Table 1.
Polarisome components in yeast and filamentous fungi.
Figure 1.
BUD-6 localization in conidia, conidial germlings and sites of septum formation.
(A) Intense and locally defined clusters of BUD-6 were localized at the cell poles of dormant macroconidia. Scale bars, 5 µm. (B–C) Specific recruitment of BUD-6 during isotropic expansion, cell symmetry breaking and germ tube outgrowth was not observed. Scale bars, 2.5 µm. (D) Localized recruitment of BUD-6-GFP to apical caps of growing germ tubes (arrowhead), as well as to septa (arrow), occurred in germlings ≥35 µm in length. FM4–64 stained vesicle clusters were observed at the same positions. (E) Enlarged view of the germ tube tip highlighted in (D). The merged image shows that BUD-6-GFP fluorescence only partially colocalizes with the FM4–64 stained apical cap which extends over a larger crecent. (F) Staining with FM4–64 revealed that BUD-6 recruitment to the incipient septation site preceded plasma membrane invagination (arrowheads), and that it constantly remained associated with the leading edge of the closing septum. Scale bar, 5 µm. See Movie S1 for time course sequences. Note: the bright FM-4–64 stained spot appearing at time point 0 sec does not colocalize with the cortical BUD-6 accumulation indicated by the arrowhead. (G) Reconstruction of BUD-6-GFP localization at the inner perimeter of the closing septal pore. Scale bar, 2.5 µm.
Figure 2.
BUD-6 and SPA-2 recruitment during CAT-mediated cell fusion.
(A) Clusters of BUD-6-GFP became recruited to CAT tips, concentrated at the incipient fusion site upon contact, and formed an opening ring of fluorescence during fusion pore formation (arrowhead). See Movie S2 for time course sequences. Scale bar, 5 µm. (B) Typical changes in the BUD-6 localization pattern accompanied distinct stages of the cell-cell fusion process in germling networks: (1) pronounced accumulation shortly after CAT attachment, (2) ring formation during fusion pore opening, and (3) BUD-6 disappearance shortly after cytoplasmic continuity was established. Scale bar, 5 µm. (C) Z-projections of confocal optical sections through conidial germlings expressing SPA-2-GFP during CAT homing and fusion. Shortly after cytoplasmic continuity was successfully established and SPA-2 disappeared from the fusion site, a new cluster of SPA-2 became recruited to the germ tube tip (arrowhead) that resumed polarized growth (transient arrest of germ tube growth during cell fusion, and resumed tip extension post-fusion is indicated with dotted line). Scale bars, 5 µm. See Movie S3 for full time sequence. (D) Shows an enlarged view of BUD-6 recruitment during the continuation of germ tube tip growth as shown in (C).
Figure 3.
BUD-6 recruitment to polarized growing mature hyphal tips and branches.
(A) BUD-6-GFP accumulated as a subapical cloud with a distinct exclusion zone at the very tip apex (arrowhead). FM4–64 staining revealed that this exclusion zone was occupied by the Spk. Scale bars, 10 µm; in inset, 5 µm. (B) Recording BUD-6-GFP dynamics during hyphal tip growth showed that the BUD-6-GFP exclusion zone (arrowheads and inset for magnified view) consistently remained at the extending tip apex. Scale bars, 10 µm. See Movie S4 for time course sequences. (C) Recruitment of BUD-6 to incipient lateral branch points was not observed. Detectable BUD-6 accumulation occurred after the Spk became stained with FM4–64 in newly established branches (arrowheads).
Figure 4.
BUD-6 and SPA-2 concentrated at the septal plug prior to reinitiation of polarized tip growth.
(A) Upon physical injury a Woronin body occluded the septal pore which was surrounded by pre-existing BUD-6 fluorescence (arrow). At the same time, a new septum was being initiated 25 µm further back (arrowheads), again leaving BUD-6 at the septal pore after its completion. (B) At the onset of repolarization from the septum at the severed end of the hypha, BUD-6-GFP fluorescence became concentrated at the sealed pore, in the vicinity of the Woronin body and within an accumulation of lipophilic, possibly membranous, material stained by FM4–64 (arrows). See Movies S5 for full sequence. (C) The BUD-6 cluster often remained in place while the new tip emerged (arrowhead). Subsequently and coinciding with the condensation of a recognizable Spk, apical BUD-6 fluorescence became increasingly diffuse (arrowheads). See Movie S6 for full sequence. (D) SPA-2 rapidly became recruited to the occluded septal pore forming an intense spot associated with the Woronin body (arrow). The protein did not reside at consolidated septal pores, but rather relocated into an apical cap of the emerging tip (arrowhead) which accompanied extension of the new hyphae (E). See Movie S7 for full sequences of D. Scale bars A to D, 5 µm. Scale bar E, 10 µm.
Figure 5.
BUD-6 dynamics during vegetative hyphal fusion.
(A) BUD-6 became recruited to the tips of homing fusion hyphae, then concentrated at the attachment point and surrounded the opening fusion pore. Shortly after the pore was fully established BUD-6 fluorescence disappeared from this site. Scale bar, 5 µm. See Movie S8 for time course sequence. (B) Transient BUD-6 fluorescence accumulated at incipient fusion sites in the mature colony (arrowhead) and persistent BUD-6 signal at septal pores (all other fluorescently marked sites). Scale bar, 10 µm. (C) SPA-2-GFP became recruited to vegetative hyphal fusion sites (arrow). As it was never seen at completed fusion connections (arrowheads) it must follow the transient dynamics of BUD-6 in this context. Scale bar, 10 µm.
Figure 6.
BUD-6 accumulation during conidiogenesis.
(A) BUD-6-GFP accumulated at septation sites in developing macroconidiophores (arrowhead). Scale bars, 10 µm. (B) In cytologically separated, but physically still attached conidia, BUD-6 fluorescence persisted at the cell poles; either at both or only at one pole in case of the terminal conidium. Scale bars, 10 µm. (C) In addition to strong fluorescence at the cell poles, bright clusters of BUD-6-GFP also accumulated at other locations of the cell cortex. Scale bar, 5 µm.
Table 2.
N. crassa strains used in this study.
Figure 7.
Loss of BUD-6 resulted in a reduced colony extension rate and hyperbranching.
(A) Hyperbranching and polar extension defects resulted in very slowly and extremely dense developing mycelial colonies of Δbud-6, in comparison to the wild type after 24 hours of incubation. (B) Hyphal morphology of wild type and Δbud-6 at the colony margin. Scale bars, 0.5 mm and 0.25 mm, respectively. (C) Quantification of branching frequency, which on average was more than doubled in the mutant compared to the wild type. (D) Comparison of average colony extension speed between Δbud-6 and wild type.
Figure 8.
BUD-6 was required to organize the polarized growth apparatus at the hyphal tip.
(A and B) Comparison of FM4–64 staining pattern of mature hyphae at the leading edge of the colonies in the wild type and Δbud-6 mutant confirmed the absence of septa in the mutant (arrows in A indicate septa in the wt), as well as the absence of the Spk at the hyphal tips of Δbud-6 (arrowheads in A point toward wt Spk). Scale bars, 5 µm. (C and D) Close up of the apical and subapical area of polarized growing mature hyphae of wt and Δbud-6. The arrowheads in C indicate the Spk, which shows up as a dark sphere in the phase contrast image and was brightly stained by FM4–64. No such structure was observed in hyphae of the Δbud-6 mutant. The squared bracket marks the subapical nuclear exclusion zone in the wild type, which is not established in the Δbud-6 mutant. Here, nuclei (arrows) reach further up into the hyphal tip (also seen in E). Scale bars, 5 µm. (E) Apical branching and lack of hyphal tip organization in Δbud-6. Scale bar, 5 µm. (F) Immature and malformed conidiophores in Δbud-6. Scale bar, 10 µm.
Figure 9.
A Δbni-1 strain generated through vegetative homokaryon selection phenocopied growth defects of Δbud-6.
(A and B) Wild-type like phenotype of the heterokaryotic Δbni-1 strain FGSC11490, including conidia and septa (arrows). Scale bars, 50 µm and 10 µm respectively. (C) The lack of septa in the homokaryotic Δbni-1 strain was confirmed by FM4–64 staining. Scale bar 50 µm. (D) FM4–64 staining also confirmed the absence of an organized apical tip growth apparatus, including the Spk. Scale bar, 10 µm. These defects closely resembled phenotypic key features of Δbud-6 (Figures 7 and 8).
Figure 10.
Δbni-1 and Δbud-6 strains displayed derepressed hyphal fusion at the colony edge.
(A) The lack of septa in Δbni-1 resulted in extensive accumulation of vacuoles at the leading edge of the colony. Scale bar, 50 µm. (B) Hyphal fusion at the leading edge of the colony, which is usually suppressed in the wild type through apical dominance, was observed in Δbni-1. Several fusion pores are indicated with arrowheads. Scale bar, 10 µm. See Movie S9 for time course sequence showing vacuolar passage through fusion connections. (C) After closer inspection, the same phenotype could be observed at the leading edge of Δbud-6 colonies. Scale bar 10 µm. (D–F) In the wild type, derepression of hyphal fusion at the leading edge occurred in the presence of conidial germlings. The establishment of cytoplasmic continuity – here visualized through the transfer of nuclei fluorescently labeled with histone H1-GFP (green) from conidial germlings into mature hyphae - involved either CAT-mediated cell fusion (D), or fusion pegs from the mature hypha (E and F) induced through the presence of conidial germlings. Arrowheads indicate fusion sites; C denotes the spore body; GT denotes the germ tube; H denotes mature hypha. Note that fluorescently labeled nuclei originating from the germling have only migrated into the upper part of the unlabeled wild type hypha. The arrowheads in (F) mark fusion pegs emerging from the mature hypha. Scale bar in D, 10 µm.
Figure 11.
BNI-1 recruitment to sites of polarized growth, septum formation and cell fusion.
(A) In conidial germlings recruitment of fluorescently labeled BNI-1 was observed during three different cellular processes. (1) During cell symmetry breaking BNI-1 appeared at the cell cortex. (2) During CAT-mediated cell fusion, very bright accumulations of BNI-1 could be seen at the tips of interacting CATs. Due to a spore torque response upon cell-cell attachment, the left cell moved out of the focal plane. This movement is commonly observed when imaging germling fusion in liquid medium. (3) During septum formation BNI-1 was part of contractile actomyosin rings. Scale bar, 5 µm. (B) BNI-1 also accumulated in apical crescents at growing germ tube tips (arrow). Recruitment to septal pores and a new sites of cell symmetry breaking (arrowhead) showed up as well. Also note that BNI-1 is associated with septum formation (asterisk) at the base of the germ tube. (C) Consistent with its participation in CAT-mediated cell fusion, BNI-1 also became recruited to sites of vegetative hyphal fusion, and disappeared shortly after cytoplasmic continuity was established. The arrow marks the opening fusion pore. Scale bar, 5 µm.
Figure 12.
BNI-1 is a constituent of the Spk, but also localized to an apical cap.
(A) BNI-1-GFP colocalized with the Spk, but also was present in an apical cap (see inset in A). (B) Time sequence of BNI-1 dynamics during mature hyphal tip growth and lateral branch initiation. 0–2 min: in the straight growing hyphal tip small crescents (arrowheads) of BNI-1 were located on either side of the Spk. 4–6 min: shortly before branch initiation, tip extension transiently ceased – evident by rounding-off of the tip - and apical BNI-1 fluorescence disappeared. 8–10 min: extension of the main tip resumed with a brighter cluster of BNI-1 at the left hand side of the apex, followed by displacement of the Spk and left-orientation of the tip. A small crescent of formin fluorescence localized to the tip of the emerging branch (arrow). 12–14 min: a brighter cluster of the formin accumulated at the right hand side preceding reorientation of tip extension into this direction. Scale bars, 10 µm.
Figure 13.
BNI-1 localization during septal plugging, tip repolarization and septum formation.
(A) Two minutes after damaging leading hyphae BNI-1 became recruited to the septal plug (position of the Woronin body is indicated with an arrow, 0 min). Fluorescence focused into a smaller area from which a new hyphal tip repolarized, and shortly after condensed into an subapical spot (arrowhead, 16 min) with flanking BNI-1 crescents on either side (inset, 20 min). In parallel, a new septum was being formed about 25 µm behind the severed end, and an additional site of polarity was established (arrowhead, 20 min). Scale bar, 5 µm. See Movie S11 for full sequence. (B) Continuation of (A) but with an extended field of view including an old septum (asterisk). The part of the hypha shown in (A) is outlined with a dashed box. A selection of individual optical slices shows the formation of several septa. Upon septum completion, BNI-1 gradually disappeared from the septal pore. Barely visible remains are indicated with circles at the 68 min time point. BNI-1 fluorescence was usually not observed at ‘old’ septa (asterisk). Recruitment of BNI-1 to a vegetative hyphal fusion site is indicated by an arrowhead.
Figure 14.
BNI-1 localization during conidiogenesis.
(A) During cytological compartmentalization of conidiophores BNI-1 localized to forming septa (arrowheads). Apart from occasional cortical clusters no specialized localizations (e.g. to cell poles) of the formin could be observed at mature stages of conidial development. (B) In contrast, apart from weak cytoplasmic fluorescence, no specific accumulation of SPA-2 could be observed during conidiophore formation, cytokinesis or (C) upon physical separation of mature conidia. Scale bars, 5 µm.
Figure 15.
Schematic representation of the subcellular localization and dynamics of the three polarisome components SPA-2, BUD-6 and BNI-1 during key developmental stages of N. crassa.
(A) Prior to the constitution of the entire polarisome complex during germ tube growth and CAT-mediated cell fusion, BNI-1 accumulates at the incipient sites of cell symmetry breaking. (B) Equivalent to its dynamics during CAT fusion, the polarisome complex is present during homing and fusion of fusion hyphae, and removed once cytoplasmic continuity is achieved. (C) BNI-1 accumulates at the incipient site of branch formation, and together with BUD-6 and SPA-2, subsequently constitutes the complete polarisome complex as an apical crescent at the tip of the emerging branch. Finally, the polarisome adopts the mature hyphal tip configuration (D). (D) Configuration of the polarized tip growth apparatus in mature hyphae, including an apical cap and Spitzenkörper core of BNI-1, a fan-shaped distribution of SPA-2 inside the apical dome, and the subapical BUD-6 cloud. (E) Septal plugging and consolidation involves the Woronin body and all three polarisome proteins. During repolarization, a polarisome crescent is constituted at the emerging hyphal tip, and ultimately rearranged into its mature form (D). (F) Septum formation requires BNI-1 and BUD-6 – but not SPA-2 - as components of the CAR. Upon septum completion, only BUD-6 remains associated with the inner perimeter of the septal pore. (G) During cytokinesis BNI-1 and BUD-6 become recruited to the CAR and to forming secondary septa. Upon physical separation of macroconidia BNI-1 disappears, whereas BUD-6 remains at the cell poles. As during septum formation, SPA-2 has no role in this process.
Table 3.
Subcellular localization patterns of polarisome components during key developmental stages in N. crassa.