52. Vogel, H. J. 1956. A convenient growth medium for Neurospora (medium N). Microbial Gen. Bull. 13: 42–43.
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Apical Branching in a Temperature Sensitive Mutant of Aspergillus niger1
Cristina G. Reynaga-Pen ˜ a and Salomon Bartnicki-Garcia2 Department of Plant Pathology, University of California, Riverside, California 92521
Accepted for publication June 30, 1997
Reynaga-Pen˜a, C. G., Bartnicki-Garcia, S. 1997. Apical Branching in a Temperature Sensitive Mutant of Aspergillus niger. Fungal Genetics and Biology 22, 153– 167. An apical branching, temperature-sensitive, mutant of Aspergillus niger (ramosa-1) was isolated by UV mutagenesis. Ramosa-1 has a wild type morphology at 23°C, but branches apically when shifted to 34°C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenko¨rper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210–240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenko¨rper two new Spitzenko¨rper appeared, each giving rise to an apical branch. The second branch Spitzenko¨rper appeared with a 60- to 1
A video sequence of the actual apical branching process is available through the Internet: http://math3130.ucr.edu/fungus/homepg1.html. 2 To whom correspondence should be addressed. Fax: (909)787-5113. E-mail: [email protected]. 3 Abbreviations used: ACM, Aspergillus complete medium; VCM, Vogel’s complete medium; VCMPG, VCM 1 17% gelatin and 0.36% PhytaGel.
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100-s delay. We did not observe the original Spitzenko¨rper dividing in two; instead, the new Spitzenko¨rper arose de novo from vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenko¨rper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenko¨rper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization. r 1997 Academic Press
Index Descriptors: apical branching; Aspergillus niger mutant; ramosa-1 ; cytoplasmic contraction; Spitzenko¨rper role; Spitzenko¨rper ontogeny. Mycelial fungi grow by production of long tubular cells (hyphae) which branch and extend through the nutrient medium to form colonies. Fungal hyphae elongate at their apices by a process of polarized exocytosis. The wallbuilding vesicles involved in this polarized secretion accumulate in large numbers at the apices of growing hyphae (McClure et al., 1968; Girbardt, 1969; Grove and Bracker, 1970). The aggregate of vesicles forms a visible structure called the Spitzenko¨rper, long suspected of playing a key role in fungal growth (Brunswick, 1924; Girbardt, 1955, 1957). Hyphal branching makes possible mycelium development. In most instances, branches arise laterally at some distance from the primary tip, but in certain fungi, e.g., Aspergillus nidulans or Geotrichum candidum, apical
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branching is common (Trinci, 1970). Apical branching can also be part of a stress response (Robertson, 1958, 1959; Robinson and Smith, 1980; Reissig and Kinney, 1983). Although the kinetics of branching has been extensively studied in several fungi (Trinci, 1970, 1984), the details of the cytology of branching have not been elucidated, largely because of the limitations of observing unstained live specimens by ordinary light microscopy. These can be largely overcome with the help of video-enhanced phase contrast microscopy, which allows examination of fine intracellular details (1003 phase-contrast objective lens) and tape recording of fast-occurring events in growing cells. This methodology gave us a unique opportunity to study Spitzenko¨rper physiology and ontogeny. In this paper, we report the isolation and characterization of a temperature sensitive mutant of Aspergillus niger (ramosa-1). We have used this mutant to elucidate physiological and cytological details of the apical branching process. Because apical branching takes place within the hyphal apex, it was feasible to track the future site of branch formation at the high magnification needed to resolve cytological details. At the final magnification on the video screen (75003), the field of view was restricted to only 30 µm.
Reynaga-Pen˜a and Bartnicki-Garcia
Spores of ramosa-1 mutant and wild type strains were stored in silica gel at 4°C (Perkins, 1962).
Mutagenesis Conidia of A. niger were suspended in 50 mM phosphate buffer at a concentration of 5 3 107 conidia/ml and irradiated with a short-wave (254 nm) UV lamp (Fisher Scientific, Pittsburgh, PA) with a dose necessary to attain a ,10% survival rate. Mutants affected in hyphal tip morphology were selected after a filtration enrichment procedure modified from Boschloo et al. (1990). Irradiated conidia were incubated in liquid ACM at 40°C for a total of 3 days. Every 24 h, mycelia that formed in the culture were removed by filtration through four layers of cheesecloth. After three filtrations, any ungerminated conidia were collected by centrifugation, spread on ACM agar-plates, and incubated at the permissive temperature (23°C) for 48–72 h. When the colonies were about 2–3 mm in diameter, the plates were placed in an incubator at 40°C for 2 h. Individual colonies with abnormal apical growth were selected by microscopic observation with a 103 objective lens immediately after the 40°C incubation. Selected colonies were transferred to fresh ACM–agar plates for phenotype confirmation.
MATERIALS AND METHODS Culture Conditions Organism We used A. niger wild type, T312, from the fungal collection of P. H. Tsao (Department of Plant Pathology, University of California, Riverside).
Culture Media Wild type and mutant strains were grown and maintained in Aspergillus complete medium (ACM)3 (MackIntosh and Pritchard, 1963) at 23°C. All video microscopy was done with hyphae grown on VCMPG medium: Vogel’s complete medium (VCM) (Vogel, 1956) solidified with 17% gelatin (DIFCO) and 0.36% PhytaGel (Sigma Chemical Company, St. Louis, MO, USA). Gelatin was essential to minimize phase haloes around the cells (Mason and Powelson, 1956; Girbardt, 1957; Lo´pez-Franco, 1992); since the gelatin medium melts at temperatures higher than 30°C, we added PhytaGel as a second solidifying agent. For lower-magnification observations on colony growth and morphology we used both VCMPG and VCM solidified with 1.5% agar.
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For observation of growing fungi by phase-contrast microscopy, we used the slide culture chamber described by Lo´pez-Franco (1992). This chamber was a modification of the chamber developed by Girbardt (1956). The chambers consisted of 1-mm-thick microscope slides (Carolina Biological Supply Co., Burlington, NC, catalog No. 632000) on which two strips of fingernail polish were painted near the long edges. The fingernail polish strips supported a coverslip (Fisher Scientific, Pittsburgh, PA; No. 1, 22 3 50 mm) positioned in the center of the slide. Silicone sealant was used to attach the coverslip to the microscope slide and to provide a flexible hinge. A thin layer of warm VCMPG was applied to sterile slide chambers and inoculated with a mycelial plug from the edges of colonies. The chambers were incubated inside a humid petri dish at 23°C for 48 h. During incubation, the hinged coverslip was maintained open so it would not come in contact with the medium or the fungus. Once the colony extended onto the middle of the slide chamber (i.e., it had grown 20 to 30 mm), the inoculum plug plus excess mycelia with aerial growth were removed with a razor blade. After 20 min, the
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coverslip was carefully lowered to contact the gel medium. Hyphae growing in the slide chamber were observed by phase contrast optics 10 to 20 min after the coverslip was lowered to allow the fungus to adapt to the conditions of the chamber.
Video Microscopy Microscopic observations were made with an Olympus Vanox microscope (Olympus Optical Co., LTD, Tokyo, Japan) fitted with a phase contrast 1003 oil-immersion objective lens (n.a. 1.25) and a 253 W.F. eye piece (American Optical, No. 186) in the TV port. Video images were obtained with a Hamamatsu C2400-07 camera and enhanced with an Argus-10 image processor (Hamamatsu Photonic Systems Corp., Bridgewater, NJ) and displayed on a black and white, 12-inch monitor (Sony Corporation of America, San Jose, CA; Model PVM-122). Final magnification obtained on the video screen was 75003. Images were recorded in real time on S-VHS videotapes. Apical branching was induced by increasing the temperature of the slide chamber directly on the microscope stage using a household electric hair dryer (Conair Corporation, Model 187A) with its nozzle positioned 13 cm from the specimen. Air flow and temperature were regulated with a rheostat to maintain the air temperature in the vicinity of the chamber at 40 6 1°C. Slide chamber temperature was determined with an ultrathin thermocouple (made from 0.00059 5 12 µm thick foil embedded in a polymer glass laminate; Model CO1-T, Omega Engineering Inc., Stamford, CT) placed inside the chamber over a layer of VCMPG.
Measurements Colony growth rate was measured on petri plate cultures. The plates of VCM agar were inoculated with a spore suspension of either the wild type or mutant (ramosa-1) strains. Plates were incubated at 23°C (permissive temperature) for the first 24 h, until a small (3–5 mm) colony was established. For incubation at the restrictive temperature, the plates were shifted to incubators at 34 or 40°C. Colony diameter was measured in duplicate plates approximately every 24 h until the colonies reached the edge of the plate. Growth rate was calculated by linear regression. The diameter of peripheral hyphae was determined in colonies growing in petri plates or in slide chambers, at final magnifications of 15003 or 75003, respectively. Readings were made on the monitor screen with the
Argus-10 image processor. Diameter was measured at about 15–20 µm from the tip, at which distance, and for all practical purposes, diameter had reached its maximum.
RESULTS Isolation of Morphological Mutants UV mutagenesis of A. niger conidia yielded a variety of morphological mutants, several of which were branching mutants with a small, compact, colony phenotype. From 18 mutants isolated, only 3 maintained the mutant phenotype after reisolation. Among them, we recovered two temperature-sensitive mutants capable of apical branching when shifted to 40°C, and we named them ramosa-1 and ramosa-2. Since ramosa-1 underwent apical branching consistently when shifted to the restrictive temperature, we chose it to analyze the process of apical branching by video-enhanced phase contrast microscopy. Although the restrictive temperature initially used was 40°C, we subsequently found that the apical branching phenotype was also expressed at a lower temperature (34°C).
Colony Growth and Morphology At the permissive temperature (23°C), in any of the culture media we tested, both colony morphology (Fig. 1) and radial growth rate (Fig. 2) of the ramosa-1 mutant were similar to those in the parental wild type strain. In fact, the radial growth of mutant colonies was about 5% higher than wild type. The average growth rate of wild type and ramosa-1 on VCM plates was 9 to 10 µm/min. Both strains conidiated profusely. At the restrictive temperature (34–40°C), colony morphology and growth rate of wild type and mutant differed sharply. While the wild-type strain maintained its colony appearance (Fig. 1C), the overall aspect of ramosa-1 changed dramatically. It turned button-like or ‘‘colonial’’ (as defined by Barratt and Garnjobst, 1949). At this temperature, ramosa-1 did not conidiate (Fig. 1D). At either 34 or 40°C, the radial growth rate of ramosa-1 was drastically reduced to only 11 or 7%, respectively, of that at 23°C (Fig. 2). In contrast, when the wild-type strain was grown at 34 or 40°C two different responses were observed. At 34°C, the growth rate doubled but at 40°C it was reduced to 80% of that at 23°C (Fig. 2).
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Reynaga-Pen˜a and Bartnicki-Garcia
FIG. 1. Colony morphology of A. niger wild type and ramosa-1 grown at the permissive and restrictive temperatures on VCM for 5 days. (A) Wild type strain at 23°C. (B) Ramosa-1 at 23°C. (C) Wild type strain at 34°C. (D) Ramosa-1 at 34°C. Magnification 1.123.
FIG. 2. Colony growth of A. niger wild type and ramosa-1. Radial growth rate of wild type (W) and ramosa-1 (X) strains at the permissive (23°C) and restrictive (34 and 40°C) temperatures. Agar plates of VCM were inoculated in the center with a spore suspension, preincubated for 24 h at 23°C, and then transferred to 34 or 40°C or maintained at 23°C. Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Observed at low magnification, the wild-type phenotype consisted of long hyphae with widely spaced lateral branches. Typically, the first lateral branch emerged at 150–300 µm from the growing tip (Fig. 3A). The same phenotype was maintained after a change to the restrictive temperature (Fig. 3C). At the permissive temperature, ramosa-1 phenotype was similar to the wild type, with only a slight reduction (5–7%) in hyphal diameter (Fig. 3A vs Fig. 3B). After transfer to the restrictive temperature (34 to 40°C), all leading hyphae of ramosa-1 branched apically within 45–75 min (Fig. 3D). When colonies of ramosa-1 were kept at the restrictive temperature for additional 20 to 30 min after the first branching, each new tip branched again within 20 µm of the previous branch (Fig. 3E). Incubation at the restrictive temperature for 90 to 120 min also produced increased septation and formation of small lateral branches next to
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the septa (Fig. 3F). Subsequently, these lateral branches developed apical branches (not shown). Expression of the apical branching phenotype was reversible; mutant colonies that were returned to the permissive temperature ceased producing apical branches and grew long hyphae with lateral branches similar to the wild-type strain.
of about 0.4 µm, although at times this zone was not evident. Numerous mitochondria were found just outside this phase-light zone, mostly aligned with the longitudinal axis of the hypha. Mitochondria were in continuous motion; individual mitochondria were seen to dart frequently into the apical region and interact momentarily with the Spitzenko¨rper.
Hyphal Tip Organization4
Response of Ramosa-1 during Shift to the Restrictive Temperature
At 23°C, hyphae of ramosa-1, observed at high magnification by phase contrast optics, were similar to those of the wild type (Figs. 4A and 4B). The overall distribution of organelles in the apical and subapical region followed the pattern typical of Spitzenko¨rper-bearing fungi (Girbardt, 1969; Grove and Bracker, 1970). The Spitzenko¨rper appeared as a single, phase-dark, ellipsoidal structure within the apical dome. Its size, measured in 30 different hyphae, varied from 0.9 to 1.5 µm in its short axis (1.1 µm average) and 1.2 to 1.9 µm in its long axis (1.5 µm average). This phase-dark structure corresponds to the vesicle cluster described by Lo´pez-Franco and Bracker (1996). The two other components of the Spitzenko¨rper of A. niger described by Lo´pez-Franco and Bracker were less evident: we could see a diffuse vesicle cloud surrounding the vesicle cluster but, except on rare occasions, we could not detect a core inside the vesicle cluster. In single, undisturbed growing hyphae of wild type or mutant, Spitzenko¨rper shape and size remained relatively constant. The Spitzenko¨rper position varied slightly; most of the time, we could see a small but distinct separation (median value 5 0.26 µm, n 5 30) between the Spitzenko¨rper and the apical pole (Fig. 4). During hyphal elongation, the Spitzenko¨rper moved along a straight path but with minor transverse oscillations (up to 6 pixels 5 0.35 µm from the longitudinal axis). The Spitzenko¨rper lies in an exclusion zone described by Lo´pez-Franco and Bracker (1996) as ‘‘a region in the apex where other large organelles were scarce or absent, except for occasional intruding mitochondria.’’ In our observations of A. niger, both wild type and ramosa-1, we found that behind the Spitzenko¨rper there was a phase-light zone 4 The nomenclature for the different parts of a hypha is the one adopted by Lo´pez-Franco and Bracker (1996) from an earlier proposal (Bartnicki-Garcia, 1990). The different hyphal regions are subdivided on the basis of d values, d being the distance between the VSC and the apical pole in the hyphoid equation (Bartnicki-Garcia et al., 1989). The apical dome extends between 0 and 1d; the hyphal apex between 0 and 2d; the subapical region begins at 2d. For the parent hypha in Fig. 7A, d 5 1.3 µm.
During a shift from permissive (23°C; room temperature) to restrictive temperature (34°C), the temperature in the slide chamber first rose rapidly to 30°C and then climbed more gradually between 30 and 34°C (Fig. 5). Within 20 min, the temperature inside the chamber had reached 34°C and remained at 34 6 1°C as long as the heat source was on. We recorded the behavior of 120 individual hyphae of ramosa-1 during the temperature shift to 34°C. The following responses were noted: (1) In about half of all hyphae (52%) the Spitzenko¨rper displayed abnormal behavior accompanied by abnormal cell morphology. Within this group, there were two categories: (i) About 21% of all hyphae underwent cytoplasmic contractions (see description below), and these contractions were followed by a sharp reduction in elongation rate. In most cases, the Spitzenko¨rper disappeared from view. Preliminary observations indicated occasional apical branching in such hyphae but the large number of nongrowing, seemingly dead hyphae indicated that, in the confinement of the slide chamber, heating had a deleterious effect not observed in petri plate cultures. We circumvented this problem by discontinuing the heating of the slide chamber as soon as the Spitzenko¨rper disappeared. Accordingly, about half of the hyphae that underwent cytoplasmic contractions branched apically (the temperature in the slide chamber dropped an average of 4°C within the first 5 min after the heating source was turned off). We analyzed a total of 13 videotaped sequences of apical branching to follow the behavior of the Spitzenko¨rper and related cytological events (see below). (ii) Of all hyphae, 31% showed morphological alterations without manifesting cytoplasmic contractions. The shapes produced departed significantly from the normal hyphoid5 shape; the beaded morphology observed in Fig. 6A was a distinct phenotype shown by 5% of the total number of hyphae. 5
A hyphoid shape is one that equals, or approximates closely, the shape described by the hyphoid equation y 5 x cot (xV/N) from the VSC model for fungal morphogenesis (Bartnicki-Garcı´a et al., 1989). Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 3. Effect of temperature on the morphology of peripheral (leading) hyphae in colonies of A. niger wild type and ramosa-1. (A) Wild type strain at 23°C. (B) Ramosa-1 at 23°C. (C) Wild type strain at 40°C. (D–F) Ramosa-1 at 40°C.
(2) In the other half of the hyphae observed (46%) there were no gross abnormalities and growth simply ceased within 10–30 min; typically, the apical zone of exclusion increased accompanied by a gradual reduction in growth rate, cell diameter, and Spitzenko¨rper size (Fig. 6B). No cytoplasmic contractions were detected in these hyphae. (3) Only 2% of the hyphae observed continued elongating normally during the entire treatment period (60 to 90 min).
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Events during Apical Branching in Ramosa-1 The following observations on hyphal cytology and physiology pertain to the apex and subapical region 20 µm long, which was the average length of hypha within the field of view. Apical branching was accompanied by marked changes in Spitzenko¨rper behavior and internal
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organization of the hyphal apex and subapex. The following description of events is a composite picture made from 13 examples of apical branching. However, the illustrations (Figs. 7 to 10) and the description of Spitzenko¨rper ontogeny and branch development come from a single representative sequence of apical branching where all the described events were clearly visible. We recognized six stages in this morphogenetic process: (a) Cytoplasmic contraction. The first discernible event preceding apical branching was a cytoplasmic contraction; it occurred within 20 to 60 min after the heat source was turned on, by the time the chamber temperature had stabilized at 33 to 35°C (Fig. 5). The contraction included two simultaneous events: (1) a flash created by a sharp change in refractive index (darkening) in a localized area inside the cell with a corresponding lightening of the
FIG. 5. Temperature increase inside the slide chamber placed on the microscope stage during the heating procedure described under Materials and Methods.
FIG. 4. Hyphal tips of A. niger wild type (A) and ramosa-1 (B) growing at 23°C, observed by phase contrast microscopy. Arrows point to the Spitzenko¨rper.
adjacent zone outside the cell (Fig. 7B) and (2) a sudden synchronous movement of organelles toward the apex lasting about 1 s (Reynaga-Pen˜a, 1996). The overall aspect of the hyphal segment under observation was affected by the contraction. The cytoplasm darkened, and the organelles (notably mitochondria) became ‘‘compacted’’ (Fig. 7C); these changes were reversed within 5–10 s. (b) Spitzenko¨rper retraction. Within 5 s following the cytoplasmic contraction, the Spitzenko¨rper retracted from its polar position in the apical dome (Fig. 7D) and moved backward up to 0.4 µm. During retraction, the Spitzenko¨rper shape appeared more spheroidal compared to the ellipsoidal shape when it was next to the apical pole (Figs. 7A and 7B vs Fig. 7D) and measured an average of 1.4 µm in diameter. During retraction, a phase-light zone of exclusion was maintained around the Spitzenko¨rper and it was possible to see thin filaments radiating from the Spitzenko¨rper region (Fig. 7D), crossing the zone of exclusion, and extending forward to the cell wall and backward to nearby mitochondria. (c) Spitzenko¨rper disappearance. After its momentary retraction, the Spitzenko¨rper migrated back toward the apical pole (Fig. 7E), and its size decreased gradually as it meandered within a confined area at the apex, and finally vanished (Figs. 7F and 7G). The time elapsed between the contraction and the disappearance of the Spitzenko¨rper varied from 20 to 58 s. As the Spitzenko¨rper disappeared, mitochondria migrated toward the subapical region and clustered in a subapical zone between 5 and 10 µm from the apical pole (Fig. 7G); this caused the hyphal apex to acquire an ‘‘empty’’ appearance. This organelle-depleted
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FIG. 6. Abnormal morphology and behavior of some hyphae of ramosa-1 growing at the restrictive temperature of 34°C. (A) Beaded appearance. (B) Reduction in Spitzenko¨rper size and hyphal diameter and increase in exclusion zone size prior to cessation of growth.
zone extended up to 6 µm from the apical pole. Organelle clustering occurred only in a small subapical zone (within 15 µm from the apical pole). Occasionally during the period when no Spitzenko¨rper was visible, the cluster of mitochondria dispersed for a few seconds to later cluster again. (d) Transition phase. During the period when no Spitzenko¨rper was visible (60–260 s) elongation ceased (Fig. 11) but some growth continued, albeit at a much slower rate (Reynaga-Pen˜a et al., 1997). During this time, the apex lost its characteristic hyphoid shape and became enlarged and blunt (Fig. 8). From opposite sides of this deformed apex, the two branches later emerged with their corresponding Spitzenko¨rper.
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Reynaga-Pen˜a and Bartnicki-Garcia
(e) Spitzenko¨rper ontogeny. At the sites where the new apical branches originated, there was no evidence of a specific, discrete structure preceding the appearance of the new Spitzenko¨rper. Small clouds of irregular outline and medium density were commonly seen moving in the area where new Spitzenko¨rper formed, sometimes aggregating into a denser cloud that gave the appearance of an incipient Spitzenko¨rper (Fig. 7H). The Spitzenko¨rper-like body did not seem to be stable; it moved along the cell periphery and rapidly dissolved. Frequently, but momentarily, mitochondria migrated into the area where new Spitzenko¨rper formed. Details of the ontogeny of the two Spitzenko¨rper formed in the apically branching hypha are shown in Figs. 9 and 10. The first new Spitzenko¨rper became established and remained distinctly visible 263 s after the cytoplasmic contraction (Fig. 9C). This Spitzenko¨rper increased in size rapidly and within 20 to 30 s it reached a maximum size of 1.1 µm by 1.3 µm (Fig. 9G). As the Spitzenko¨rper developed, the nearby cell wall expanded and the first apical branch emerged (Figs. 7I–7L and 8). In the five apical branching sequences where we could follow the development of the two Spitzenko¨rper simultaneously, we found that they did not develop in unison. One Spitzenko¨rper appeared first, the second one sometime later (60 to 100 s after the first) (Figs. 7J, 7L). The second Spitzenko¨rper was first visible at 360 s (Fig. 10B); it was smaller than the first branch Spitzenko¨rper and measured 0.8 µm by 1.0 µm when it reached a maximum size (Fig. 10F). (f) Recovery of normal tip organization. Recovery of the typical organelle distribution at the hyphal tip occurred gradually, and was simultaneously accompanied by recovery of apical elongation. First, mitochondria moved slowly toward the apex and became gradually positioned close to the area where the first of the two new apices later appeared (Fig. 7I). As branches developed, organelle distribution in the emerging tips gradually attained that of normal growing hyphae (Figs. 7K–7N). (g) Branch development. Two branches were formed on opposite sides of the main hyphal axis at angles of 50° to 60°. In the two instances when both apical branches grew on the same focal plane, it became feasible to simultaneously compare their development. As judged by comparison of cell profiles, the two emerging branches were clearly evident at 310 s (Fig. 8). From the outset, one branch became dominant. By 270 s there was already evidence of two incipient branches, one slightly larger than the other. Because of this size discrepancy it was hard to tell if both
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branches began to grow at exactly the same time or whether the dominant branch preceded the other; in any case, we estimate the time difference to be less than 20 s. The first branch was of a larger size and by the end of the observations (Fig. 7N), it had attained a diameter of 6.0 µm at its base, while the diameter of the second branch was 4.7 µm (78% of the diameter of the first branch). Occasionally, more than two branches formed at the apex of the ramosa-1 mutant after a shift to the restrictive temperature.
Changes in Cell Wall Aspect As the Spitzenko¨rper began to disappear, the appearance of the apical cell wall changed (Fig. 7). Its outline became progressively darker and wider, suggesting thickening of the wall. The change in appearance was more perceptible in the vicinity of the area where the cytoplasmic contraction occurred. As the branches began to develop, the newly formed wall at the two apical poles had the appearance of a normal growing hypha.
enhanced phase-contrast microscopy allowed us to define more precisely the cytological events in apical branching. Although the physiology of apical branching had been studied earlier in A. nidulans (Trinci, 1970; Trinci and Morris, 1979), this seems to be the first time that cytological details in hyphal tip organization and Spitzenko¨rper behavior have been described during apical branch formation. The recording of microscopic images on videotape was essential for the subsequent qualitative and quantitative analysis of events. Videotape analysis made it possible to disclose important short-term events associated with apical branching in ramosa-1 such as cytoplasmic contractions, the retraction of the Spitzenko¨rper, its disappearance, and the appearance of two new Spitzenko¨rper. The profound changes in growth rate and hyphal morphology observed during apical branching were accompanied by drastic changes in Spitzenko¨rper behavior. These findings reinforce the idea that this structure plays a decisive role in apical growth and morphogenesis (Girbardt, 1957, 1969; McClure et al., 1968; Grove and Bracker, 1970; Bartnicki-Garcı´a, 1990).
Growth Rates Spitzenko¨rper Morphology
The apical branching process was accompanied by profound changes in elongation rate of hyphae (Fig. 11). Within 2 s after the cytoplasmic contraction, the elongation rate began to drop. By 30 s, the rate had diminished to 35% of the original value (from 5.60 to 1.98 µm/min). Between 30 and 60 s it dropped to 20% of the original rate, 1.12 µm/min. During the time when the Spitzenko¨rper was not visible (60 to 260 s) the average elongation rate dropped further to 7.5% of the original rate, 0.42 µm/min. From 150 to 180 s, no elongation was detected. As the first and second Spitzenko¨rper appeared, the cell began to recover and the elongation rate of the branches slowly started to rise. From 263 s (the time when the first Spitzenko¨rper was visible) to 330 s, the first branch elongated at 2.22 µm/min, and attained a rate of 2.76 µm/min by the end of our observations (570 s). Similarly, from 360 s (when the second Spitzenko¨rper appeared) to 420 s, the second branch elongated at 1.61 µm/min, and by 570 s it reached 2.46 µm/min (Fig. 11).
The Spitzenko¨rper of A. niger corresponds to pattern 4 described by Lo´pez-Franco and Bracker (1996). Our observations on the Spitzenko¨rper morphology of A. niger, both wild type and ramosa-1, correspond to what Lo´pezFranco and Bracker described as the vesicle cluster (the phase-dark spheroidal body in close proximity to the apical pole). We could not distinguish the central dark core from the outer vesicle cluster, and there was usually a distinct phase-light zone of separation between the Spitzenko¨rper and the apical cell membrane. Generally, the Spitzenko¨rper of our strain of A. niger maintained an ellipsoidal shape, but during retraction its shape changed from ellipsoidal to spherical. This could be another example of Spitzenko¨rper pleomorphism (Lo´pez-Franco and Bracker, 1996); on the other hand, the ellipsoidal shape could be the result of optical distortion by the high curvature of the apical dome, in which case, the spheroidal shape might be the true shape of the Spitzenko¨rper.
DISCUSSION
Spitzenko¨rper Behavior
The isolation of temperature sensitive mutants of A. niger (ramosa-1 and ramosa-2) and the use of video-
The Spitzenko¨rper is a dynamic structure that advances continuously as the hypha elongates (Girbardt, 1957; Lo´pez-Franco and Bracker, 1996). As it moves on along the
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FIG. 7. Cytological events during apical branching of ramosa-1. (A) Normal hyphal growth before the cytoplasmic contraction. (B) Cytoplasmic contraction; arrow points to sharp change in refractive index. (C) Hyphal tip after the contraction. (D) Spitzenko¨rper retraction; arrows indicate filaments radiating from the Spitzenko¨rper. (E) Spitzenko¨rper returning to the apical pole. (F–G) Spitzenko¨rper disappearance; arrow in G points to cluster of mitochondria. (H) Transient vesicle cloud (arrow) appeared to form incipient Spitzenko¨rper but later disappeared. (I) Blunt hyphal tip prior to Spitzenko¨rper appearance. (J–L) Formation of new branch Spitzenko¨rper. (M–N) Branch elongation. Bar 5 5 µm.
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Apical Branching in A. niger Mutant
FIG. 7—Continued
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FIG. 9. Ontogeny of the first Spitzenko¨rper (Fig. 7, upper branch) formed during the ramosa-1 apical branching sequence. Arrows indicate the vesicle cloud (B) that gave rise to a Spitzenko¨rper (C).
FIG. 8. Progressive changes in hyphal tip morphology preceding the appearance of apical branches. (A) The traced cell profiles at 0 and 246 s were superimposed on the image captured at 310 s. Note the transformation of the apex from a typical hyphoid-like shape (0 s) to a blunt shape tip prior to the emergence of the two incipient branches (310 s). (B) A hyphoid plot was superimposed on the image of the blunt apex (246 s) to show the departure from hyphoid morphology.
longitudinal axis of the hypha, it also shows minor oscillations in the transverse direction. The factors responsible for advancing and maintaining the position of the Spitzenko¨rper are not well understood. Our observations on cytoplasmic contractions (see below) suggest the involvement of the cytoskeleton in Spitzenko¨rper dynamics. A number of studies provide evidence for a Spitzenko¨rper–cytoskeleton connection. Examination of hyphal tips of Magnaporthe grisea (Bourett and Howard, 1991) and Allomyces macrogynus (Vargas et al., 1993) revealed actin filaments and microtubules in the Spitzenko¨rper region. Although the role of cytoskeletal elements in Spitzenko¨rper structure and function is not well established, several
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reports suggest that the cytoskeleton may propel and control the motion of the Spitzenko¨rper. Howard and Aist (1977) showed that the Spitzenko¨rper of Fusarium acuminatum vanished after treatment with MBC, an anti-tubulin agent. Similarly, Grove and Sweigard (1981), Roberson and Vargas (1994), and Srinivasan et al. (1996) showed that disruption of either microtubules or actin filaments caused the detachment and disappearance of the Spitzenko¨rper of Rhizoctonia solani or A. macrogynus. An association between cytoskeletal filaments and the
FIG. 10. Ontogeny of the second Spitzenko¨rper (Fig. 7, lower branch) formed during the ramosa-1 apical branching sequence. Arrows in B and C point to the incipient Spitzenko¨rper.
Apical Branching in A. niger Mutant
FIG. 11. Hyphal elongation during apical branching. Time zero corresponds to the cytoplasmic contraction. X, Main branch; W, first branch; M, second branch. Measurements started at 226 s for the main hypha, and at 246 s for the branches.
Spitzenko¨rper is also suggested by our observations of phase-dark filaments radiating from the Spitzenko¨rper of ramosa-1 during its detachment from the apical pole. Similar filaments were reported by Lo´pez-Franco and Bracker (1996) for the Spitzenko¨rper of R. solani.
Spitzenko¨rper Ontogeny From our findings on apical branching, we can make some tentative conclusions about Spitzenko¨rper ontogeny. Although it seems certain that the original Spitzenko¨rper did not split into two to form the Spitzenko¨rper of the apical branches, we cannot rule out that invisible remnants (core?) of the original Spitzenko¨rper may have contributed or given rise to the two new Spitzenko¨rper. We favor the view that the two branch Spitzenko¨rper were formed de novo from vesicle accumulations around an invisible nucleation site. Whether this presumed nucleation site becomes, or forms part, of the Spitzenko¨rper core remains an intriguing question, and one that is particularly difficult to address since the nature and structure of the Spitzenko¨rper core have yet to be elucidated. The first recognizable event in Spitzenko¨rper formation was the appearance of a vesicle cloud in the apical region. Although we could not identify, unequivocally, these accumulations of medium density material as being vesicle populations, such explanation is likely, at least for those which later condensed and became a Spitzenko¨rper. Note
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that some of these clouds did not necessarily produce a ‘‘new’’ Spitzenko¨rper, they dissolved or drifted away. Perhaps Spitzenko¨rper development from a vesicle cloud may require an ‘‘anchoring’’ step to stabilize it in place. Conceivably, actin and tubulin could be involved in anchoring the Spitzenko¨rper and/or in maintaining its integrity. The idea that ‘‘Spitzenko¨rper integrity might require fully functional microtubules’’ was previously suggested by Howard and Aist (1977). Although our observations and conclusions on Spitzenko¨rper ontogeny pertain exclusively to apical branching, the question arises as to whether they apply to lateral branching which is, by far, the most prevalent mode of branching in fungi. A de novo mechanism for Spitzenko¨rper formation seems likely in lateral branching, especially since the main Spitzenko¨rper lies too far away from the branching site to have any direct effect on its formation. Whether or not a localized cytoplasmic contraction also precedes lateral branching remains an intriguing possibility.
Apical Dominance and Branch Ontogeny The apical branching we observed in ramosa-1 is not a perfectly dichotomous branching in which the parental hypha would split evenly into two branches of equal standing. The apical branches of ramosa-1 were uneven in three respects: time of emergence, growth rate, and size. One branch, the first one to appear, became dominant, it grew faster and wider than the other. This raises intriguing questions about the basis of this dominance and also questions about definitions. On the latter, Trinci (1970) acknowledged the difficulty in determining whether the branching in A. nidulans was truly apical or subapical (if it occurred beyond 4 µm). With the much higher magnification we employed and with the subsequent image analysis, we were able to distinguish more precisely the origin of the two branches in A. niger. Figure 8 shows clearly that both branches arise from equivalent but opposite sites on the foremost part of the hyphal apex, which by this time had become enlarged and its shape was no longer hyphoid but blunt. Therefore, on the basis of location, the branching we observed is truly apical. As to the dominance exerted by the first branch, presumably this arises from cytoskeletal connections that give the first Spitzenko¨rper a larger share of the supply of cytoplasmic vesicles from the parental hypha. Once this is set, the second branch Spitzenko¨rper can marshal only a minor share of the flow of wall-building vesicles.
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Kinetics of Apical Branching Our observations on the kinetics of apical branching in the ramosa-1 mutant of A. niger agree with previous conclusions made for apical branching in Fusarium oxysporum (Robertson, 1958) and A. nidulans (Trinci, 1970). In both instances, there was a drastic but transient reduction in elongation rate prior to appearance of apical branches. Unlike Trinci’s (1970) findings on A. nidulans, the branches in ramosa-1 did not recover the elongation rate of the parent hypha previous to branching; this discrepancy, however, could be due to the temperature shift down of the slide chamber before the appearance of the apical branches. The presumed alteration in cytoskeleton organization during a cytoplasmic contraction may also affect vesicle travel from the subapex, where vesicles originate (Grove and Bracker, 1970), to the apex. A drastic reduction in the flow of wall-building vesicles reaching the tip would account for the momentary but sharp reduction in cell expansion following the cytoplasmic contraction.
Phenotype Expression Ramosa-1 expressed its apical branching phenotype readily and consistently on solid media in ordinary petri dishes. However, phenotype expression inside closed slide chambers, placed under a 1003 oil-immersion objective, was a more difficult challenge. On petri plates essentially all peripheral hyphae branched apically when shifted from 23 to 34°C, whereas only 11% of the hyphae examined underwent apical branching inside the chambers. To understand this low response, we have to consider that the hyphae were already under considerable stress caused by the limiting environment inside the slide chambers. For instance, Lo´pez-Franco et al. (1994) found that elongation rates of hyphae growing in closed slide chambers were only 27 to 69% of those measured in cells growing in petri plates. Presumably, the temperature increase required to express the apical branching phenotype compounded other stresses created by confinement in the slide chambers. The high incidence of cell death (46%) and abnormal morphology (31%), observed within the first few minutes of heating indicate a highly stressful environment.
Cytoplasmic Contractions Cytoplasmic contractions are not unique to the ramosa-1 mutant. We have also observed them in the wild-type strain of A. niger, though at a much lower
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Reynaga-Pen˜a and Bartnicki-Garcia
frequency. Localized cytoplasmic contractions can also be detected in hyphae of other fungi, e.g., Neurospora crassa and Trichoderma harzianum (Reynaga-Pen˜a, unpublished results). In R. solani, ‘‘cytoplasmic waves’’ with some similarity to the cytoplasmic contractions in A. niger have also been reported (Lo´pez-Franco and Bracker, 1996). Although the cause and nature of the cytoplasmic contractions is unclear, it seems safe to conclude that the observed unidirectional movement of organelles involved a contraction of a cytoskeletal network that links all these structures together. Cytoplasmic contractions of longer duration were induced in hyphae of Basidiobolus magnus (McKerracher and Heath, 1986) and Saprolegnia ferax (Jackson and Heath, 1992; Kaminskyj et al., 1992). These reports suggested that calcium and actin–myosin interactions were involved in the mechanism causing the contractions. In all instances where cytoplasmic contractions were detected in ramosa-1 (21% of total hyphae), we found a significant drop in hyphal elongation rate. In the majority of these hyphae (16% of the total hyphae analyzed), Spitzenko¨rper behavior was also affected: it either retracted from its polar position and disappeared from view, or momentarily stopped advancing. Our finding that cytoplasmic contractions were consistently followed by Spitzenko¨rper dislocation and/or disappearance suggests the existence of a dynamic connection between the cytoskeletal network and the Spitzenko¨rper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization, manifested by the cytoplasmic contraction. A logical next step in understanding the biochemical nature of the mechanism responsible for apical branching would be the characterization of the genetic locus affected in ramosa-1.
ACKNOWLEDGMENTS Supported in part by grants from the National Institutes of Health (GM-48257), the National Science Foundation (IBN-9204541), and a predoctoral fellowship from the ‘‘Consejo Nacional de Ciencia y Tecnologı´a’’ (CONACYT, Me´xico) to the senior author.
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