Rab22A recruits BLOC ‐1 and BLOC ‐2 to promote the biogenesis of recycling endosomes
2018; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês
10.15252/embr.201845918
ISSN1469-3178
AutoresSaurabh Shakya, Prerna Sharma, Anshul Milap Bhatt, Riddhi Atul Jani, Cédric Delevoye, Subba Rao Gangi Setty,
Tópico(s)CRISPR and Genetic Engineering
ResumoScientific Report7 November 2018Open Access Transparent process Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes Saurabh Shakya Saurabh Shakya Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Prerna Sharma Prerna Sharma Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Anshul Milap Bhatt Anshul Milap Bhatt Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Riddhi Atul Jani Riddhi Atul Jani Structure and Membrane Compartments, CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Cédric Delevoye Cédric Delevoye Structure and Membrane Compartments, CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Subba Rao Gangi Setty Corresponding Author Subba Rao Gangi Setty [email protected] orcid.org/0000-0003-4035-2900 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Saurabh Shakya Saurabh Shakya Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Prerna Sharma Prerna Sharma Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Anshul Milap Bhatt Anshul Milap Bhatt Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Riddhi Atul Jani Riddhi Atul Jani Structure and Membrane Compartments, CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Cédric Delevoye Cédric Delevoye Structure and Membrane Compartments, CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Subba Rao Gangi Setty Corresponding Author Subba Rao Gangi Setty [email protected] orcid.org/0000-0003-4035-2900 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Search for more papers by this author Author Information Saurabh Shakya1, Prerna Sharma1,‡, Anshul Milap Bhatt1,‡, Riddhi Atul Jani2, Cédric Delevoye2,3 and Subba Rao Gangi Setty *,1 1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 2Structure and Membrane Compartments, CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France 3Cell and Tissue Imaging Facility (PICT-IBiSA), CNRS, UMR 144, Institut Curie, PSL Research University, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +91-80-22932297/+91-80-23602301; Fax: +91-80-23602697; E-mail: [email protected] EMBO Reports (2018)19:e45918https://doi.org/10.15252/embr.201845918 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Recycling endosomes (REs) are transient endosomal tubular intermediates of early/sorting endosomes (E/SEs) that function in cargo recycling to the cell surface and deliver the cell type-specific cargo to lysosome-related organelles such as melanosomes in melanocytes. However, the mechanism of RE biogenesis is largely unknown. In this study, by using an endosomal Rab-specific RNAi screen, we identified Rab22A as a critical player during RE biogenesis. Rab22A-knockdown results in reduced RE dynamics and concurrent cargo accumulation in the E/SEs or lysosomes. Rab22A forms a complex with BLOC-1, BLOC-2 and the kinesin-3 family motor KIF13A on endosomes. Consistently, the RE-dependent transport defects observed in Rab22A-depleted cells phenocopy those in BLOC-1-/BLOC-2-deficient cells. Further, Rab22A depletion reduced the membrane association of BLOC-1/BLOC-2. Taken together, these findings suggest that Rab22A promotes the assembly of a BLOC-1-BLOC-2-KIF13A complex on E/SEs to generate REs that maintain cellular and organelle homeostasis. Synopsis Recycling endosomes are transient tubular intermediates that maintain cellular/organelle homeostasis. Rab22A promotes the assembly of a BLOC-1-BLOC-2-KIF13A complex on sorting endosomes to generate the recycling tubular endosomes that are required for the biogenesis of lysosome-related organelles. Rab22A acts as a novel regulator of recycling endosome biogenesis. Rab22A recruits BLOC-1 and BLOC-2 on sorting endosomes and form a complex with KIF13A. Rab22A-BLOC-1-BLOC-2-KIF13A complex modulates recycling endosome dynamics for lysosome-related organelle biogenesis and cellular homeostasis. Introduction The endosomal network consists of early (EE), sorting (SE), recycling (RE) and late (LE) endosomes 1. These organelles are produced ubiquitously through the endocytic/secretory process, and they contribute to the maturation/function of lysosomes in all cells 2 and lysosome-related organelles (LROs) in specialized cells 3. Moreover, these endosomal intermediates maintain several cellular homeostasis pathways, including those involved in secretion, signalling, nutrient/growth factor uptake, pathogen clearance, neurotransmitter storage and release 4. The mechanism of RE biogenesis is poorly understood while the formation of EE/SE/LE has been extensively studied. Biochemical and electron microscopy (EM) studies revealed that REs originate either from E/SEs or the trans-Golgi network (TGN) as long tubular intermediate structures carrying cargoes or receptors towards the cell surface 1, 2, 5 and partly to LROs such as melanosomes in melanocytes 3. Several Rab GTPases (Rabs), including Rab4A, 8A, 11A, 14 and 22A, have been shown to regulate the trafficking of RE containing clathrin-dependent (TfR, EGFR, ATP7A, GLUT4, etc.) and clathrin-independent (MHC1, integrin β1 receptor, β2-adrenergic receptor, etc.) endocytosed cargoes to the cell surface in non-melanocytes 6. Moreover, Rab11A and 22A are shown to localized to these REs and maintain their structures 7, 8. However, the regulation of Rabs in the biogenesis and maintenance of REs and their targeting towards melanosomes are largely unknown. The formation, length and stability of RE tubular structures require several cytosolic factors such as coat/adaptor proteins, kinesin motors and actin- or microtubule-remodelling proteins 9, 10. It has been shown that a multimeric protein complex, retromer in association with the WASH (Wiskott–Aldrich syndrome protein and scar homologue) complex, regulates the biogenesis of a set of REs 11. In contrast, another set of REs is dependent on the kinesin-3 family motor KIF13A, cargo adaptor AP-1 and the endosomal eight-subunit (pallidin, muted, dysbindin, cappuccino, snapin, BLOS1, BLOS2 and BLOS3) protein complex BLOC (biogenesis of lysosome-related organelles complex)-1 in a retromer-independent fashion 8, 12, 13. Interestingly, KIF13A-BLOC-1-dependent tubular REs control the trafficking of melanocyte-specific cargo like TYRP1 (tyrosinase-related protein-1) 14, ATP7A (copper transporter) 15 and OCA2 16 to melanosomes in melanocytes and Tf/TfR (transferrin/its receptor) to the cell surface in all cell types 8. Additionally, our earlier studies have shown that tri-subunit (HPS3, HPS5 and HPS6) protein complex BLOC-2 interacts 17 and functions downstream of BLOC-1 in these transport pathways 14, 18. Moreover, the stability of KIF13A-BLOC-1-dependent RE tubules has been shown to be dependent on the actin-cytoskeletal protein annexin A2 and ARP2/3 complex 13. However, the membrane recruitment of BLOC-1 and BLOC-2 and their coordination with KIF13A motor in regulating the formation/function of REs remain unknown. To connect the individual functions of KIF13A, BLOC-1 and BLOC-2 in regulating RE length/stability (dynamics) or biogenesis, we hypothesize a role of Rabs in coordinating these molecules on the endosomal membranes. Our small RNAi screen limited to endosomal Rabs identified Rab22A as a potential regulator of RE biogenesis. Quantitative fluorescence microscopy, live cell imaging, EM and biochemical approaches demonstrated that Rab22A localized to REs and its depletion dramatically reduced their length and number that lead to a defect in cargo delivery to the cell surface in non-melanocytic (HeLa) cells and to the melanosomes in melanocytes, concurrently causes hypopigmentation. Moreover, we found that Rab22A regulates the recruitment and stabilization of BLOC-1 and BLOC-2 on the E/SE membranes and biochemically forms a complex by associating with KIF13A motor. This Rab22A-BLOC-1-BLOC-2-KIF13A complex might maintain biogenesis of REs and loss of any of these components result in decreased RE dynamics and increased E/SE size. Overall, our study demonstrated that Rab22A acts as a key organizer in controlling the formation of REs that regulates cargo delivery to cell surface or LROs during their biogenesis. Results and Discussion Rab22A regulates RE dynamics and pigment synthesis in melanocytes The mechanism of RE formation from E/SEs is poorly understood. Rab GTPases act as master regulators of vesicle biogenesis/transport 19. We postulated that small Rab GTPases might also regulate the length and number of REs (referred to here as RE dynamics). REs are characterized by the localization of the SNARE STX13 (syntaxin 13) 20 and the kinesin motor KIF13A 8. We visualized REs in HeLa cells by expressing KIF13A-YFP, which was localized (i) to enlarged E/SEs (appeared as clusters) near the cell periphery and (ii) as long tubular structures throughout the cytosol, resembling the REs 8 (Fig 1A, control sh). These KIF13A-puncta/tubular structures were also positive for RE proteins STX13, Rab11A, AP-1 and internalized Tf (Fig EV1A). Figure 1. Selected endosomal Rab RNAi screen identified Rab22A as a regulator of RE dynamics A. IFM images of KIF13A-YFP-transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean ± SEM, n = 3). B, C. Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean ± SEM). n = 3. nc: total number of cells. nt: total number of tubules. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test). D. IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A-transfected HeLa cells. E. Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 s) are shown separately. F. Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead-bound His-KIF13A in each pull-down was shown on the Coomassie-stained gel. *, non-specific bands. Note, part of this experiment was shown in Fig 5F. Data information: In (A, D, E), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs, respectively. Scale bars: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysis of endosomal Rab RNAi screen and the localization of Rabs to REs A. IFM images of HeLa cells to study the localization of KIF13A-YFP to REs. B. PCR analysis of Rab-knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels. C. Subcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP-1. D, E. IFM analysis of HeLa cells that were transfected with different constructs as indicated. F. Live cell imaging of mCherry-Rab22A with respect to GFP-Rab9A in HeLa cells. Arrowheads point to the Rab22A-positive tubular structures. Magnified view of insets (at 0, 16, 40 s) are shown separately. Scale bars: 10 μm. G. IFM images of Rab22A sh, BLOC-1 sh and control sh HeLa cells that were transfected with indicated constructs. H. Graphs represent the measurement of corrected total cell fluorescence (CTCF) in HeLa cells of Fig 2A. n = 6–8 cells. Average CTCF values (AU: arbitrary units) and their respective fold changes (mean ± SEM) are indicated. Data information: In (A), arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs, respectively. In (D, E, G), arrowheads point to the STX13- or KIF13A-positive tubular REs or E/SEs. Scale bars: 10 μm. Download figure Download PowerPoint A small shRNA screen against nine endosomal Rabs showed defective KIF13A-YFP-positive REs in Rab5C-, 7A-, 9A-, 11A- and 22A-knockdown HeLa cells compared to control or Rab4A-, 5A-, 5B-, 14A-knockdown cells (Fig 1A). In these experiments, cells were transiently transfected with pooled gene-specific shRNAs, which reduced the respective Rab transcript levels equivalent to 50% or more (Fig EV1B). A similar depletion of Rab7A-, 11A- and 22A- significantly reduced the average number of KIF13A-YFP-positive tubules per cell (referred to here as TN) compared to control cells. However, Rab4A-, 5B-, 5C- and 9A- depletion only moderately reduced the average TN (Fig 1B and Table 1). In addition, quantification of KIF13A-YFP-positive tubule length (referred to here as TL, measured in μm) showed a significant loss in average TL in Rab11A- and 22A-knockdown and a moderate effect in Rab5C-, 7A- and 9A- depleted cells compared to control cells (Fig 1C and Table 1). These results suggest that Rab7A, 11A and 22A regulate RE dynamics either independently or cooperatively along the same pathway. We hypothesize that the modest RE defect observed in the knockdown cells of other Rabs is possibly due to the alteration in endosomal morphology. Table 1. Quantification of RE dynamics in HeLa cells Transfection of DNA/shRNA Avg. no. of tubules/cell (TN) Avg. length/tubule (TL in μm) Control sh 22.84 ± 2.30 3.03 ± 0.10 Rab4A sh 13.96 ± 2.69 3.02 ± 0.13 Rab5A sh 29.14 ± 3.97 3.01 ± 0.10 Rab5B sh 16.13 ± 2.00 3.10 ± 0.13 Rab5C sh 10.19 ± 2.51 2.43 ± 0.10 Rab7A sh 2.70 ± 0.41 2.17 ± 0.17 Rab9A sh 14.00 ± 2.31 2.63 ± 0.10 Rab11A sh 4.05 ± 1.14 1.82 ± 0.08 Rab14A sh 36.68 ± 4.82 3.36 ± 0.11 Rab22A sh 2.54 ± 0.37 1.73 ± 0.09 Rab22AWT 24.85 ± 1.30 2.92 ± 0.10 Rab22AQ64L 26.86 ± 3.30 3.18 ± 0.14 Rab22AS19N 1.35 ± 0.25 1.84 ± 0.25 Control sh 24.48 ± 2.80 2.93 ± 0.08 Rab22A sh 3.90 ± 0.46 2.27 ± 0.10 BLOC-1 sh 4.39 ± 0.48 2.53 ± 0.09 BLOC-2 sh 3.24 ± 0.32 2.19 ± 0.10 HeLa cells were transfected with respective shRNAs or Rab22A constructs as indicated. Cells were further transfected with KIF13A-YFP, fixed with methanol and then imaged. Images were analysed for RE dynamics using Fiji Macro programme plug-in as described in Materials and Methods. Data are derived from three independent experiments (n = 3, mean ± SEM) and represented as graphs in Figs 1B and C or 2B and C or 3B and C. In a recent study, we have shown that Rab9A functions in targeting REs towards melanosomes 21. Thus, we evaluated the regulatory role of Rab9A along with Rab7A, 11A and 22A on RE dynamics in HeLa cells. At steady state, Rab7A, 9A, 11A and 22A localize equally to the STX13 or AP-1-positive subcellular membrane fractions 12 (Fig EV1C, data not shown for Rab9A) or to the STX13-labelled endosomes in HeLa cells (Fig EV1D and Table 2 for colocalization studies). However, the localization of Rab11A and 22A to the KIF13A-YFP-positive REs was moderately higher than that of Rab7A or 9A in HeLa cells (Figs 1D and EV1E). Similar to Rab11-positive tubules 8, Rab22A (tagged with GFP/mCherry) also appeared as long tubular structures resembling REs in live cell imaging experiments (Figs 1E and EV1F, and Movies EV1, EV2 and EV3). Further, Rab22A-positive tubules colocalized with a cohort of Rab11A, but not with Rab7A/9A-positive vesicles (Figs 1E and EV1F, and Movies EV1, EV2 and EV3). These results suggest that Rab22A-positive tubules are either generated from Rab11A-positive vesicles or represent a subpopulation of Rab11-positive RE tubules. Next, we examined the KIF13A-Rab interactions by pull-down assay using four different domains of His-tagged KIF13A (Fig 1F; motor: 1–359 aa, stalk-1: 360–800 aa, stalk-2: 801–1,306 aa and tail: 1,307–1,770 aa). Rab22A but not Rab7/9/11 showed an interaction with the stalk domain of KIF13A in HeLa cell lysate (Fig 1F). Interestingly, Rab11-KIF13A interaction has been reported previously and studied extensively using various approaches 8. Rab11 interacts with KIF13A in live cells through a binding site encompassing likely the stalk and the tail domains of KIF13A 8. Therefore, both Rab11 and Rab22A could associate with KIF13A at two distinct motifs. We thus tested whether Rab11A compensates the loss of Rab22A function in maintaining the KIF13A-positive REs. However, overexpressing Rab11A did not rescue the formation of long KIF13A-positive REs in Rab22A-inactivated cells (Fig EV1G), suggesting that Rab22A is required for a subpopulation of RE dynamics or biogenesis. Table 2. Quantification of Pearson's coefficient values (r) Cell type Colocalization study between r value HeLa STX13 and Rab7A 0.75 ± 0.01 STX13 and Rab9A 0.72 ± 0.01 STX13 and Rab11A 0.80 ± 0.02 STX13 and Rab22A 0.72 ± 0.03 HeLa mCherry-Rab22AWT and KIF13A-YFP 0.42 ± 0.03 mCherry-Rab22AQ64L and KIF13A-YFP 0.48 ± 0.05 Melanocytes (melan-Ink) mCherry-Rab22AWT and EEA1 0.30 ± 0.05 mCherry-Rab22AWT and TYRP1 0.09 ± 0.02 mCherry-Rab22AWT and LAMP-2 0.07 ± 0.01 mCherry-Rab22AQ64L and EEA1 0.31 ± 0.04 mCherry-Rab22AQ64L and TYRP1 0.16 ± 0.02 mCherry-Rab22AQ64L and LAMP-2 0.23 ± 0.03 Melanocytes (melan-Ink) Control sh: TYRP1 and EEA1 0.13 ± 0.01 Rab22A sh-1: TYRP1 and EEA1 0.40 ± 0.03 Rab22A sh-2: TYRP1 and EEA1 0.37 ± 0.02 Control sh: TYRP1 and LAMP-2 0.14 ± 0.01 Rab22A sh-1: TYRP1 and LAMP-2 0.45 ± 0.03 Rab22A sh-2: TYRP1 and LAMP-2 0.44 ± 0.02 The degree of colocalization between the two markers measured as Pearson's coefficient value "r" (mean ± SEM) using cellSens Dimension software (Olympus). To test whether Rab22A regulates RE biogenesis, we expressed mCherry-Rab22A wild-type (Rab22AWT), constitutively active (Rab22AQ64L) and dominant negative (Rab22AS19N) mutants along with KIF13A-YFP in HeLa cells. Both, Rab22AWT and Rab22AQ64L, appeared as tubular and punctate structures that were colocalized with KIF13A-YFP (Fig 2A and Table 2 for colocalization studies). Further, the corrected total cell fluorescence (CTCF) of Rab22AQ64L was moderately higher than that of Rab22AWT (1.13-fold), suggesting an increased membrane association of Rab22AQ64L compared to Rab22AWT (Fig EV1H). Correspondingly, the CTCF of KIF13A was also enhanced (1.57-fold) in Rab22AQ64L compared to Rab22AWT expressing HeLa cells (Fig EV1H). These results indicate that Rab22AQ64L possibly stabilizes KIF13A association with the endosomal membranes. Consistently, mCherry-Rab22AS19N localized to the cytosol and abolished only the KIF13A-positive tubular structures but not the peripheral E/SEs (see below; Fig 2A). Likewise, the average TN/cell and TL of KIF13A-YFP were dramatically reduced in HeLa cells expressing Rab22AS19N compared to Rab22AWT/Q64L. Interestingly, these parameters showed a modest increase in cells expressing Rab22AQ64L mutant compared to Rab22AWT that was statistically insignificant (Fig 2B and C, and Table 1). These findings suggest that Rab22A regulates the TN and TL of KIF13A-positive REs similar to the MHC-1-positive tubules shown previously 7. Additionally, subcellular membrane fractionation showed that Rab22A was enriched in the membrane (1–1.4 M sucrose) fractions positive for the RE markers STX13 20, KIF13A 8 and AP-1 12 (Fig 2D). As a control, the membrane fractions were also probed for other organelle-specific makers such as GM130 (cis-Golgi), LIMPII (LE and TGN) and LAMP-2 (lysosome) (Fig 2D). Together, these results indicate that Rab22A localizes to REs and regulates their dynamics. Figure 2. Rab22A localizes to the E/SE and REs and regulates RE dynamics A. IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs, respectively. The colocalization coefficient (r, in mean ± SEM) between two markers was indicated separately. Scale bars: 10 μm. B, C. Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A (mean ± SEM). n = 3. nc: total number of cells. nt: total number of tubules. **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test). D. Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle-specific proteins. *, non-specific bands. Download figure Download PowerPoint In contrast to the HeLa cells, mCherry-Rab22AWT in wild-type mouse melanocytes (melan-Ink) localizes to STX13-positive enlarged vacuolar endosomes 20 and also to the buds (arrowheads, persisted for < 20 s in live cell imaging) emanating from these endosomes in live cell imaging experiments. However, mCherry-Rab22AWT does not appear as extended tubular structures as seen in HeLa cells (Fig EV2A and Movie EV4). Consistently, mCherry-Rab22AWT showed colocalization with both EEA1 (EE marker) and STX13 (RE marker, data not shown), but not with TYRP1 or LAMP-2 (melanosomal or lysosomal proteins) or melanosomes imaged in bright-field microscopy (BFM) (Fig EV2B and Table 2). The expression of mCherry-Rab22AQ64L mutant in melanocytes results in the formation of enlarged EEA1-positive vacuolar endosomes that were colocalized with a cohort of both TYRP1 and LAMP-2 proteins (Fig EV2B and Table 2). Whereas the cytosolic mCherry-Rab22AS19N mutant expression in melanocytes moderately reduced the EEA1-, LAMP-2- and TYRP1-positive organelles (Fig EV2B). These results suggest that Rab22A overexpression possibly cause a defect in cargo sorting on E/SEs. Consistent with these results, both Rab22AQ64L- and Rab22AS19N- expressing compared to Rab22AWT- or GFP- expressing cells showed a moderate reduction in melanocyte pigmentation without affecting the cargo (TYRP1) stability (Fig EV2B for BF images, EV2C for melanin content and EV2D for immunoblotting). Altogether, these findings indicate that RE localized Rab22A regulates cargo transport to melanosomes in melanocytes similar to cell surface trafficking of Tf/MHC-1 in HeLa cells 7, 22. Click here to expand this figure. Figure EV2. Analysis of mouse melanocytes expressing different constructs of Rab22A A. Live cell imaging of mCherry-Rab22A with respect to GFP-STX13 in wild-type melanocytes. Arrows point to Rab22A-positive buds or tubular structures arising from STX13-positive E/SEs (arrowheads). Magnified view of insets (at various seconds) is shown separately. Scale bars: 10 μm. B. IFM analysis of mCherry-Rab22AWT/Q64L/S19N-transfected melanocytes. Arrowheads point to Rab22A localization, and arrows indicate the melanocyte pigmentation. Nuclei are stained with Hoechst 33258. The colocalization coefficient (r, in mean ± SEM) between two markers was indicated separately. Scale bars: 10 μm. C, D. Quantification of melanin content (C, n = 5) and the protein levels of TYRP1 (judged by immunoblotting in D, n = 3) in melanocytes that were transfected with GFP-Rab22AWT/Q64L/S19N. In (D), graph indicates the relative levels (mean ± SEM) of TYRP1 with respect to overexpressed Rab22A. Protein band intensities were quantified and indicated on the gels. **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test). Download figure Download PowerPoint Rab22A depletion resembles the RE defect of BLOC-1- and BLOC-2-deficient cells The depletion of BLOC-1 or KIF13A activity in HeLa cells has been shown to cause a defect in cargo recycling and accumulation of Tf in the vacuolar endosomes 8, 13. Further, studies have shown that BLOC-2 functions downstream of BLOC-1 14 by forming a complex 17. However, the role of BLOC-2 in regulating RE dynamics remains unknown. Our membrane fractionation experiments showed a pool of muted/pallidin (BLOC-1 subunits) and HPS6 (BLOC-2 subunit) 14, 18 co-fractionate with Rab22A (Fig 2D), indicating that BLOC-1 and BLOC-2 associate with the Rab22A-enriched membrane fractions. We reported earlier that depleting any of the BLOC-1 or BLOC-2 subunits destabilizes the integrity and function of entire BLOC complex 14, 18. Here, we knockdown muted (BLOC-1) and HPS6 (BLOC-2) subunits in HeLa cells by lentiviral transduction, confirmed their depletion by immunoblotting (see below), henceforth referred as BLOC-1Mu sh and BLOC-2HPS6 sh cells. Immunofluorescence microscopy (IFM) analysis of these cells showed a dramatic reduction in KIF13A-positive RE tubules, which appeared as punctate structures similar to Rab22A-depleted cells (Fig 3A). Compared to control cells, the average TN/cell and TL in both BLOC-1- and BLOC-2-deficient cells were significantly reduced, similar to that in Rab22A-knockdown cells (Fig 3B and C, and Table 1). In addition, the decreased KIF13A-RE dynamics in these cells was not due to an instability or alteration in the cytoskeletal network (microtubules were labelled anti-α-tubulin antibody and actin with phalloidin–Alexa Fluor 594; Fig EV3A). Furthermore, immunoblotting analysis revealed that Rab22A-knockdown in HeLa cells moderately reduced the stability of both BLOC-1 (muted, pallidin, dysbindin) and BLOC-2 (HPS6) subunits. In contrast, Rab22A levels in BLOC-1/BLOC-2-depleted HeLa cells were unaffected (Fig 3D). As expected, the stability of BLOC-1 subunits in BLOC-2 sh cells and BLOC-2 subunit in BLOC-1 sh cells was only modestly altered (Fig 3D). These findings suggest that membrane recruitment of Rab22A and/or BLOC-1/BLOC-2 might play a role in RE generation. Interestingly, IFM analysis showed KIF13A-positive punctate structures in Rab22A-/BLOC-1-/BLOC-2-depleted cells colocalized with AP-1 (γ subunit) 12, 23, STX13 and Rab11A (Figs 3A and EV1G, data not shown for BLOC-2− cells), those correspond to E/SEs. Moreover, AP-1 localization to these peripheral E/SE clusters was enhanced with a concomitant decrease in the perinuclear area in the Rab22A/BLOC-1/BLOC-2-knockdown HeLa cells compared to control cells (Fig 3A). Consistently, KIF13A, AP-1 complex, STX13 and Rab11A protein levels (Fig 3D, data not shown for Rab11) or KIF13A membrane association (Fig EV3B, data not shown for BLOC-1− and BLOC-2− cells) were also grossly unchanged in the knockdown cells. Together, these results indicate that KIF13A and AP-1 recruitment to the endosomal membranes is independent of Rab22A or BLOC-1/BLOC-2. Figure 3. Rab22A functions upstream in the pathway of BLOC-1 and BLOC-2 and regulates RE dynamics A. IFM images of KIF13A-YFP-transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6-knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa Fluor 594. B, C. Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A (mean ± SEM). n = 3. nc: total number of cells. nt: total number of tubules. *P ≤ 0.05 and ***P ≤ 0.001 (unpaired Student's t-test). D. Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels. E. IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6-knockdown HeLa cells. Data information: In (A, E), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs, respectively. Scale bars: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Analysis of cargo recycling in control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells IFM images of KIF13A-YFP-transfected control and knockdown HeLa cells as indicated. Arrowheads point to the localization of KIF13A to E/SEs or REs. Membrane-cytosol fractionation of HeLa cell homogenate for the localization of KIF13A. *, non-specific bands. IFM images of control and Rab22A-, BLOC-1-knockdown HeLa cells that were stained with LAMP-2 or internalized with fluorescein–dextran. Cell surface levels of LAMP-1 and M6PR in control and Rab22A-, BLOC-1-knockdown HeLa cells measured using flow cytometry. Normalized mean fluorescence intensity (MFI) was calculated (mean ± SEM) and then plotted. n = 3. IFM images of HeLa cells that were subjected to Tf-Alexa Fluor 594 recycling kinetics. Fluorescence intensities Tf in the images of (E) were quantified and plotted (mean ± SEM). n = 3. nc = total number of cells. Immunoblotting analysis of Tf receptor in HeLa cells as indicated. Protein band intensities were quantified and indicated on the gels. Data information: In (A, C, E), arrows point to the localization of cytoskeletal proteins (A) or internalized dextran or lysosomes (C) or accumulation of Tf to the intracellular vesicles (E). Scale bars: 10 μm. In (D, F), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ns = not significant (unpaired Student's t-test). Download figure Download PowerPoint
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