Fig 1.
(A) domain organization of ChsB. CS-N, Chitin_synth_1N (PF08407); CS, Chitin_synth_1 (PF01644); the C-terminal transmembrane region includes 7 predicted helices (gray boxes). (B) Growth phenotype of chsBΔ microcolonies compared to the wt; plates incubated for 3 days at 37°C. (C) Subcellular localization of endogenously tagged GFP-ChsB. Arrows in the magnified inset indicate the Spitzenkörper (SPK). The image is a MIP of a deconvolved z-stack. (D) The synaptobrevin homologue SynA and ChsB strictly colocalize in the apical crescent, besides the SPK. Images are MIPs of deconvolved z-stacks. The scheme shows an interpretation of endocytic recycling.
Fig 2.
Endocytosis required to maintain ChsB polarity.
(A) The basal limit of the GFP-ChsB apical crescent coincides with the position of the endocytic collar labeled with mCh-AbpA (actin binding protein1). Right graph, linescans along the longitudinal hyphal axis for the green (ChsB) and red (AbpA) channels. (B) Latrunculin B (100 μM) treatment facilitates the diffusion of GFP-ChsB to plasma membrane regions located far away from the apex. Arrows indicate the limits of the PM region occupied by ChsB in untreated and treated examples. The perimeters of the regions occupied by GFP-ChsB in the PM of treated and untreated hyphae are plotted on the right (n = 15 tips). Error bars indicate S.D. The two populations, which passed normality tests, are significantly different (P < 0.0001) in an unpaired t-test with Welch’s correction. (C) Localization of ChsB in a fimAΔ hypha compared to the wt. (D) Scheme: slaB1 drives expression of SlaB under the control of the nitrite reductase promoter (niiAp); Images show the localization of ChsB in a strain carrying the conditional expression allele slaB1 as the only source of SlaBSla2 and its comparison with the wt. slaB1 drives expression of this key endocytic regulator on nitrate as N source but not on ammonium. The germlings derived from conidiospores continuously cultured on medium containing nitrate or ammonium, as indicated. All images represent MIPs of deconvolved z-stacks.
Fig 3.
SlaB downregulation results in ChsB depolarization.
(A) GFP-ChsB localization in hyphae derived from germlings that had been pre-cultured on medium containing nitrate as sole N source (left) and subsequently shifted to medium containing ammonium (right). (B) Promoter down-shift experiment with slaB1. The same nutritional regime used in (A) results in downregulation of SlaB levels (scheme), markedly affecting the polarization of GFP-ChsB in the PM. Class I (‘ruffled’) and class II (‘normal’) hyphae are depicted. For class I the inset shows a characteristic GFP-ChsB ‘clump’ associated with the PM (arrowed). (C) Quantitation of the perimeter of PM occupied by ChsB in wt (n = 19) and slaB1 (n = 15) cells pre-cultured on nitrate and shifted to ammonium. The two datasets were significantly different (P < 0.0001) in an unpaired t-test. All images represent MIPs of deconvolved z-stacks and are shown at the same magnification, with the exception of the inset, which is magnified 2.5 times.
Fig 4.
ChsB localizes to the tip-proximal cisternae of the TGN.
(A) colocalization of internal puncta containing GFP-ChsB with the TGN marker mRFP-PHOSBP, and absence of colocalization with the early Golgi marker mCh-SedVSed5 (syntaxin 5). Note the characteristically fenestrated structures of the TGN puncta in the left panels. (B) Quantitation of internal ChsB structures that contain Golgi markers for 178 puncta in n = 10 mRFP-PHOSBP hyphae and 122 puncta in n = 11 mCh-SedVSed5 hyphae. Error bars indicate mean ± SD. The two datasets were significantly different (P< 0.0001) in an unpaired t-test. (C) Plot of fluorescence intensities in the PHOSBP and the ChsB channels vs. distance to the apex. Data of n = 60 TGN cisternae were pooled from 6 hyphae. The fluorescence of ChsB negatively correlates with the distance to the apex (Pearson’s r = -0.748, P = 6E-12) whereas that of PHOSBP does not (r = -0.08, P = 0.53).
Fig 5.
Recycling of ChsB from endosomes necessitates dynein (A) Left, schematics summarizing the rationale of these experiments. Middle images: localization of GFP-ChsB in hyphal tips of the wt and of strains carrying ts mutations in nudA encoding the dynein heavy chain, before and after shifting cells from 28°C to 37°C. Note the aggregate of ChsB in the nudA mutants at 37°C (see scheme), which forms apparently at the expense of the signal in the apical dome and the SPK (which is not detectable in the mutants at 37°C). Right, linescans of the ChsB channel for the hyphae displayed in the images (1 px, 0.103 μm). (B) Hyphal tip of a nudA2 cell shifted to 42°C, stained with the endocytosed membrane tracker FM4-64.
Fig 6.
(A) GFP-ChsB is a transient resident of TGN cisternae: kymograph traced along the longitudinal axis of a growing hypha showing multiple events of transient ChsB recruitment to TGN cisterna. The strong signal at the apex is the SPK. (B) Selected examples of events of ChsB recruitment to TGN cisternae and statistical analysis of the average residence time of ChsB on them (58 ± 4 S.D., n = 36); (C) region of a kymograph showing two examples of RabERAB11 being recruited at the end of the ‘ChsB cycle’. The green and red channels of a cell co-expressing mCh-ChsB and GFP-RabERAB11 were filmed simultaneously with a beam splitter at 1 fps time resolution (D) hypA1ts cells (hypA encodes A. nidulans Trs120 in TRAPPII) expressing GFP-ChsB were filmed at 28°C and at different times after shifting the culture to 37°C on the microscope stage. The time required for the culture medium to reach 37°C is ~15 min. Note the delocalization of ChsB at the apical dome and the SPK to internal structures.
Fig 7.
A hypB5 (= sec7ts) mutation strands ChsB in the TGN.
(A) Images of a wt strain co-expressing mCh-ChsB and GFP-TlgB cultured at 28°C and following a shift to 37°C. (B) Images of the corresponding hypB5 strain before and 14 min after the temperature shift. (C) The hypB5 mutant at a later time-point. Right graph, plot of Li’s intensity correlation coefficient (ICQ) used to estimate colocalization. ‘Ai’ and ‘a’ are the ChsB channel’s current and mean intensity, whereas ‘Bi’ and ‘b’ indicate the same values for the TlgBTlg2 channel. Colocalization results in a pixel cloud spread on the right side of the plot. ICQ ranges from −0.5 (exclusion) to 0.5 (complete colocalization). All images are MIPs of deconvolved z-stacks.
Fig 8.
ChsB, stranded at the TGN by hypB5 (= sec7ts), is rescued by geaA1.
Images of hypB5 mCh-ChsB strains carrying or not geaA1-GFP (encoding GFP-tagged GeaAY1022C), photographed at 28°C or after a shift to 37°C for 27 min (hypB5), or 37 min (hypB5 GFP-GeaAY1022C). Note that GeaAY1022C-GFP was the only source of GeaAGea1 in the double mutant. Also note the colocalization of GeaAY1022C with ChsB in the apical dome at both temperatures.
Fig 9.
RabCRAB6- and GARP-dependent recycling of ChsB from an endosome located upstream of the RaBRAB5 domain.
(A) Scheme of the effectors subordinated to RabBRAB5 in early endosomes (EEs)(see text). (B) Normal localization of ChsB in a rabBΔ mutant. (C) Normal localization of ChsB in a vps33ts strain at 28°C and following a shift to 42°C. (D) Left, delocalization of ChsB in rabCΔ; Middle, plot of average intensities of ChsB in wt and rabCΔ apical domes determined from 2 x 50 pixel arch-shaped linescans as in the scheme. P values estimated with an unpaired t-test; Right plot, linescans (mean values ± S.E.M. bars) of maximal intensities across the whole width of the same tips used for the plots. (E) Delocalization of ChsB from the apical dome in the vps52Δ mutant; middle and right plots and statistical analysis as in (D). Note that for (D) and (E), hyphal tip images were not deconvolved to better display the differences between the wt and the mutants.
Fig 10.
Characterization of A. nidulans GARP.
(A) Growth of the wt and indicated mutant strains after 60 h of incubation. (B) Silver staining of proteins retained after passing extracts of a strain expressing endogenously tagged Vps54-S-tag compared with an untagged strain control. Proteins were eluted from S-tag columns. The indicated bands were excised and their identity determined by MS/MS. (C) Comparison of S-tag affinity purifications as in (B) but using Vps54-S-tag and Vps51-S-tag baits and colloidal Coomassie staining. Note the shift in mobility of Vps51 due to the S-tag.
Fig 11.
Delocalization of ChsB to the vacuolar system by vps52Δ Wt and vps52Δ strains expressing GFP-ChsB photographed at 28°C or following a shift to 37°C.
Cells were stained with the vital dye CMAC (7-amino-4-chloromethylcoumarin) to reveal late endosomes and vacuoles (CMAC shown in magenta in color composites). The main images are MIPs of unprocessed z-stacks, but the insets were deconvolved to remove apical haze. Note that the contrast of the 28°C vps52Δ image has been adjusted to reveal the cytosolic haze, with ‘empty holes’ corresponding to the nuclei. Colocalization of ChsB with CMAC in the 37°C vps52Δ sample was complete.
Fig 12.
A model for ChsB recycling ChsB is transported with SVs (red) that accumulate at the SPK before being transported and tethered to the apical PM to undergo fusion.
Once inserted into the PM ChsB undergoes diffusion away from the apex until it is captured and endocytosed by the subapical endocytic collar. Endocytic vesicles containing ChsB reach a mosaic of sorting endosomes. Here domains enriched in RabBRAB5 acquire EE identity (blue), engage dynein by means of the Hook complex and undergo basipetal transport and maturation across the degradative endocytic pathway. ChsB segregates into ‘recycling’ domains (green) that are delivered to the TGN in a RabCRAB6-, GARP- and dynein-dependent manner. Once at the TGN ChsB is selected into RabERAB11 SVs (red), perhaps with cooperation of RabORAB1, and delivered to the SPK. Alternative minor pathways (thinner yellow arrows) between degradative endosomes and either the TGN or the early Golgi (EG) that ensure the robustness of this crucial circuitry must exist, accounting for the proportion of ChsB that persists in the apical dome in the absence of RabCRab6 /GARP.