. 2015 Apr 20;33(2):176-88.
BORC, a Multisubunit Complex That Regulates Lysosome Positioning
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BORC, a Multisubunit Complex That Regulates Lysosome Positioning
Free PMC article
The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multisubunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the kinesin-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes.
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Figure 1. Proteomic Identification of BORC
(A) Proteins that interact with BLOS2, LOH12CR1, or C17orf59 appended with OSF or FOS tags were identified by TAP-MS from HeLa (BLOS2) or H4 cells (LOH12CR1 and C17orf59). Selected prey proteins and their numbers of peptides detected in the MS are shown. The results suggest the existence of two complexes, BLOC-1 and BORC, that share BLOS1, BLOS2, and Snapin. KXD1 could also be a component of BLOC-1. The possibility that there could be variants of these complexes having different subunit combinations cannot be ruled out. (B) Properties and orthologs of BORC subunits. AA, amino acid. (C) Schematic of the overlapping subunit composition of BLOC-1 and BORC. New names for the previously uncharacterized subunits are shown in parenthesis. See also Figure S1 and Table S1.
Figure 2. Biochemical Characterization of BORC
(A) HeLa cell extracts were subjected to immuno-precipitation (IP) followed by immunoblotting (IB) with the indicated antibodies. (B) HeLa cells stably expressing Myc-, FOS-, or OSF-tagged subunits of BORC and BLOC-1 were extracted with lysis buffer and subjected to immunoprecipitation with antibody to the Myc epitope or pull-down with Strep-Tactin beads. Endogenous myrlysin and Snapin were then detected by immunoblotting. The ~20-kDa band in the Snapin IB of the Myc-Snapin IP lane (arrow) corresponds to Myc-Snapin. (C) cDNAs encoding BORC subunits, including His
6-Snapin and myrlysin-GST, were cloned into the E. coli expression plasmid pST39 (top). Purified BORC was cleaved with TEV protease to remove the tags and analyzed by gel filtration on Superose 6. Fractions 25–35 were resolved by SDS-PAGE, and proteins were visualized by Coomassie blue staining (bottom) and their identity confirmed by MS (Table S2). (D) H4 cell extracts were analyzed by gel filtration under the same conditions as in (B). Endogenous myrlysin, pallidin, Snapin, and the adaptor protein 4 (AP-4) ε subunit (control) were detected by immunoblotting. (E) siRNA-KD of BORC and BLOC-1 subunits was performed in HeLa cells, and myrlysin, pallidin, clathrin heavy chain (CHC), and actin (the latter two as controls) were detected by immunoblotting. The positions of molecular mass (Mr) markers (in kilodaltons) are indicated.
Figure 3. Myrlysin Regulates Lysosome Positioning
(A) Sanger sequencing of the genomic PCR product from a CRISPR/Cas9 myrlysin-KO HeLa cell clone shows a 99-bp deletion (residues 434–532 of cDNA) between two protospacer-adjacent motifs (PAM) (left). The agarose gel shows the PCR products from WT and myrlysin-KO cells (right). (B) No myrlysin was detected in the KO cells by immunoblotting using antibodies to a synthetic peptide (residues 167–196) (left two lanes) or to a GST fusion protein (right two lanes). (C) Confocal microscopy of WT and myrlysin-KO cells immunostained for Lamp-1, CD63, LAMTOR4, or TfR or stained with MitoTracker or ER-Tracker. Scale bar, 10 μm. (D) The distance of lysosomes from the nucleus boundary in WT (solid line and black bar) and myrlysin-KO cells (dashed line and gray bar) was measured using ImageJ. Twenty-five cells in each group were analyzed. Bar graphs represent the mean ± SD. The p value was calculated using Student’s t test. (E) myrlysin-GFP was transiently expressed in myrlysin-KO cells, and lysosomes were visualized by immunostaining with antibody to Lamp-1. Scale Bar, 10 μm. Notice the rescue of the lysosome-positioning phenotype in the myrlysin-GFP transfected but not in the untransfected cells. The bottom row shows 3.9-fold-magnified views of the insets. Arrows indicate co-localization. See also Figure S2.
Figure 4. BORC Recruits Alr8b to Lysosomes
(A) Schematic representing the involvement of BORC in the Arl8b-SKIP-Kinesin-1 pathway of lysosome movement toward the plus end of microtubules. PH, pleckstrin homology; RUN, RPIP8, UNC-14, and NESCA. (B) WT and myrlysin-FOS-expressing H4 cells were transiently transfected with plasmids encoding Arl8b-GFP, Myc-SKIP, KLC1-GFP, KIF5A-GFP, or GFP (control). After 72 hr, cells were treated with dithio-bis-succinimidyl propionate, extracted, and subjected to pull-down with StrepTactin beads. Eluted proteins were analyzed by immunoblotting with the indicated antibodies. myrlysin. The positions of molecular mass markers (in kilodaltons) are indicated. Myrl, myrlysin. (C and D) Confocal microscopy of WT and myrlysin-KO HeLa cells transiently transfected with plasmids encoding Arl8b-GFP (C) or Rab7-GFP (D) and immunostained for GFP and Lamp-1 or CD63. Scale bar, 10 μm.
Figure 5. Requirement of BORC, but Not BLOC-1, for Lysosome Dispersal and Arl8b Recruitment
siRNA-mediated KD of BORC and BLOC-1 subunits was performed in HeLa cells. Non-targeting siRNA was used as a control. Arl8b-GFP was expressed by transfection 48 hr after KD. Fixed permeabilized cells were stained with antibodies to GFP and Lamp-1 and examined by confocal microscopy. KD of myrlysin, lyspersin, Snapin, KXD1, BLOS1, and BLOS2 resulted in juxtanuclear clustering of lysosomes and dissociation of Arl8b in 65%–90% of the cells. The extent of Arl8b dissociation in these KD cells was 84%–97% as quantified by image analysis. Scale bar, 10 μm. See also Figure S3.
Figure 6. BORC Enables Kinesin-1-Dependent Motility of Lysosomes
(A and B) WT and myrlysin-KO HeLa cells were transiently transfected with plasmids encoding Myc-SKIP (A) or Lamp-1-KBS-GFP (B) and fixed 48 hr thereafter (KBS, KLC-binding sequence). Cells were immunostained for Myc, GFP, LAMTOR4, and/or CD63 and examined by confocal microscopy. Scale bar, 10 μm. (C) WT and myrlysin-KO cells were transiently transfected with a plasmid encoding Lamp-1-GFP and imaged live 48 hr later. The images show the first frames from Movie S1 (left). Anterograde (red) and retrograde (green) movements were tracked using ImageJ over a 19-s period. The number and distance of lysosome movements were quantified from the movies for five cells in each group. Values are mean ± SD. The p values were calculated using Student’s t test (right). Scale bar, 5 μm.
Figure 7. BORC Regulates Cell Spreading and Migration
(A) WT HeLa cells and myrlysin-KO HeLa cells expressing GFP or myrlysin-GFP (rescue) were stained with phalloidin-Alexa 647, antibody to LAMTOR4, and 4′6-diamidino-2-phenylindole, and imaged by confocal microscopy. Scale bar, 10 μm. (B) The footprint area was calculated using ImageJ from 100 phalloidin-stained cells in each group in (A). Values are mean ± SD. The p values were calculated using Student’s t test (right). (C) WT, myrlysin-KO, and myrlysin-GFP rescue HeLa cells were detached from culture plates using 10 mM EDTA in PBS, and forward-scatter was measured by flow cytometry of 50,000 cells in each group. Error bars indicate the coefficient of variation. (D) Two-dimensional cell migration was analyzed using a circular gap closure assay. Images were captured at the indicated time points. (E) The empty area was measured using ImageJ, and cell migration velocity was calculated from the images at 0 and 24 hr in five independent experiments. Values are mean ± SD. The p values were calculated using Student’s t test (right). See also Figure S4.
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