Fig 1: MICAL-L1-marked tubular recycling endosomes are impaired in the absence of either Rab10 or its effector, EHBP1.A–I, HeLa cells on coverslips were either Mock treated (A–C), or treated with either Rab10 siRNA (D–F) or EHBP1 siRNA (G–I) prior to fixation and immunostaining with antibodies directed against endogenous MICAL-L1 (A, D, G) or endogenous Rab10 (B, E, H). Dual-channel merged images are shown in C, F, and I. J–M, immunoblots and quantification depicting knock-down efficiency for Rab10 (J, K) and EHBP1 (L, M). N–O, the mean apparent TRE volume was measured for MICAL-L1- and Rab10-containing structures, using Imaris software. Serial z-section imaging was done on random fields of cells on the coverslip, and pixel values were converted into surfaces using a preset threshold (see Experimental procedures). The total volume per field was calculated and then divided by the number of cells per field to derive the volume per cell for each fluorescent channel. Equivalent thresholds were used for WT and KD cells. At least ten fields of cells were measured and quantified in each experiment, and graphs are derived from three independent experiments. Error bars denote standard deviation, and p-values are derived from one-tailed Student's t-tests. P, HeLa cells were either mock treated or treated with EHBP1 siRNA. Knock-down efficiency and specificity are shown in the right panel (EHPB1 total lysate in mock versus knock-down). Mock and knock-down cells were also lysed and subjected to fractionation to membrane and cytosolic fractions, and Rab10 levels were detected by immunoblotting (right panel). Controls include GAPDH as an exclusively cytosolic protein and EEA1 as a primarily membrane-associated protein. The experiment displayed is a representative one from six individual experiments showing a similar trend (although the ratios of cytosolic to membrane proteins varies from experiment to experiment). The scale bar represents 10 µm (inset, 5 µm). KD, knock-down.
Fig 2: Introduction of WT Rab10 to knockout MEFs increases the volume of MICAL-L1-containing TRE.A, B, WT MEF cells (A) or Rab10 KO MEF cells (B) were plated on coverslips, fixed, immunostained with antibodies to MICAL-L1, and imaged by confocal microscopy. Representative images from three separate experiments are depicted. C–H, Rab10 KO MEF cells were plated on coverslips and transfected with either GFP (control; C–E) or RFP-Rab10 (F–H). After 24 h, cells were fixed and immunostained with antibodies to detect MICAL-L1. I, J, (I) WT MEF and Rab10 KO MEF cells or (J) transfected MEF Rab10 KO cells (transfected with GFP or RFP-Rab10) were imaged by serial z-sections, and the mean apparent TRE volume was measured for MICAL-L1- and Rab10-containing structures, using Imaris software. Pixel values were converted into surfaces using a preset threshold (see Experimental procedures). The total volume per field was calculated and then divided by the number of cells per field to derive the volume per cell for each fluorescent channel. At least 10 fields of cells were measured and quantified in each experiment, and graphs are derived from three independent experiments. Error bars denote standard deviation, and p-values are derived from one-tailed Student's t-tests. The scale bars represent 10 µm. KO, knockout; MEF, mouse embryonic fibroblast; TRE, tubular recycling endosome. GFP, green fluorescent protein; RFP, red fluorescent protein.
Fig 3: MICAL-L1 and Rab10 are residents on the same tubular recycling endosome.A–C, HeLa cells were cultured on cover slides, fixed, and immunostained with antibodies against endogenous MICAL-L1 (A, green) and Rab10 (B, red). A series of serial sections were obtained and the representative image is a snapshot from a 3D reconstitution. C, depiction of the merged image from A and B, showing the overlap between MICAL-L1 and Rab10 in yellow. D–F, the dashed boxes from A–C are shown as magnified insets in the zoomed regions depicted in D–F. G, the graph represents three independent experiments with at least 10 images each that were subjected to imaging and quantification of surface volume overlap. The scale bars represent 10 µm. Error bars denote standard deviation.
Fig 4: Rab10-marked tubular recycling endosomes are not impaired in the absence of MICAL-L1 or Syndapin2.A–I, HeLa cells on coverslips were either Mock treated (A–C) or treated with either MICAL-L1 siRNA (D–F) or Syndapin2 siRNA (G–I) prior to fixation and immunostained with antibodies directed against endogenous MICAL-L1 (A, D, G) or endogenous Rab10 (B, E, H). Dual-channel merged images are shown in C, F, and I. J–M, immunoblots and quantification depicting knock-down efficiency for MICAL-L1 (J, K) and Syndapin2 (L, M). N–O, the mean apparent tubular recycling endosome volume was measured for MICAL-L1- and Rab10-containing structures, using Imaris software. Serial z-section imaging was done on random fields of cells on the coverslip, and pixel values were converted into surfaces using a preset threshold (see Experimental procedures). The total volume per field was calculated and then divided by the number of cells per field to derive the volume per cell for each fluorescent channel. Equivalent thresholds were used for WT and knock-down (KD) cells. At least ten fields of cells were measured and quantified in each experiment, and graphs are derived from three independent experiments. Error bars denote standard deviation, and p-values are derived from one-tailed Student's t-tests. The scale bar represents 10 µm (inset; 5 µm).
Fig 5: Rab10 is a crucial protein for TRE biogenesis.A–L, Mock-treated HeLa cells were dimethyl sulfoxide (DMSO)-treated (A, B, G, H), treated with a phospholipase D (PLD) inhibitor (C, D, I, J), or treated with a PLD inhibitor followed by a washout of the inhibitor (E, F, K, L). Cells were then fixed and immunostained with antibodies directed against MICAL-L1 (A–F) or Rab10 (G–L). Enlarged insets are depicted in B, D, F and H, J, L. M–P, immunoblotting (M) and quantification (N) of Rab10 knock-down by siRNA and immunoblotting (O) and quantification (P) of EHBP1 knock-down by siRNA. Q–S, in addition to Mock-treated HeLa cells, HeLa cells treated with Rab10 siRNA or EHBP1 siRNA were similarly subjected to DMSO treatment, PLD inhibitors, or PLD inhibitors followed by inhibitor washout prior to fixation and immunostaining with MICAL-L1 (Q, R) and Rab10 (S) antibodies. Volumetric analysis was done as described in Figures 3 and 4. Serial z-sections were obtained by confocal imaging, and Imaris software was used to compare the mean volume of MICAL-L1 tubular recycling endosome (TRE) in mock-treated cells upon Rab10 knock-down (Q) or upon EHBP1 knock-down (R), or to compare the mean volume of Rab10 TRE in Mock-treated and EHBP1 knock-down cells (S). Each graph shows the TRE volume for mock and knock-down cells with DMSO treatment, PLD inhibitor treatment, and following PLD inhibitor washout. The scale bars represent 10 µm. Graphs are based on three independent experiments measuring at least five fields of cells per experiment. p-Values are derived from one-tailed Student's t-tests and are as follows: for MICAL-L1 TRE in Rab10 KD: (1) Mock DMSO versus Rab10 KD DMSO p < 0.0001, (2) Mock PLD inhibitors versus Rab10 KD DMSO p = 0.0069, (3) Mock washout versus Rab10 KD washout p < 0.0001, (4) Mock DMSO versus Mock PLD inhibitors p < 0.0001, (5) Mock DMSO versus washout p = 0.000304, (6) Mock PLD inhibitors versus Mock washout p < 0.0001, (7) Rab10 KD DMSO versus Rab10 KD PLD inhibitors p = 0.00036, (8) Rab10 KD DMSO versus Rab10 KD washout p = 0.17, (9) Rab10 KD PLD inhibitors versus Rab10 KD washout: p = 0.0034. For MICAL-L1 tubules in EHBP1 KD: (1) Mock DMSO versus EHBP1 KD DMSO p < 0.0001, (2) Mock PLD inhibitors versus EHBP1 KD DMSO p = 0.056, (3) Mock washout versus EHBP1 KD washout p = 0.41, (4) Mock DMSO versus Mock PLD inhibitors p < 0.0001, (5) Mock DMSO versus washout p = 0.000304, (6) Mock PLD inhibitors versus Mock washout p < 0.0001, (7) EHBP1 KD DMSO versus EHBP1 KD PLD inhibitors p < 0.0001, (8) EHBP1 KD DMSO versus EHBP1 KD washout p = 0.00073, (9) EHBP1 KD PLD inhibitors versus EHBP1 KD washout p = 0.00038. For Rab10 tubules in EHBP1 KD: (1) Mock DMSO versus EHBP1 KD DMSO p = 0.0056, (2) Mock PLD inhibitors versus EHBP1 KD DMSO p = 0.17, (3) Mock washout versus EHBP1 KD washout p = 0.26, (4) Mock DMSO versus Mock PLD inhibitors p = 0.00040, (5) Mock DMSO versus washout p = 0.029, (6) Mock PLD inhibitors versus Mock washout p = 0.0021, (7) EHBP1 KD DMSO versus EHBP1 KD PLD inhibitors p = 0.055, (8) EHBP1 KD DMSO versus EHBP1 KD washout p = 0.0019, (9) EHBP1 KD PLD inhibitors versus EHBP1 KD washout p = 0.00058. KD, knock-down.
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