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Molecular and Cellular Biology, February 2009, p. 771-783, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.01150-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Of Bars and Rings: Hof1-Dependent Cytokinesis in Multiseptated Hyphae of Ashbya gossypii{triangledown} ,§

Andreas Kaufmann and Peter Philippsen*

Biozentrum der Universität Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland

Received 21 July 2008/ Returned for modification 18 August 2008/ Accepted 12 November 2008


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ABSTRACT
 
We analyzed the development of multiple septa in elongated multinucleated cells (hyphae) of the filamentous ascomycete Ashbya gossypii in which septation is apparently uncoupled from nuclear cycles. A key player for this compartmentalization is the PCH protein Hof1. Hyphae that are lacking this protein form neither actin rings nor septa but still elongate at wild-type speed. Using in vivo fluorescence microscopy, we present for the first time the coordination of cytokinesis and septation in multiseptated and multinucleated cells. Hof1, the type II myosin Myo1, the landmark protein Bud3, and the IQGAP Cyk1 form collars of cortical bars already adjacent to hyphal tips, thereby marking the sites of septation. While hyphae continue to elongate, these proteins gradually form cortical rings. This bar-to-ring transition depends on Hof1 and Cyk1 but not Myo1 and is required for actin ring assembly. The Fes/CIP4 homology (FCH) domain of Hof1 ensures efficient localization of Hof1, whereas ring integrity is conferred by the Src homology 3 (SH3) domain. Up to several hours after site selection, actin ring contraction leads to membrane invagination and subsequent cytokinesis. Simultaneously, a septum forms between the adjacent hyphal compartments, which do not separate. During evolution, A. gossypii lost the homologs of two enzymes essential for cell separation in Saccharomyces cerevisiae.


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INTRODUCTION
 
Cytokinesis is an essential process for cell proliferation. Early studies showed that a contractile actomyosin ring drives cytokinesis that finally leads to cell division (6, 17). The principle of contracting rings and the timing of cytokinesis with other cell cycle events are conserved in many biological systems, but mechanistic or molecular details can markedly differ between species. Some good examples are the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. In these two unicellular ascomycetes, a conserved set of proteins is involved in three processes at the cell division plane: cytokinesis (the separation of the cytoplasm), septation (primary and secondary septum formations), and cell separation (the controlled degradation of the primary septum and the connecting cell wall). The formation of a contractile actomyosin ring is tightly coupled to the cell cycle. After nuclear division, the ring contracts, thereby generating two cytoplasmic compartments with one nucleus each, and simultaneously, a septum forms at the division site (4, 23). Finally, the partial degradation of the septum leads to the separation of the two daughter cells. The main difference between the systems is the site selection for the contractile ring and, thus, the septum. In fission yeast, this site is selected in the midzone of the cell after the positioning of the nucleus (12, 49), whereas in budding yeast, this site is predetermined by the selection of the bud site (7, 13).

Compared to unicellular ascomycetes, very little is known about the control of cytokinesis and septation in filamentous ascomycetes. These fungi proliferate as fast-spreading mycelia, i.e., networks of continuously elongating and branching cells called hyphae. Septation leads to subdivisions of hyphae but is not followed by cell separation. The process of septation in filamentous ascomycetes is most likely similar to cytokinesis and septum formation in yeasts, because the components seem to be conserved (59), but the process of cell separation is either repressed or absent (19). Since hyphal compartments of many filamentous ascomycetes carry multiple nuclei, nuclear divisions proceed without (or only rarely) triggering septum formation (24). This indicates that, in contrast to unicellular yeasts, septation and the nuclear cycle are not coupled in filamentous ascomycetes. One exception is Aspergillus nidulans that performs waves of synchronized mitoses and for which a strong influence of mitotic nuclei and nuclear density on septum formation was reported (64, 66).

In filamentous ascomycetes, the best-studied proteins with conserved roles in septation are septins (28, 43, 51). Septins make up a protein family that is essential for cytokinesis, which was first described in budding yeast (21, 22, 27, 41). Septin homologs have been found throughout eukaryotes, with the exception of plants. Strikingly, septins of filamentous fungi form higher-order structures with different appearances and potentially different functions within one hypha (19, 54). Apart from septins, few other proteins that play conserved roles in cytokinesis have been characterized in filamentous ascomycetes. In Ashbya gossypii, the IQGAP Cyk1 protein is essential for actin ring formation and septation, but hyphae lacking Cyk1 grow as quickly as the wild type (63). The landmark protein Bud3 is involved in restricting Cyk1 to the sites of septation (60). Deletion of the PAK-like protein kinase Cla4, a putative downstream effector of the well-conserved GTPase Cdc42 (62), severely impairs actin ring formation and septation (2). In Neurospora crassa, the RHO-4 GTPase regulates vegetative septation (45). Interestingly, A. gossypii cells that lack the formin Bni1 have no actin cables, but they are still able to form aberrant septa (48), whereas in A. nidulans, formin mutants are blocked in cytokinesis (26), again indicating the differences among filamentous ascomycetes.

In many species, pombe Cdc15 homology (PCH) proteins have emerged as important coordinators of actomyosin ring assembly and membrane dynamics. PCH proteins are characterized by a conserved domain composition, and many of them are involved in actin-based processes, especially in cytokinesis (39). S. pombe Cdc15, the founding member of the PCH protein family (16), plays an essential role in actomyosin ring maintenance (57). Similarly, disruption of S. cerevisiae HOF1/CYK1 results in actin ring disassembly during contraction, leading to incomplete cytokinesis (38). Hof1 is thought to function in parallel with type II myosin Myo1 to promote septum formation even in the absence of an actomyosin ring (53). It was shown that PCH proteins are involved in membrane binding and deformation (31, 52). This indicates that PCH proteins can couple membrane deformation to the actin cytoskeleton, as is required during cytokinesis when the actin ring contracts. Only recently, Ustilago maydis Cdc15 was found to form a cytokinetic contractile ring during yeast-like growth in the dimorphic fungus Ustilago maydis (9).

Here, we investigate, mainly by three-dimensional (3-D) time-lapse fluorescence microscopy, the initiation and maturation of multiple sites of septation in the model fungus A. gossypii. Despite the similarities of the A. gossypii and S. cerevisiae genomes with mostly syntenic genes (14), A. gossypii exclusively grows filamentously. We used the conserved cytokinesis protein Hof1, the first PCH protein analyzed in a filamentous fungus, to establish a complete picture of the structure of emerging sites of septation and the dynamics of the structural changes during the formation of multiple septa. Next, we analyzed the contributions of Hof1 to actin ring formation and to the localization of other cytokinesis proteins like Cyk1, Bud3, Myo1, and Sep7. The contributions of these proteins to Hof1 function were also studied. The findings allow us to dissect spatially and temporally the process of septation during the development of multiple septa in a fungal mycelium. Finally, by comparing the genomes of A. gossypii and S. cerevisiae, we found evidence for the loss of genes that are essential for cell separation during the evolution of A. gossypii. The results we present here will contribute to the understanding of how two systems composed of basically the same components can give rise to such fundamentally different morphologies as filamentous and unicellular growth.


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MATERIALS AND METHODS
 
A. gossypii strains, media, and transformation. Strains ATCC 10895 and {Delta}l{Delta}t (1) will be referred to as the wild types. All strains were cultured as previously described (50, 67). Strains were constructed either by PCR-based gene targeting (61) or by transformation with linear DNA fragments with long flanking homology regions (approximately 200 to 1,000 bp) made from plasmids, which carry the gene with the mutation that will be introduced, as previously described (48). All strains with descriptions of construction (PCR templates and primer names or plasmids and restriction enzymes) are given in Table 1. All oligonucleotides are listed in Table S8 in the supplemental material. The descriptions of plasmids used for strain constructions are listed in Table S9 in the supplemental material. Strains were verified by analytical PCR as previously described (61). All fusion proteins, except Hhf1-green fluorescent protein (GFP), were expressed from their native promoters at their chromosomal loci.


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  		TABLE 1. A. gossypii strains used in this study

Plasmids and DNA manipulations. All plasmids used and details of construction (primers, templates, and restriction enzymes used) are listed in Table S9 in the supplemental material. All DNA manipulations were carried out according to reference 47, with Escherichia coli strain DH5{alpha}F' as the host (25). For the recombination of plasmids and PCR products, both were cotransformed into the yeast host strain FY1679 (65) derivative DY3 (MAT{alpha} his3{Delta}200 trp1{Delta}63 leu2{Delta}1 ura3-52{Delta}), according to reference 18. Plasmids were isolated from yeast using the High Pure plasmid purification kit (Roche Diagnostics, Rotkreuz, Switzerland) with a modified protocol as previously described (48).

Actin, chitin, membrane, and immunofluorescence stainings. Actin staining with either Alexa Fluor 568 or rhodamine phalloidin (Molecular Probes, Eugene, OR) and chitin staining with calcofluor white (fluorescent brightener 28; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were done as previously described (2, 32). Membrane staining with FM 4-64 (Invitrogen, Carlsbad, CA) was done as previously described (58). The septins Cdc11a and Cdc11b were stained as previously described (20). Primary antibody rabbit anti-ScCdc11 (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:20 dilution. Secondary antibody Alexa Fluor 568 goat anti-rabbit immunoglobulin G (H+L) (Invitrogen, Carlsbad, CA) was used at 1:200.

Microscopy. The microscope used was an Axioplan 2 imaging microscope equipped with the objectives Plan-Apochromat 100x 1.40-numerical-aperture oil differential interference contrast (DIC) and Plan-Apochromat 63x 1.40-numerical-aperture oil DIC (Carl Zeiss AG, Feldbach, Switzerland) and appropriate filters (Zeiss and Chroma Technology, Brattleboro, VT). The light source for fluorescence microscopy was either a 75-W XBO lamp (OSRAM GmbH, Augsburg, Germany), controlled by a MAC2000 shutter and filter wheel system (Ludl Electronics, Hawthorne, NY), or a Polychrome V monochromator (TILL Photonics GmbH, Gräfelfing, Germany). Images were acquired at room temperature using a CoolSNAP HQ cooled charge-coupled device camera (Photometrics, Tucson, AZ) with MetaMorph 6.2r6 software (Molecular Devices Corp., Downingtown, PA). Out-of-focus shading references were used for DIC image acquisitions. The distance between two planes in stack acquisitions was set between 0.2 and 1 µm. Brightness and contrast were adjusted using MetaMorph's "scale image" command. Stacks were deconvolved with MetaMorph's "2-D deconvolution" module and flattened by maximum projection with the "stack arithmetic" function. Tilted views of stacks were made with MetaMorph's "3-D reconstruction" function. Images were colored and overlaid using MetaMorph's "overlay images" command. Kymographs were made with MetaMorph's "montage stacks" function. Images were exported from MetaMorph as 8-bit grayscale or RGB TIFF files. Lateral-branching mycelia were cultured in liquid medium and mounted onto microscopy slides. For time-lapse acquisitions or the imaging of tip-splitting mycelia, small pieces of mature mycelium were cultured on agarose slides as previously described (32). The acquisition frequency was set to 0.2 min–1. Time-lapse picture series were processed as described above and converted into QuickTime MPEG-4 movies (QuickTime Player Pro, Apple Inc.).


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RESULTS
 
The PCH protein Hof1 is required for actin ring formation and septation. Mycelia of Ashbya gossypii show two distinct growth patterns: young and slowly growing hyphae (10 to 50 µm h–1) form lateral branches perpendicular to the main hyphae, whereas mature and fast-growing hyphae (80 to 200 µm h–1) display tip splitting, i.e., the symmetrical division of the growing hyphal tip, also referred to as apical or dichotomous branching (48). Schematic presentations of these two growth patterns are shown in Fig. 1A. In lateral-branching mycelia, septa form at the neck of the germ bubble, along the main hyphae, and at the bases of lateral branches. In tip-splitting hyphae, septa form at tip-splitting sites and at approximately regular intervals along the hyphae. The sites of septation in A. gossypii can be seen as cross walls by DIC microscopy or as actin rings after it is stained with fluorescent phalloidins (5, 32).


Figure 1
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  		FIG. 1. Septation in A. gossypii and the PCH protein Hof1. (A) Schematic septation pattern in A. gossypii lateral-branching and tip-splitting mycelia. Empty ovals indicate actin rings; filled ovals indicate septa. (B) ABR082W, which encodes the PCH protein A. gossypii Hof1, was annotated as a syntenic homolog of S. cerevisiae YMR032W (HOF1/CYK2) and as a putative homolog of S. pombe SPAC20G8.05C (cdc15) and SPBC11C11.02 (imp2) (14). ClustalW (37) pairwise alignments of Abr082w with S. cerevisiae Hof1, S. pombe Cdc15, and S. pombe Imp2 resulted in scores of 31, 16, and 14, respectively. An N-terminal FCH domain and a C-terminal SH3 domain were detected with PROSITE (30). COILS (42) predicted two coiled-coil (CC) regions after the FCH domain, with a probability of 1 and 0.982, respectively. PESTfind analysis (46) detected a potential PEST sequence (a region rich in proline, glutamic acid, serine, and threonine) with a score of 5.27. Domain composition of Hof1 and the constructs bearing the domain deletions were used in this study. For functional analyses, all constructs shown were C-terminally tagged with either GFP, YFP, or cyan fluorescent protein and expressed at the chromosomal locus of HOF1 under the control of its native promoter. (C) DIC images and fluorescence images of the Alexa Fluor 568 phalloidin-stained actin cytoskeleton of lateral-branching reference (HOF1-GFP) and hof1{Delta} mycelia. Septation sites were identified as closed septa (black arrowhead) in the DIC images or as Alexa Fluor 568 phalloidin-stained actin rings (white arrowheads). Neither structure was found in hof1{Delta} mutants. Bars, 10 µm.

The A. gossypii open reading frame ABR082W was annotated as a syntenic homolog of S. cerevisiae HOF1/CYK2 (14). Sequence analysis confirmed that ABR082W encodes a PCH protein (Fig. 1B). To test if A. gossypii Hof1 was involved in either actin ring formation or actin ring stability and whether its inactivation would, thus, completely prevent septation or lead to aberrant septa, respectively, the complete open reading frame of HOF1 was deleted. In homokaryotic hof1{Delta} mycelia, cortical actin patches and actin cables appeared to be wild type-like, but actin rings and septa were never observed (Fig. 1C). In 23 fixed and Alexa Fluor 568 phalloidin-stained lateral-branching hof1{Delta} mycelia with lengths of 162 ± 5 µm (standard error of the mean [SEM]) and 5.1 ± 0.2 branches (SEM), no actin rings or septa were found. In contrast, in 23 mycelia of a reference strain (HOF1-GFP) with similar lengths and numbers of branches (152 ± 14 µm and 5.5 ± 0.4 branches; SEM), 72 actin rings or septa were found, resulting in a septation index of 0.6 ± 0.04 (SEM). The septation index is calculated as the number of septation sites (rings and mature septa) divided by the number of branches per mycelium. Apart from the cytokinesis and septation defect, no other polarity defects were discernible: radial colony and tip growth speeds, tip splitting, and the branching index (total mycelial length per number of tips) were not affected by the lack of Hof1, and the mycelial developmental pattern was indistinguishable from that of the wild type (not shown).

Formation of multiple septa in multinucleated tip-splitting hyphae. Since Hof1 was required for actin ring formation and septation in A. gossypii, we tested whether it localizes to the sites of septation and whether it could be used as a marker for different stages of septum formation. To this end, we constructed a HOF1-GFP fusion at its chromosomal locus expressed under the control of its native promoter. Hof1 formed collars of cortical bars and single cortical rings, and at some sites, bars and rings were overlapping (Fig. 2A). When septa were seen in DIC images, Hof1 was found on either side of the septa as cortical spots and ill-defined structures (Fig. 2A). The cortical Hof1 rings were contractile and colocalized with actin rings, as will be analyzed in detail in the next section. Since Hof1 clearly marked septation sites, we could determine how regularly these sites were spaced within hyphae. We found significant growth speed-dependent differences. In lateral-branching mycelia, the distance between the septation sites ranged from 12 to 61 µm and was 38 µm on average (n = 72), whereas in tip-splitting mycelia, it varied between 32 and 112 µm, with an average of 71 µm (n = 135) (Fig. 2B). Next, we analyzed if the different forms of Hof1 localization (bars, rings, or spots) displayed a spatial pattern. We traced 11 tip-splitting hyphae from the hyphal tip to as far back into the mycelium as possible (Fig. 2A). The distances ranged between 290 and 750 µm, which allowed us to categorize five to nine septation sites in each hypha. In all 11 hyphae, Hof1 formed collars of bars at the first two or three sites, counting from the tip. At the forth site, Hof1 localized in five hyphae as bars, in three as bars and a ring, and in three only as a ring. At the fifth site, Hof1 formed bars in only one hypha, a ring in six hyphae, and a contracting ring in two hyphae, and the septa were closed in two hyphae. Further back, Hof1 formed either cortical or contracting rings or the septa were closed. Only in rare cases, it was observed that one septum was closing while the next one further back in the hypha was still open. In these 11 hyphae, the smallest compartment with a continuous cytoplasm, i.e., from the hyphal tip to the first closed septum, was 290 µm, and the biggest one was 570 µm in length. The fact that contracting Hof1 rings and closed septa only formed that far away from hyphal tips, but Hof1 formed collars of bars in tip regions, strongly indicated that the septation sites were selected at or close to growing tips. For lateral-branching mycelia, it was previously suggested that future septation sites are marked at tips when tip growth slowed down concomitant with the emergence of a subapical lateral branch (32). As the localization pattern of Hof1 was similar to those of septins in A. gossypii (28), we acquired time-lapse movies of hyphae expressing either SEP7-GFP or HOF1-GFP to determine how septation sites close to the tip were selected. Hof1 bars emerged immediately behind the growing tip (Fig. 2C; see also Movie S1 in the supplemental material). Similarly, Sep7 localized to a collar of bars right behind the growing tip (Fig. 2C; see also Movie S2 in the supplemental material). This clearly showed that septation sites were indeed selected at the growing tip.


Figure 2
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  		FIG. 2. Hof1 localization at septation sites in multinucleated tip-splitting hyphae. (A) Montage of seven DIC and fluorescence micrographs of a tip-splitting hypha expressing HOF1-CFP and HHF1-yEVenus to visualize the sites of septation and nuclei, respectively. Bar, 50 µm. Blow-ups of the Hof1-cyan fluorescent protein (CFP) signal at the six septation sites and a schematic drawing of the hyphae indicating cortical bars (green lines), rings (empty green ovals), closed septa (filled black ovals), and nuclei (small red ovals) are shown below the composite micrograph. Bars, 5 µm. (B) Distances between two septation sites in lateral-branching and tip-splitting mycelia. (C) Selected frames from Movies S1 and S2 in the supplemental material showing the localization of Hof1 and Sep7, respectively, as collars of bars as the growing tips pass the site of septation. Arrowheads indicate where GFP signals first become visible. Bars, 5 µm.

We also determined the distribution of nuclei in tip-splitting hyphae between the still-open septation sites and between closed septa (Fig. 2A). In 11 hyphae, we found 43 to 86 nuclei that were in a continuous cytoplasm, i.e., between the tip and the first closed septum. The nuclear density decreased from the tip toward the first closed septum. Between the tip and the first septation site, nuclei were spaced 4.5 ± 0.2 µm (SEM; n = 144) apart from each other, with 15.4 ± 0.8 nuclei per section (SEM; n = 11). In the section before the first closed septum, the distance between nuclei was 8.5 ± 0.4 µm (SEM; n = 82), with 10.1 ± 0.9 nuclei per section (SEM; n = 9). A very similar nuclear distribution was observed in compartments between two closed septa: 7.8 ± 0.3 µm between nuclei (SEM; n = 96) and 10.6 ± 0.7 nuclei per section (SEM; n = 10). In parallel to this decrease in nuclear density from the tip to the first closed septum, we saw a strong vacuolization in hyphae, visible in DIC images, starting 200 to 300 µm behind the tip (Fig. 2A).

From Hof1 bars to contracting Hof1 rings. Closer analyses of the Hof1 collar of cortical bars revealed that the bars were rather spotted in appearance and aligned in parallel to the hyphal growth axis (Fig. 3A, column 1). The bars were not associated with F-actin structures (Fig. 3B, column 1). The length of the bars ranged from 2.4 to 6.7 µm (n = 73). Although these values varied considerably, they correlated with the hyphal diameter (3.0 to 5.6 µm) such that wider hyphae displayed longer bars. The ratio between bar length and hyphal diameter was 1.01 ± 0.02 (SEM; n = 73). This indicates that faster-growing hyphae form longer bars, since hyphal width correlates with growth speed as well (33). At sites where Hof1 localized simultaneously as a collar of bars and a cortical ring, the bars tended to be shorter than the hyphal diameter (Fig. 3A, column 2). Additionally, the portion of Hof1 that was localizing as a ring colocalized with F-actin structures (Fig. 3B, column 2). Apparently, this marks a transitional state between a collar of bars and a ring and will further be referred to as bar-to-ring transition. At sites without Hof1 bars, single continuous cortical Hof1 rings colocalized with actin rings (Fig. 3B, column 3). At sites where the septum was growing inward, the Hof1 ring diameter was smaller than the hyphal diameter (Fig. 3A, columns 4 and 5), indicating that Hof1 forms a contractile ring during septation. At completely closed septa, some Hof1 still localized to cortical spots on either side of each septum (Fig. 3A, column 6).


Figure 3
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  		FIG. 3. Hof1 dynamics at the sites of septation. (A) Maximum projections and 90°-tilted views of six independent septa show the different localization patterns of Hof1-GFP. Arrowheads indicate closing septa visible in the DIC images. (B) The localization of Hof1-GFP and rhodamine phalloidin-stained actin in fixed hyphae. (C) Selected frames from Movie S3 in the supplemental material show the dynamics of Hof1-GFP, the FM 4-64-stained plasma membrane, and the calcofluor white-stained cell wall before and during septation. (D) Maximum projections and 90°-tilted views of a closed septum with a FM 4-64-stained plasma membrane and a calcofluor white-stained cell wall. (E) Representative DIC images of young (top) and old (bottom) septa. (F) Ring diameter of the Hof1-GFP signal (see Movies S3 to S6 in the supplemental material) at six future septa plotted versus time. Arrows indicate the time points when the bar-to-ring transition has completed and a continuous ring has formed. Complete closure of the rings was chosen as time point 0. (G) Representative kymographs for the ring contractions of Hof1-GFP (gray line in panel F) and hof1{Delta}PEST-GFP. Time points are 5 min apart. Bars, 5 µm (A to E) and 2.5 µm (G).

The distribution of the different Hof1 localization patterns within hyphae suggested that there was a gradual maturation process for each septation site, starting with Hof1 localizing as bars and ending with ring contraction and septum formation. The coordination of this process with plasma membrane invagination and the deposition of chitin was monitored in 3-D time-lapse movies of HOF1-GFP hyphae growing in the presence of the lipophilic membrane dye FM 4-64 and calcofluor white (Fig. 3C; see also Movie S3 in the supplemental material). Representative frames show the collar of bars (0 min), shortening of the bars (90 min), ring formation (145 min), and the beginning of ring contraction (195 min). Simultaneously with the early phase of Hof1 ring contraction, the plasma membrane started to invaginate and chitin accumulated as a ring around the plasma membrane (195 min). Concomitant with further ring contraction and membrane invagination, the chitin-rich septum grew inward (210 min). Upon complete contraction of the Hof1 ring, a septum had formed that separated the plasma membranes of the two adjacent hyphal compartments (250 min). This chitin-rich septum formed a continuous disc between the two compartments (Fig. 3D). In DIC images, closed septa initially appeared as cross walls of equal width but, as seen in older hyphae, expanded in the center by the deposition of cell wall material, leading to the bulging of septa (Fig. 3E). The thickness of closed septa increased from 0.9 to 4.5 µm, and the average thickness was 2.4 µm (n = 84).

To determine whether the time for the individual processes of septation is fixed or variable, the diameters of Hof1 collars of bars and rings at six future septation sites were measured in four independent movies and plotted against time (Fig. 3F; see also Movies S3 to S6 in the supplemental material). The GFP signal at a septation site could be monitored for up to 325 min (Fig. 3F; see also Movie S5, middle frame, in the supplemental material). Due to bleaching, it was not possible to follow septation from the initial Hof1 localization at the tip until the closure of the ring. In Movie S5 in the supplemental material, it took over 135 min until bar-to-ring transition had finished. The rings persisted between 60 to 160 min. During this time, the ring diameter remained constant. In only one case, a slow decrease in ring diameter of about 1.5 µm over 160 min was observed (Fig. 3F; see also Movie S5, middle frame, in the supplemental material). The actual contraction phase of the ring was rather fast and occurred in all movies within 30 min. The average ring closure rate over the last 20 min was calculated for these six septa and was 0.18 µm min–1. This is close to the rates of 0.20 µm min–1 and 0.14 µm min–1 measured in budding and fission yeast, respectively (15, 44). In S. cerevisiae, actin ring contraction depends on the degradation of Hof1, which interacts via its PEST domain with the SCF component Grr1 (8). To test if the predicted PEST domain in Hof1 was also required for efficient ring contraction in A. gossypii, we constructed a strain expressing hof1{Delta}PEST-GFP (Fig. 1B). The localization pattern and ring contraction of hof1{Delta}PEST was indistinguishable from those of Hof1 (Fig. 3G). Ring persistence (up to 130 min) and the average ring closure rate (0.16 µm min–1) did not differ significantly from those of the strain expressing the Hof1 wild-type allele (not shown).

Association of Cyk1, Bud3, and Myo1 with cortical bars. Previous studies with A. gossypii have shown that the IQGAP protein Cyk1 and the landmark protein Bud3 form rings at the sites of septation (60, 63) and that the type II myosin Myo1 (ACR068W) is essential for actin ring formation (H.-P. Helfer, unpublished data). These studies did not reveal whether these proteins would already localize as collars of bars or only as rings. Therefore, strains expressing pairs of fluorescent fusion proteins were analyzed. Clearly, Cyk1, Bud3, and Myo1 colocalized with Hof1 collars of bars and with Hof1 rings (Fig. 4A to C). In addition, single rings of Bud3 split into double rings, as previously reported (60), that delimited the single ring formed by Hof1 (Fig. 4B) and, presumably, by Myo1, Cyk1, and actin. When septation took place (Fig. 4A to C), the single rings of Hof1, Myo1, Cyk1, and actin contracted, whereas the double rings of Bud3 did not (shown for Bud3 and Hof1 in Fig. 4B). The septin Sep7 was previously reported to form collars of bars and cortical rings (Fig. 4D) (28). Additionally, we found Sep7 to form very thin and long cortical filaments that run in parallel to the growth axes of the hyphae (Fig. 4E). The filaments were distributed over the whole cortex and became denser and had higher signal intensity at septation sites where Sep7 actually formed a collar of bars (Fig. 4E). Such filaments were not observed for any of the other proteins analyzed. To our knowledge, there are no reports about S. cerevisiae or S. pombe on septin rings that contract during septation. Surprisingly, we found that Sep7 formed an apparently contractile ring, i.e., the diameter of the ring was smaller than the hyphal diameter at the closing septum (Fig. 4D).


Figure 4
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  		FIG. 4. Proteins involved in septation localize to bars and rings. Bars, 5 µm. (A to C) Hof1 colocalizes with the IQGAP Cyk1, landmark protein Bud3, and myosin II Myo1 as bars and rings at future septa. Bud3 rings split into double rings that delimit the single Hof1 ring. Hof1, Cyk1, and Myo1 rings are contractile, whereas Bud3 double rings are not. CFP, cyan fluorescent protein; RFP, red fluorescent protein. (D) Sep7 localizes as a collar of cortical bars to a future septum. During septum formation, it is found as an apparently contractile ring. Black arrowheads in panels A to D indicate closing septa. (E) Sep7 localizes as thin and long cortical filaments (white arrows) and as collars of bars. max, maximum intensity projection.

Hof1 localization in strains deleted for cytokinesis genes. As Bud3, Myo1, Cyk1, and presumably Sep7 colocalized with Hof1, we tested if Hof1 depended on these proteins to localize to the sites of septation. In lateral-branching bud3{Delta} mycelia, Hof1 was found to colocalize with actin rings and partially colocalize with aberrant actin filaments (Fig. 5A). Hyphae lacking Bud3 display either normal septa or aberrant chitin accumulations (60). Septa formed normally when Hof1 localized as contractile rings in bud3{Delta} hyphae, whereas when Hof1 formed filaments, abnormal cell wall accumulations were visible in DIC images (Fig. 5B). Similar to hof1{Delta} mutants, cyk1{Delta} mycelia lack actin rings (63). Nevertheless, Hof1 was able to localize to septation sites in lateral-branching cyk1{Delta} mycelia (Fig. 5A). In myo1{Delta} mutants, Hof1 localized to septation sites as well and, even though actin rings were lacking, aberrant septa could still form (Fig. 5A and B). No actin rings were found in 17 lateral-branching myo1{Delta} mycelia with lengths of 145 ± 10 µm (SEM) and 6.1 ± 0.3 branches (SEM). Nevertheless, a total of 71 Hof1 sites and septa were found, resulting in a wild-type-like septation index of 0.71 ± 0.05 (SEM), indicating that septation site selection was normal in myo1{Delta} mutants. In DIC images of wild-type hyphae, closed septa initially appear as thin cross walls of equal width, and they widen by bulging out in the center when they become older (Fig. 3E). In contrast, the cross walls of closing septa in myo1{Delta} hyphae were much wider, and mature septa were relatively slim in the center compared to the wide base at the cortex. Hof1 did not form a sharp ring in closing septa of myo1{Delta} mutants but was lining the invaginating septum (Fig. 5B).


Figure 5
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  		FIG. 5. Hof1-dependent bar-to-ring transition in tip-splitting hyphae. Bars, 5 µm. (A) Hof1 localizes to septation sites along main hyphae and at the bases of branches in lateral-branching bud3{Delta}, myo1{Delta}, cyk1{Delta}, and sep7{Delta} mycelia. Septation occurs in the absence of actin rings in myo1{Delta} hyphae. CFP, cyan fluorescent protein. (B) Hof1 forms either normal contractile rings or abnormal cortical filaments in bud3{Delta} hyphae. Instead of forming a sharp contractile ring, Hof1 lines the ingrowing septum in myo1{Delta} hyphae. (C) Bud3, Myo1, Cyk1, and Hof1 do not depend on Sep7 to form cortical rings at the sites of septation. (D) Hof1 forms abnormal cortical filaments in cdc3{Delta} and cdc12{Delta} hyphae, whereas Hof1 localization is mostly normal in cdc11a{Delta} hyphae. (E) Bud3, Myo1, Cyk1, and Sep7 do not depend on Hof1 for localization to the sites of septation in lateral-branching mycelia. (F) Schematic drawing of the localization pattern of Sep7, Bud3, and Hof1 to bars and rings at the first and second tip-splitting site as counted from the hyphal tip. Quantification of these localization patterns in tip-splitting hof1{Delta} and cyk1{Delta} hyphae. (G) Representative images of the localization of Sep7 and Bud3 as bars at the first tip-splitting site and as rings at the second, respectively. Both proteins are unable to form rings at the second tip-splitting site in hof1{Delta} hyphae. (H) Representative images of Hof1 localizing as bars and rings at the first and second tip-splitting sites, respectively. Hof1 depends on neither Bud3 nor Myo1 but does depend on Cyk1 to form rings at the second tip-splitting site.

In A. gossypii, the septins Cdc3, Cdc10, and Cdc12 are nonessential but are required for the localization of Sep7 (28), and deletion of either CDC3 or CDC10 abolishes actin ring formation (Helfer, unpublished). Therefore, we wanted to know whether deletion of septin genes interfered with the localization of Hof1 and other cytokinesis proteins. In lateral-branching sep7{Delta} mycelia, actin rings formed normally and colocalized with Hof1 rings (Fig. 5A). Furthermore, the localization of Bud3, Myo1, Cyk1, and Hof1 to cortical bars (not shown) and rings was barely affected (Fig. 5C), since aberrant cortical Bud3, Myo1, and Hof1 filaments were seen at less than 5% of the septation sites analyzed (n > 185; not shown). Deletion of CDC3 resulted in a severe phenotype: at 22 of 53 septation sites analyzed, Hof1-GFP was not detectable; at 29 sites, Hof1 formed cortical filaments (Fig. 5D); and at only 2 sites, Hof1 formed a cortical ring (not shown). Similarly, in cdc12{Delta} mutants, Hof1-GFP was detectable forming filaments at only 10 of 37 sites (Fig. 5D) and only in one case forming a ring (not shown). Cortical Hof1 bars were neither observed in cdc3{Delta} nor cdc12{Delta} mutants. However, Hof1 localized normally as either bars or rings at 62 of 71 septation sites in cdc11a{Delta} mutants and formed filaments in the other 9 cases, frequently in close proximity to rings (Fig. 5D).

Hof1-dependent bar-to-ring transition in tip-splitting hyphae. The early appearance of Hof1 at the septation site (Fig. 2C) could suggest that it was involved in septation site selection. To test if Hof1 was required to recruit other proteins than actin to the septation site, we deleted HOF1 in strains expressing BUD3-GFP, MYO1-GFP, CYK1-YFP, or SEP7-GFP. In lateral-branching hof1{Delta} mycelia, all four proteins localized to septation sites at the neck of the germ bubble, in the main hyphae, and at the bases of lateral branches (Fig. 5E). Even though actin rings and septa did not form, septation site selection was not affected in hof1{Delta} mutants: in 22 lateral-branching hof1{Delta} mycelia with lengths of 157 ± 6 µm (SEM) and 4.8 ± 0.2 branches (SEM), a total of 56 myosin rings were found, resulting in a septation index of 0.54 ± 0.05 (SEM). This is comparable to the aforementioned ratio determined for HOF1-GFP mycelia. Myo1-GFP and Cyk1-yellow fluorescent protein (Cyk1-YFP) signals were much weaker in hof1{Delta} mycelia, and they were not detectable in fixed mycelia (n > 30 for each strain). In contrast to lateral-branching hof1{Delta} mycelia, Myo1-GFP and Cyk1-YFP signals were not detectable in tip-splitting hof1{Delta} hyphae (n > 60 for each strain). Furthermore, neither Sep7 nor Bud3 were found to localize as rings in tip-splitting hof1{Delta} hyphae. To quantify these defects, the localization of Sep7 and Bud3 was compared at the first and second tip-splitting sites counted from the hyphal tip (Fig. 5F and G). In the wild-type background, Sep7 localized mainly to collars of bars at the first site (69%; n = 32) and to rings at the second tip-splitting site (69%; n = 39). Similarly, the localization of Bud3 changed from bars at the first site (77%; n = 30) to rings at the second tip-splitting site (85%; n = 33). In the remaining cases, at the first and second tip-splitting sites, bar-to-ring transition was progressing. At the first tip-splitting site in hof1{Delta} hyphae, Sep7 and Bud3 localized as bars in 100% and 92% of the cases, respectively (n > 33 for each strain). This localization pattern did not change at the second tip-splitting site, where Sep7 and Bud3 still localized as bars in 97% and 94% of the cases, respectively (n > 37 for each strain). These results clearly show that Hof1 is required for bar-to-ring transition in tip-splitting hyphae. Next, we analyzed the localization of Hof1 at the tip-splitting sites of bud3{Delta}, myo1{Delta}, and cyk1{Delta} mutants. In the wild-type background, Hof1 localized at the first tip-splitting site as bars in 79% of the cases (n = 34) and at the second as rings in 68% of the cases (n = 31) (Fig. 5F and H). Neither deletion of BUD3 nor MYO1 affected the bar-to-ring transition of Hof1 (Fig. 5H). However, Hof1 was unable to undergo bar-to-ring transition in cyk1{Delta} mutants (Fig. 5F and H). In all analyzed cases, Hof1 was found to localize as bars at the first as well as at the second tip-splitting site (n > 30 for each site). This was intriguing, since on one hand, Hof1 was required to efficiently localize Cyk1 to the septation sites in tip-splitting hyphae, and on the other hand, Cyk1 and Hof1 were required to promote the bar-to-ring transition.

The Hof1 FCH domain mediates efficient targeting to the septation site. PCH proteins have been shown to mediate interactions between membranes and the actin cytoskeleton through their N-terminal Fes/CIP4 homology (FCH) and coiled-coil domains (extended FCH [EFC] or FCH-Bin- amphiphysin-Rvs [F-BAR]) (31, 52). We tested if the FCH domain or the whole EFC of Hof1 was required for its localization and thus, for its function during bar-to-ring transition and for recruitment of the actin ring to septation sites. We constructed FCH and EFC domain deletions tagged with GFP and exchanged the wild-type allele of HOF1 with these constructs (Fig. 1B). In lateral-branching hof1{Delta}FCH and hof1{Delta}EFC mycelia, actin rings and septa were formed (Fig. 6A) but did so less frequently than HOF1 mycelia. In 29 lateral-branching hof1{Delta}EFC mycelia with lengths of 149 ± 11 µm (SEM) and 5.0 ± 0.3 (SEM) branches, only 26 actin rings and septa were found, resulting in a septation index of only 0.19 ± 0.02 (SEM). For hof1{Delta}EFC mycelia, we determined an identical ratio of 0.19 ± 0.02 (SEM) septation sites per branch. These ratios were significantly lower than the septation index of 0.6 ± 0.04 (SEM) measured for HOF1-GFP mycelia. In tip-splitting mycelia, the GFP signals of both constructs were barely detectable (Fig. 6B). The maximal signal intensity of the Hof1-GFP ring at the second tip-splitting site was 699 ± 35 arbitrary units (AU) (SEM; n = 29) and was well above the cytoplasmic background fluorescence. The maximal signal intensities measured for hof1{Delta}FCH-GFP and hof1{Delta}EFC-GFP were with 285 ± 6 AU (SEM; n = 31) and 304 ± 4 AU (SEM; n = 31), only slightly above the background (276 ± 4 and 293 ± 3 AU) (SEM; n = 29), respectively. Nevertheless, we were able to visualize both constructs in tip-splitting mycelia as bars and rings, and septa also formed (Fig. 6C). As the effects of both truncations were virtually identical, the FCH domain alone is presumably the major, but not sole, contributor to efficiently target Hof1 to the sites of septation.


Figure 6
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  		FIG. 6. Analyses of the Hof1 FCH and EFC domains. Bars, 5 µm. (A) N-terminal truncations of Hof1 lacking either the FCH domain alone or the FCH and coiled-coil domains (EFC) together are able to form actin rings at the sites of septation. (B) The maximum GFP signal intensity of hof1{Delta}FCH-GFP and hof1{Delta}-EFC-GFP at the second tip-splitting site counted from the tip is significantly lower than of Hof1-GFP and barely above the cytoplasmic fluorescence. (C) Both hof1{Delta}FCH and hof1{Delta}EFC localize to the collars of bars and cortical rings in tip-splitting hyphae, and septation occurs.

The Hof1 Src homology 3 (SH3) domain is required for ring integrity. In a two-hybrid assay, Hof1 was found to interact via its SH3 domain with the three formins present in A. gossypii (not shown). If this interaction was responsible for recruiting formins to the septation sites to nucleate the actin ring, then cells expressing Hof1 without its SH3 domain should lack actin rings like hof1{Delta} cells. To test this, we deleted the C-terminal SH3 domain by the fusion of YFP to residue 620 (Fig. 1B). Cells expressing hof1{Delta}SH3-YFP were able to form actin rings and septa, and this truncated version of Hof1 localized seemingly correctly as bars and rings to septation sites (Fig. 7A). Bar-to-ring transition appeared to be normal, but in some cases, hof1{Delta}SH3 rings opened and formed cortical filaments, thus preventing septum formation (see Movie S7 in the supplemental material). In other cases, hof1{Delta}SH3 did not form closed rings but formed cortical filaments (see Movie S7 in the supplemental material) that colocalized partially with aberrantly thick actin filaments (Fig. 7A), similar to filaments observed in septin or bud3{Delta} mutants. These filaments had about the same length as the circumference of normal Hof1 rings. This suggests that these filaments are of the same origin as normal rings. The filaments displayed sliding movements along the cortex and became shorter over time (see Movie S7 in the supplemental material). The shortening of theses filaments occurred more slowly than the ring closure rates measured for Hof1 rings. In Movie S7 in the supplemental material, the filaments constantly became shorter during more than 9 h but did not disappear completely, indicating that this shortening cannot be compared with normal ring contraction and may rather be due to slow filament disassembly at their ends. We tested whether these filaments influenced the structural organization of septation sites in addition to the formation of abnormal actin filaments. Immunofluorescence stainings of septins showed that septin rings colocalized with closed hof1{Delta}SH3 rings (Fig. 7B, column 1). Even at sites where once closed hof1{Delta}SH3 rings had opened, septin rings appeared normal (Fig. 7B, column 2). In contrast, no septins were found in proximity to hof1{Delta}SH3 filaments that probably never formed a closed ring (Fig. 7B, column 3). The absence of septin rings at sites with hof1{Delta}SH3 filaments but the presence of septin rings at sites with hof1{Delta}SH3 rings indicate that the SH3 domain of Hof1 is involved in normal bar-to-ring transition. Furthermore, the SH3 domain of Hof1 is not essential for actin ring formation but fulfills a role for the structural integrity of the Hof1 ring.


Figure 7
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  		FIG. 7. Analyses of the Hof1 SH3 domain. Bars, 5 µm. (A) Hof1 lacking its SH3 domain localizes as normal bars at septation sites (left column) and colocalizes with actin rings after bar-to-ring transition (middle column). Additionally, hof1{Delta}SH3 forms abnormal cortical filaments that partially colocalize with thick actin filaments (right column). (B) Immunofluorescence staining of septins using anti-ScCdc11 in mycelia expressing hof1{Delta}SH3-YFP. Septins colocalize with hof1{Delta}SH3 when the latter forms a closed ring (left column) but not when the rings are open (middle column) or when hof1{Delta}SH3 forms cortical filaments (right column).


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DISCUSSION
 
We report on the identification and characterization of A. gossypii Hof1, a novel member of the PCH protein family. To date, no other PCH protein has been functionally characterized during the filamentous growth of fungi. We show that the transition of septins from bars to rings depends on this PCH protein and that it is required for actin ring formation. Using Hof1 as a marker, we analyzed the events that lead to cytokinesis and septation in A. gossypii. We see that cytokinesis and septation take place in five stages: (i) site selection with the formation of cortical Hof1 bars, (ii) bar-to-ring transition, (iii) ring persistence, (iv) cytokinesis and septation, and (v) septum reinforcement (Fig. 8). In addition to this basic analysis by in vivo imaging, we investigated the contributions of eight conserved proteins, i.e., Hof1, Myo1, Bud3, Cyk1, and four septins (Sep7, Cdc3, Cdc11a, and Cdc12), to the process of septation. In this complex analysis, pairwise combinations of fluorescent fusions proteins or pairwise combinations of deletions with fluorescent fusion proteins were studied.


Figure 8
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  		FIG. 8. Model of septation in A. gossypii. (1) Site selection. Polarity factors (gray spheroid) permanently localize to the growing tip (long horizontal arrow) and signal to septins to mark the site of septation. Septins, PCH protein Hof1, landmark protein Bud3, IQGAP Cyk1, and type II myosin Myo1 localize as a collar of cortical bars (black horizontal lines). (2) Bar-to-ring transition. Bars gradually become shorter, and cortical rings start to form (black curved vertical line). This transition requires Hof1 and Cyk1 but not Myo1. Actin filaments start to assemble into the actin ring in a Hof1-, Cyk1-, and Myo1-dependent manner. (3) Ring persistence. The single Hof1, Cyk1, Myo1, and actin rings (black circle) can persist for variable time periods and are delimited by split (double) Bud3 rings (gray circles). (4) Cytokinesis and septation. Triggered by an unknown signal, the actomyosin, Hof1, and Cyk1 rings contract, leading to the constriction of the plasma membrane. Concomitantly, the septum grows inward. Septum ingrowth can occur in the absence of the actomyosin ring but requires Hof1 and Cyk1. (5) Septum reinforcement. The plasma membranes of the adjacent compartments delimit the septum that bulges out in the center as it is reinforced (thick black vertical line with filled circle). No cell separation occurs. All five stages of septation take place simultaneously within a single hypha that is only spatially separated.

The first stage of septation is site selection. In S. pombe, the equatorially positioned nucleus determines the division plane through the release of the aniline-like protein Mid1 upon entry into mitosis (3, 49). However, it is unlikely that the positions of nuclei determine the sites of septation in A. gossypii, as no mid1 homolog is encoded in its genome (14) and the very hyphal tip is free of nuclei. In S. cerevisiae, the incipient bud site, which becomes the site of septation, is marked late in G1 by the bud site selection machinery and reflects the site of the previous bud. Cla4 is activated by Cdc42 and is involved in septin ring assembly by directly phosphorylating the septins Cdc3 and Cdc10 (11, 55). In A. gossypii, the hyphal tips constantly elongate, and proteins with a conserved role in cell polarization permanently localize to the hyphal tip, e.g., Cdc42 (33) and its effector Cla4 (2). We found that Sep7 not only localizes at approximately regular intervals to collars of bars but also as long thin filaments to other cortical areas. Based on the septation defect of cla4{Delta} mutants (2), it is likely that Cdc42 signals to septins via Cla4 to build up a collar of bars at the hyphal tip. Nevertheless, the actual trigger for this signaling remains elusive. This collar of bars, which marks the site of septation, presumably serves as a positional cue for other proteins. Similarly, Mid1 in S. pombe forms a broad band of cortical nodes, which mature with the additions of myosin II (myo2, cdc4, and rlc1), IQGAP (rng2), PCH protein (cdc15), and formin (cdc12) (69). The bars we observed in A. gossypii were somewhat spotted in appearance, but they were clearly aligned parallel to the hyphal growth axis, which was not observed in S. pombe (69). In budding yeast, the septins Cdc3, Cdc10, Cdc11, and Cdc12 are essential, whereas Shs1/Sep7 is not, and only Cdc3, Cdc11, and Cdc12 are necessary for septin filament formation (54, 56). In A. gossypii, septins are not essential (28; this study), presumably because cytokinesis itself is not essential. Nevertheless, Cdc3, Cdc11a, and Cdc12 but not Sep7 are required for the selection and structural organization of the septation site. In the absence of Cdc3, Cdc11a, or Cdc12 septin filaments are most likely not present. Hof1 is then only randomly targeted to septation sites and mostly forms aberrant cortical filaments instead of bars or rings. Similarly, hof1{Delta}SH3 mutants also form cortical filaments, which do not colocalize with septins, suggesting that the SH3 domain might be involved in linking Hof1 to the septin scaffold. In agreement with reports that PCH proteins are involved in membrane binding (31, 52), a parallel pathway for targeting Hof1 to the cortex at the septation site could be mediated by its FCH domain, since hof1{Delta}FCH localizes only poorly to septation sites in A. gossypii.

In the next stage, the bar-to-ring transition, the bars become gradually shorter and the assembly of cortical rings begins. In hof1{Delta} mutants, this transition is blocked, and neither Sep7 nor Bud3 can form cortical rings. Hof1 is also required to efficiently target Myo1 and Cyk1 to the septation site, and Cyk1 but not Myo1 is needed to promote the bar-to-ring transition of Hof1 itself. The requirement for Hof1 to recruit downstream components is apparently different at septation sites in young lateral-branching and mature tip-splitting mycelia: in the former, Cyk1 and Myo1 weakly localized in the absence of Hof1, while they were not detectable in the latter. This may reflect a developmental difference between young and mature mycelia, because the hyphal diameter increases from about 3 to more than 5 µm in tip-splitting hyphae with a concomitant increase in bar length, which might impose more stringent requirements for the structural organization of the septation site. Only after the onset of the bar-to-ring transition do actin filaments become visible in the middle of the bars and start assembling a ring, a process depending on the activities of Hof1, Cyk1, and Myo1.

The bar-to-ring transition as well as the persistence of the contractile ring can be very variable in time. In one instance, bar-to-ring transition took over 135 min, and ring persistence was measured to be between 60 to 160 min. This particularly highlights the differences between unicellular and filamentous fungi. Cytokinesis and septation in yeasts are tightly coupled to the nuclear cycle and happen only once per cell cycle after completion of mitosis. The generation times of S. cerevisiae and S. pombe is 1.5 to 2 h and 2 to 4 h, respectively, which sets the time frame for septation. The hyphae of A. gossypii contain several dozen nuclei in one common cytoplasm before the first septum closes, and asynchronous nuclear cycles vary from 46 to 250 min (20). Thus, septation in A. gossypii is obviously uncoupled from single nuclear division events.

Next, ring contractions of the actomyosin ring as well as the Hof1 and Cyk1 rings initiate cytokinesis and concomitant septation. In A. gossypii, the ring closure rates during cytokinesis are constant and similar to S. cerevisiae and S. pombe. In S. cerevisiae, it is unclear whether the Hof1 ring contracts completely or only partially before Hof1 forms double rings on either side of the septum (38, 53). In A. gossypii, however, the Hof1 ring clearly contracts completely (like the Myo1 ring) and then localizes to patches but not double rings on both sides of the septum. In agreement with this observation, the Cdc15 ring in S. pombe contracts completely as well (68). The actual trigger for ring contraction is still unclear. We found that ring contraction is accompanied by vacuolization of the hyphae and a lower nuclear density. Hyphae of starving A. gossypii cultures are also vacuolated, and nutrient availability regulates nuclear density (28). Together, this could indicate that nutrient availability in subapical parts of the hyphae triggers septum closure. In the absence of the actomyosin ring, septation can still occur, although aberrantly, but not without either Hof1 or Cyk1. Similar to myo1{Delta} mutants, A. gossypii mutants that lack the essential formin Bni1 and, thus, have no actin rings (and cables) form cross walls resembling septa (48). In S. cerevisiae, cytokinesis is thought to be the result of three parallel pathways: Myo1-dependent actomyosin ring contraction and Hof1- and Cyk3-dependent septum formations. Deletion of any pathway alone does not block cytokinesis; only deletion of any two pathways together is lethal (6, 7, 34, 38, 53). Iqg1/Cyk1 seems to be involved in actomyosin ring and septum formation, since iqg1/cyk1 null mutants are inviable (40). Unlike in budding yeast, actin ring formation and cytokinesis are blocked in A. gossypii hof1{Delta} mutants, presumably because Hof1 is required for Cyk1 localization and, thus, bar-to-ring transition. This, in turn, might be necessary to recruit the machinery for septum formation. Furthermore, A. gossypii Cyk3 seems to play a minor role, since it is unable to compensate for the loss of Hof1, and cyk3{Delta} mutants display only a mild defect in actin ring formation (our unpublished data).

In the last stage, septum reinforcement, the plasma membranes of the adjacent hyphal compartments become curved, bending away from the septum that bulges out in the center. The compartments stay attached because cell separation does not occur. This is crucial for filamentous fungi, as only the lack of cell separation enables them to form a continuous mycelium. In contrast, in S. cerevisiae, after cytokinesis and the formation of the primary septum, secondary septa form and new cell wall material is deposited on either side of the primary septum. Then, the primary septum and the connecting cell wall are degraded, and only this allows cell separation to occur. In the SGD database (29), 89 genes are annotated with the gene ontology term "cytokinesis." Only 4 of these 89 genes have no homolog in A. gossypii. One of the missing genes encodes Sla2, which is associated with actin patches and endocytosis (70). More interesting are the three other genes that have no homolog in A. gossypii: CTS1 which encodes an endochitinase and EGT2 which encodes a cell wall endoglucanase, both required for cell separation (35, 36), and SCW11 which encodes a cell wall protein with similarity to glucanases (10). The synteny maps of S. cerevisiae and A. gossypii (14) show that EGT2 in A. gossypii most likely got lost as a consequence of a translocation between chromosome 7 and chromosome 4. At the syntenic positions of CTS1 and SCW11, deletions occurred in the A. gossypii genome, but there is no break of synteny. This indicates that the removal of only a few components required for cell separation can lead to a dramatic change in the growth pattern of a yeast-like organism, provided that the system also acquired mutations that enforce sustained polar growth.


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ACKNOWLEDGMENTS
 
This work was supported by the University of Basel and was partly supported by the European Union Marie Curie Research Training Networks (RTN) PENELOPE project 36076.

We thank Hans-Peter Schmitz for his advice and suggestion to investigate the function of Hof1. We also thank Michal Köhli for critically reading the manuscript, Dominic Hoepfner for providing pUC19NATPS, Hans-Peter Helfer for sharing unpublished data on AgMyo1, AgCdc3, and AgCdc10; and Christian Böhmer for providing helpful discussions on the role of UmCdc15.


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FOOTNOTES
 
* Corresponding author. Mailing address: Applied Microbiology, Biozentrum, Klingelbergsrasse 50-70, CH-4056 Basel, Switzerland. Phone: 4161 267 1480. Fax: 4161 267 1481. E-mail: peter.philippsen{at}unibas.ch Back

{triangledown} Published ahead of print on 24 November 2008. Back

§ Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, February 2009, p. 771-783, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.01150-08
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