Supplemental Material for Martzoukou, Diallinas, and Amillis, 2018

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图S1。(A) 针对图2A所示菌株，在ap1σ表达或完全阻遏条件（分别为-thi、+thi）下，沿25 μm菌丝顶端（hyphal tips）的荧光强度定量分析。测量区域如右上角示意图所示。荧光强度测量的详细步骤参见材料与方法。(B) 胞液染色剂CMAC（vacuolar stain CMAC）存在下，AP-1耗竭时多种货物蛋白的亚细胞定位。货物蛋白与胞液存在轻微的共定位，以箭头标注。比例尺均为5 μm。所有图像的菌丝顶端（hyphal apex）均位于图像上方。 图S2。DnfA-GFP与内吞染料FM4-64（孵育10 min）的共定位分析，结果显示多数无运动性的内部结构未与FM4-64共染色。比例尺为5 μm。 图S3。针对图3E所示菌株，在ap1σ表达或完全阻遏条件（分别为-thi、+thi）下，沿25 μm菌丝顶端（hyphal tips）的荧光强度定量分析。荧光强度测量的详细步骤参见材料与方法。 图S4。Ap1σ与网格蛋白轻链（ClaL）和重链（ClaH）的亚细胞定位补充图像。所有图像的菌丝顶端（hyphal apex）均位于图像右侧，第二和第四幅图像除外，二者展示的是亚顶端菌丝区域（另见图4A、文件S3与文件S4）。 图S5。左图：标准野生型（wt）、携带硫胺素可阻遏thiAp-ap1β等位基因的菌株，以及在可阻遏thiAp-ap1β背景中表达ClaL-GFP与ClaH-GFP的菌株的生长测试。右图：携带"内源位点"可阻遏thiAp-ap1β等位基因的菌株，以及从质粒整合事件中表达Ap1β野生型或突变体版本的菌株，同时带有ClaL-GFP与ClaH-GFP等位基因的生长测试。值得注意的是，当thiAp-ap1β等位基因被阻遏时，看似存在网格蛋白招募缺陷的突变体Ap1β版本的表达可部分恢复生长。结合图4所示结果，这表明AP-1缺失时完全停止生长并非仅由AP-1与网格蛋白的相互作用缺陷所导致。 图S6。(A) 内吞染料FM4-64存在下，RabE-GFP定位的时间序列非合并通道图像，结果显示多数无运动性的内部结构未与FM4-64共染色。所有图像的菌丝顶端（hyphal apex）均位于图像右侧（另见图5A）。(B) 图5G所示SynA与RabE共定位分析的分通道图像。该图像序列的菌丝顶端（hyphal apex）位于图像下方。所有图像的比例尺均为5 μm。 表S1 本研究使用的菌株 表S2 本研究用于克隆的寡核苷酸序列 文件S1：图3A的视频数据。AP-1σ-GFP虽可在部分情况下围绕顺式高尔基体标记物（cis-Golgi marker）运动，但并未与SedV-mCherry发生共定位。 文件S2：图3B的视频数据。Ap1σ-GFP标记的焦点与PHOSBP-mRFP标记的晚期高尔基体结构存在显著程度的共定位。 文件S3：图4A的视频数据。ClaL-mRFP与Ap1σ-GFP标记的焦点似乎共迁移，表明两种蛋白之间存在显著的拓扑关联。 文件S4：图4B的视频数据。ClaH-GFP标记的“马蹄形”结构与Ap1σ-mRFP标记的焦点紧密相关。两种标记蛋白的运动同步性与ClaL和AP-1的情况一致。 文件S5：图6A的视频数据。AP-1σ-GFP标记的焦点可动态结合mCherry-TubA（α微管蛋白）标记的微管（microtubules）。 文件S6：图7A的视频数据。AP-1σ-GFP与RabB标记的内体（endosomes）存在动态结合。 文件S7：补充参考文献