Supplemental Material for Martzoukou, Diallinas, and Amillis, 2018
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Figure S1. (A) Quantitative analysis of fluorescence intensity of strains shown in Figure 2A, under ap1σ expressed or fully repressed conditions (-thi, +thi respectively) along 25 μm of hyphal tips. The region measured is depicted in the cartoon on the top right. For details of fluorescence intensity measurements see Materials and methods. (B) Subcellular localization of several cargoes in the presence of the vacuolar stain CMAC upon AP-1 depletion. Minor cargo-dependent colocalization with vacuoles is indicated with arrows. Scale bars represent 5 μm. Hyphal apex is in all cases at the upper side of the images.
Figure S2. Co-localization of DnfA-GFP with the endocytic dye FM4-64 (10min) indicating that most immotile internal structures are not co-stained with FM4-64. Scale bars represent 5 μm.
Figure S3. Quantitative analysis of fluorescence intensity of strains shown in Figure 3E, under ap1σ expressed or fully repressed conditions (-thi, +thi respectively) along 25 μm of hyphal tips. For details of fluorescence intensity measurements see Materials and methods.
Figure S4. Additional images on the subcellular localization of Ap1σ relative to that of clathrin light (ClaL) and heavy (ClaH) chains. Hyphal apex is in all cases at the right side of the images, except for the second and fourth where a subapical hyphal region is presented (see also Figure 4A, File S3 and FileS4).
Figure S5. Left panel: Growth test of a standard wild-type (wt), a strain carrying a thiamine-repressible thiAp-ap1β allele, and strains expressing ClaL-GFP and ClaH-GFP in the repressible thiAp-ap1β background. Right panel: Growth test of strains carrying the repressible thiAp-ap1β allele “in locus”, together with wt or mutated versions of Ap1β expressed from plasmid integration events, as well as, ClaL-GFP and ClaH-GFP alleles. Notice that expression of the mutated Ap1β versions, which seem defective for clathrin recruitment, partially rescue growth when thiAp-ap1β allele is repressed. This, together with results presented in Figure 4, indicates that total lack of growth observed in the absence of AP-1 is not simply due to defective interaction of AP-1 with clathrin.
Figure S6. (A) Non-merged channel images of the time course of RabE-GFP localization in the presence of the endocytic dye FM4-64, indicating that most immotile internal structures are not co-stained with FM4-64. Hyphal apex is in all cases at the right side of the images (see also Figure 5A). (B) Separate channel images of the co-localization analysis of SynA and RabE shown in Figure 5G. Hyphal apex is at the lower side of the image series. Scale bars in all cases represent 5 μm.
Table S1. Strains used in this study
Table S2. Oligonucleotides used in this study for cloning purposes
File S1. Video data of Figure 3A. AP-1σ-GFP
does not co-localize with SedV-mCherry, although in some cases it orbits around
the cis-Golgi marker.
File S2. Video data of Figure 3B. A significant degree of association is observed between Ap1σ-GFP labeled foci and PHOSBP-mRFP late-Golgi equivalents.
File S3. Video data of Figure 4A. Foci labeled with ClaL-mRFP and Ap1σ-GFP seem to co-migrate, indicating a significant topological association between the two proteins
File S4. Video data of Figure 4B. "Horseshoe"-like structures labeled with ClaH-GFP are closely related to Ap1σ-mRFP foci. Coherent movement of the two tagged proteins is observed, as in the case of ClaL and AP-1
File S5. Video data of Figure 6A. AP-1σ-GFP foci decorate dynamically microtubules labeled with mCherry-TubA (α-tubulin)
File S6. Video data of Figure 7A. AP-1σ-GFP exhibits dynamic association with RabB-labeled endosomes
File S7. Supplementary references