Journal: The EMBO Journal

Article Title: SKIP ‐ HOPS recruits TBC 1D15 for a Rab7‐to‐Arl8b identity switch to control late endosome transport

doi: 10.15252/embj.2019102301

Figure Lengend Snippet: A, B Hybrid Rab7/Arl8b compartment. (A) Representative confocal images of fixed HeLa cells expressing Arl8b‐GFP ( green ), immunolabelled against endogenous Rab7 (eRab7, magenta ). Zoom insets (3×) highlight select regions of colocalization ( white ), and white arrowheads point to vesicles positive for both GTPases. (B) Colocalization (Mander's overlap) of endogenous Rab7 with Arl8b‐GFP versus free GFP (EV), n EV = 4, n Arl8b = 9 images (3 ≥ cells per image) analysed from 2 independent experiments. Significance: two‐tailed Student's t ‐test, * P < 0.05. C–F Analysis of Arl8b compartment organization and dynamics as a function of Rab7 activity status. (C) Left and middle panels : representative confocal images of live HeLa cells expressing mCherry‐Rab7 or its mutants Q67L and T22N ( white ), together with Arl8b‐GFP ( green ), taken at the start of time‐lapse ( t 0 ). Right panels : tracks followed by Arl8b‐positive vesicles during the time‐lapse lasting 50 s recorded at 0.5 s per frame, with highest displacement rates for each track depicted on a rainbow colour scale (blue: immobile; red: maximum mobility per time interval). Zoom insets (2.8×) highlight select peripheral (PP) and perinuclear (PN) cell regions (see also Movies , , ). (D) Plot of Arl8b‐positive pixel distribution expressed as fractional distance along a straight line from centre of nucleus (0) to the plasma membrane (1.0), numbers of (pixels) plotted given above each scatter, n = 7 cells analysed per condition from 2 independent experiments. (E, F) Quantification of mean Arl8b vesicle displacement and maximum speed, respectively, n Rab7 = 21, n QL = 25, n TN = 24 images (3 ≥ cells per image) analysed from 2 independent experiments. Significance: one‐way ANOVA test (relative to wild type Rab7), *** P < 0.001, ns: not significant. Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines, respectively, all scale bars: 10 μm. Graphs report mean (red line) of sample values (open circles), and error bars reflect ± SD.

Article Snippet: GFP‐Rab7 and Myc‐Rab7 have been described before (Jordens et al , ), and GFP‐Rab7 Q67L and T22N were gifts from P. Chavrier (Meresse et al , ). mCherry‐Rab7 [G. Voeltz, Addgene plasmid #61804 (Rowland et al , )] was used to generate mCherry‐Rab7 Q67L and T22N mutants by site‐directed mutagenesis using standard protocols.

Techniques: Expressing, Two Tailed Test, Activity Assay, Clinical Proteomics, Membrane

Journal: The EMBO Journal

Article Title: SKIP ‐ HOPS recruits TBC 1D15 for a Rab7‐to‐Arl8b identity switch to control late endosome transport

doi: 10.15252/embj.2019102301

Figure Lengend Snippet: Representative confocal images of fixed HeLa cells ectopically expressing HA‐RILP ( blue ) in combination with Myc‐SKIP, GFP‐FYCO or Myc‐PLEKHM1 ( red ), immunolabelled against endogenous CD63 ( green ) and the indicated epitope tags. Zoom insets (3.5×) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bar: 10 μm. Plots of CD63 pixel distribution as a function of various effector perturbations shown in (A) expressed as fractional distance along a straight line from the centre of the nucleus (0) to the cell membrane (1.0), number of (pixels) plotted given above each scatter, n ≥ 4 cells per condition analysed from 2 independent experiments. Significance: one‐way ANOVA (relative to EV), *** P < 0.001, ns: not significant. Colocalization (Mander's overlap) of the indicated effectors with CD63, n ≥ 6 images (2 ≥ cells per image) per condition analysed from 2 independent experiments. Significance: 2‐tailed Student's t ‐test, * P < 0.05, **P < 0.01, ns: not significant, nd: not determined. Upper panel: wide‐field image of fixed HeLa cells harbouring endogenous CD63 tagged with GFP, co‐transfected with HA‐RILP and HA‐SKIP and labelled with SiR‐lysosome. Selected tomogram slices for peripheral (PP, middle panel ) and perinuclear (PN, bottom panel ) cell regions are shown (see also Movies and ). Arrowheads designate distinct endosomal subtypes: MVBs ( white) and endolysosomes ( yellow ), scale bars as indicated. Co‐immunoprecipitations (Co‐IP) of PLEKHM1‐FLAG with GFP‐Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP‐trap beads. Representative immunoblots against GFP and Flag are shown; EV: empty vector, TL: total lysate. InstantBlue staining of purified GST, GST‐Rab7 and GST‐Arl8b proteins. Myc‐SKIP truncation analysis for interactions with GFP‐Rab7 Q67L by Co‐IP from HEK293T cells using Myc‐trap beads. Representative immunoblots against Myc and GFP are shown, along with a schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. Co‐IP of C‐terminal RFP‐SKIP fragment (aa 537–1,019) versus its KMI motif mutants AAI and AAA with constitutively active GFP‐Rab7 Q67L from HEK293T cells using RFP‐Trap beads. Representative immunoblots against RFP and GFP are shown. Data information: cell and nuclear boundaries are demarcated with solid and dashed lines, respectively. Graphs report the mean (red line) of sample values (open circles), error bars reflect ± SD. Source data are available online for this figure.

Article Snippet: GFP‐Rab7 and Myc‐Rab7 have been described before (Jordens et al , ), and GFP‐Rab7 Q67L and T22N were gifts from P. Chavrier (Meresse et al , ). mCherry‐Rab7 [G. Voeltz, Addgene plasmid #61804 (Rowland et al , )] was used to generate mCherry‐Rab7 Q67L and T22N mutants by site‐directed mutagenesis using standard protocols.

Techniques: Expressing, Membrane, Transfection, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Staining, Purification

Journal: The EMBO Journal

Article Title: SKIP ‐ HOPS recruits TBC 1D15 for a Rab7‐to‐Arl8b identity switch to control late endosome transport

doi: 10.15252/embj.2019102301

Figure Lengend Snippet: A Electron micrograph of sections immunolabelled against RFP‐SKIP (15 nm gold) and GFP‐Rab7 (10 nm gold). Arrowheads and zoom inset (1.75×) highlight presence of RFP‐SKIP and GFP‐Rab7 on the same endosomal membrane, scale bar: 200 nm. B Co‐immunoprecipitations (Co‐IP) of HA‐RILP and RFP‐SKIP with GFP‐Rab7 (R7) versus its mutants Q67L (QL) and T22N (TN) from HEK293T cells using GFP‐trap beads. Representative immunoblots against GFP, HA and RFP are shown, EV: empty vector, IP: immunoprecipitation, TL: total lysate (see also Fig E). C Quantification of interaction between SKIP and Rab7 mutants expressed as fraction Co‐IP relative to wild‐type Rab7, n = 3 independent experiments. D, E In vitro glutathione precipitation assays. (D) Pull‐down (PD) of RFP‐SKIP or RFP‐RILP from HEK293T cell lysates using recombinant GST‐Rab7 versus GST‐Arl8b and free GST. Representative immunoblots against RFP and GST are shown (see also Fig F). (E) SKIP truncation analysis by PD against GST‐Rab7. Top panels : representative immunoblots against Myc and GST (see also Fig G). Bottom panels : schematic representation of SKIP domain organization. Regions of SKIP capable of interacting with Arl8b versus Rab7 are demarcated with solid black lines. An alignment of human ( h ) and murine ( m ) SKIP sequences to known effectors of Rab7 surrounding the conserved KML/I effector motif at residues 610–612 of SKIP is provided. F, G Co‐IP of RFP‐SKIP versus its KMI motif mutants AAI and AAA with constitutively active GFP‐Rab7 Q67L using RFP‐trap beads (see also Fig H). (F) Representative immunoblots against GFP and RFP. (G) Quantification of interaction between SKIP mutants with Rab7 expressed as fraction Co‐IP relative to wild‐type SKIP, n = 3 independent experiments. H Graphical summary of SKIP as a dual effector of Arl8b and Rab7. I–K Time‐lapse of SKIP‐mediated transport of late endosomes. (I) Schematic representation of tamoxifen‐induced activation of SKIP onto endosomal membranes. (J, K) Live HeLa cells co‐expressing GFP‐ER‐SKIP ( green ) and mCherry‐Rab7 ( magenta ) together with HA‐RILP ( unstained ) expressed at low levels (cells transfected at 1:5 RILP:SKIP ratio) were imaged in the (J) absence or (K) presence of tamoxifen, allowing on‐demand association of SKIP with endosomal membranes. Confocal frames from time‐lapses taken at the indicated time points following treatment are shown. Cell and nuclear boundaries are demarcated with solid and dashed lines, respectively, and zoom insets (3×) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10 μm (see also Movies , , ). Data information: Graphs report the mean (red line) of sample values (open circles), error bars reflect ± SD. All significance was assessed using 2‐tailed Student's t ‐test: * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available online for this figure.

Article Snippet: GFP‐Rab7 and Myc‐Rab7 have been described before (Jordens et al , ), and GFP‐Rab7 Q67L and T22N were gifts from P. Chavrier (Meresse et al , ). mCherry‐Rab7 [G. Voeltz, Addgene plasmid #61804 (Rowland et al , )] was used to generate mCherry‐Rab7 Q67L and T22N mutants by site‐directed mutagenesis using standard protocols.

Techniques: Membrane, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, In Vitro, Recombinant, Activation Assay, Expressing, Transfection

Journal: The EMBO Journal

Article Title: SKIP ‐ HOPS recruits TBC 1D15 for a Rab7‐to‐Arl8b identity switch to control late endosome transport

doi: 10.15252/embj.2019102301

Figure Lengend Snippet: A–C Consequences of effector depletion on the endogenous Arl8b/Rab7 hybrid compartment. (A) Representative confocal images of fixed HeLa cells harbouring GFP‐tagged endogenous Arl8b (G‐eArl8b, green ), transfected with the indicated siRNAs and immunolabelled against endogenous Rab7 (eRab7, magenta ). (B) Colocalization (Mander's overlap) between endogenous Arl8b and Rab7 in response to effector depletion, n siC = 10, n siRILP = 9, n siSKIP = 9 images (4 ≥ cells per image) analysed from 2 independent experiments. (C) Immunoblot analysis for depletion efficiency of SKIP and RILP, with actin as loading control. D, E Effect of Rab7 GTPase activity status on its association with the peripheral SKIP compartment. (D) Left panels : Representative confocal images of fixed HeLa cells expressing GFP‐Rab7 or its mutants Q67L or T22N ( green ) together with HA‐RILP ( red ) and Myc‐SKIP ( blue ), immunolabelled against the indicated epitope tags. Right panels : Schematic overview per condition. (E) Colocalization (Mander's overlap) between the indicated protein pairs, n Rab7 = 5, n QL = 5, n TN = 7 images (2 ≥ cells per image) analysed from 2 independent experiments. F Graphical summary of Rab7 removal from the SKIP compartment. Data information: Cell and nuclear boundaries are demarcated with solid and dashed lines, respectively, and zoom insets (3.5×) highlight select peripheral (PP) and perinuclear (PN) cell regions, scale bars: 10 μm. Graphs report the mean (red line) of sample values (open circles), error bars reflect ± SD. All significance was assessed using two‐tailed Student's t ‐test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant, nd: not determined. Source data are available online for this figure.

Article Snippet: GFP‐Rab7 and Myc‐Rab7 have been described before (Jordens et al , ), and GFP‐Rab7 Q67L and T22N were gifts from P. Chavrier (Meresse et al , ). mCherry‐Rab7 [G. Voeltz, Addgene plasmid #61804 (Rowland et al , )] was used to generate mCherry‐Rab7 Q67L and T22N mutants by site‐directed mutagenesis using standard protocols.

Techniques: Transfection, Western Blot, Control, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

doi: 10.1101/835686

Figure Lengend Snippet: ( A – C ) C18orf8 interacts with the mammalian MC1 complex. ( A ) Immune precipitation of overexpressed HA-tagged C18orf8 shows an interaction with Ccz1 and Mon1B. ( B ) Schematic for generation of an endogenous C18orf8-3xMyc fusion protein in HeLa cells. ( C ) Myc immune precipitation and immunoblotting of a knock-in clone shows the endogenous C18orf8-3xMyc fusion protein, detected by both C18orf8 and Myc antibodies. ( D ) Immune precipitation using a Ccz1-specific antibody but not an isotype control shows an interaction between Ccz1, endogenous C18orf8-3xMyc and Mon1B in C18orf8-3xMyc ki cells. ( E , F ) The C18orf8 C-terminal α-helical domain interacts with and stabilizes mammalian MC1. ( E ) Depiction of C18orf8 domain structure and mutants created. ( F ) C18orf8-deficient cells were complemented with mScarlet-labelled C18orf8 full-length (wt), C-terminal (AA 1-362) and N-terminal truncations (AA 354-657). mScarlet proteins were immune precipitated and immune precipitations and input controls were analysed by immune blotting using Mon1B, Ccz1 and mScarlet-specific antibodies. Note wild-type and N-terminal truncations (AA 354-657) stabilize Mon1B expression in input controls. ( G ) Mon1B expression in C18orf8 -deficient cells is rescued by overnight proteasome inhibition (bortezomib). ( H ) The C18orf8 C-terminus alone is insufficient to complement C18orf8 functions. C18orf8 -deficient cells were complemented with mScarlet-labelled C18orf8 full-length (wt), C-terminal (AA 1-362) and N-terminal truncations (AA 354-657) and analysed by flow cytometry for HMGCS1-Clover expression. ( I ) Complementation of Ccz1 -, Mon1A/B - and Rab7 -deficient cells. Ccz1 -, Mon1A/B - and Rab7 -deficient cells with transduced with respectively Ccz1-3xMyc; Mon1A-3xMyc, Mon1B-3xMyc or both; and 2xHA-Rab7-wt, -T22N or –Q67L and HMGCS1-Clover expression was analysed using wild-type HMGCS1-Clover cells as a control.

Article Snippet: 2xHA-Rab7 constructs were cloned from GFP-HA-Rab7 wild-type, T22N and Q67L constructs (Addgene #28047, #28048, #28049, created by Prof. Qing Zhong, University of California Berkeley, USA ( )) and inserted into pHRSIN.pCMV pGK Puro.

Techniques: Western Blot, Knock-In, Control, Expressing, Inhibition, Flow Cytometry, Transduction

Journal: bioRxiv

Article Title: A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

doi: 10.1101/835686

Figure Lengend Snippet: ( A ) The MCC complex binds an inactive Rab7 (T22N). Immune precipitations of 2xHA-tagged wild-type, T22N or Q67L Rab7 from C18orf8-3xMyc knock-in cells, were analysed by immunoblot using Myc, Ccz1 and Mon1B specific antibodies. ( B , C ) C18orf8 -, Ccz1 - and Mon1A/B -deficient cells lack activation-dependent recruitment of Rab7 effectors. ( B ) Immune precipitations of 3xFLAG-RILP from wild-type, C18orf8 -, Ccz1 - or Mon1A/B -deficient cells were analysed by immunoblotting for endogenous Rab7. ( C ) Wild-type and C18orf8 -deficient cells were transfected with HA-RILP or ORP1L ( Fig S5A ) and stained intracellularly for HA (green) and LAMP1 (magenta). Manders correlation was determined for 6-10 cells from two independent experiments (*** P<0.001). ( D – F ) Cholesterol and trafficking defects in C18orf8 deficient cells can be rescued by knockdown of Rab7GAPs or expression of a constitutively active Rab7 (Q67L). ( D ) HMGCS1-Clover expression was determined at day 8 after transduction with shRNAs against TBC1D5, TBC1D15 or both. ( E , F ) C18orf8 -deficient cells were transduced with 2xHA-tagged Rab7-T22N, -Q67L or an empty vector and either ( E ) analysed at day 10 by flow cytometry for HMGCS1-Clover expression, or ( F ) pulse-labelled with AlexaFluor 555 labelled EGF, incubated for 3 hours and stained intracellularly for LAMP1 and EEA1. Scale bars = 10µm.

Article Snippet: 2xHA-Rab7 constructs were cloned from GFP-HA-Rab7 wild-type, T22N and Q67L constructs (Addgene #28047, #28048, #28049, created by Prof. Qing Zhong, University of California Berkeley, USA ( )) and inserted into pHRSIN.pCMV pGK Puro.

Techniques: Knock-In, Western Blot, Activation Assay, Transfection, Staining, Knockdown, Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

doi: 10.1101/835686

Figure Lengend Snippet: ( A – C ) MCC -deficient cells accumulate lysosomal cholesterol and phenocopy NPC1 -deficiency. ( A ) Filipin staining of wild-type, C18orf8 -deficient and complemented C18orf8 -deficient cells; or ( C ) wild-type, Ccz1- and Mon1A/B -deficient cells. ( B ) Filipin co-staining with the LE/Ly markers Rab7 and LAMP1 in C18orf8 -deficient cells. ( D ) Theonellamides (TNM) immuno-gold labelling of C18orf8 -deficient cells, visualised by EM. ( E ) Filipin staining of wild-type, C18orf8 - and NPC1 -deficient cells. Scale bars = 10µm. ( F , G ) Rab7 interacts with the NPC1 cholesterol transporter in activation-dependent manner. ( F ) Immune-precipitation of HA-tagged NPC1 and detection of NPC1-interacting proteins using mass spectrometry. Interaction partners detected with >2 peptides are indicated by abundance. ( G ) Immune precipitations of HA-tagged wild-type, dominant-negative (T22N) or constitutively active Rab7 (Q67L) reveal an activation-dependent interaction between Rab7 and endogenous NPC1. ( H ) The Rab7-NPC1 interaction is lost in MCC-deficient cells that lack Rab7 activation. Wild-type, C18orf8-, Ccz1 and Mon1A/B-deficient cells were stably transduced with the inactive NPC1-P692S-HA. HA-tagged NPC1 was immune precipitated and immune blotted for endogenous Rab7. The inactive NPC1-P692S was used to prevent altering lysosomal cholesterol content. ( I , J ) The Rab7-NPC1 interaction is independent of NPC1 activity or lysosomal cholesterol levels. ( I ) NPC1-deficient cells were complemented with HA-tagged wild-type or inactive P692S-mutant NPC1 and NPC1-HA immune-precipitations were analysed by immune blotting for endogenous Rab7. ( J ) Wild-type NPC1-HA complemented cells were treated with LPDS to decrease, or U18666A to increase lysosomal cholesterol levels and the NPC1-Rab7 interaction was probed using immune precipitation.

Article Snippet: 2xHA-Rab7 constructs were cloned from GFP-HA-Rab7 wild-type, T22N and Q67L constructs (Addgene #28047, #28048, #28049, created by Prof. Qing Zhong, University of California Berkeley, USA ( )) and inserted into pHRSIN.pCMV pGK Puro.

Techniques: Staining, Activation Assay, Mass Spectrometry, Dominant Negative Mutation, Stable Transfection, Transduction, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

doi: 10.1101/835686

Figure Lengend Snippet: ( A, B ) Lysosomal cholesterol export is abolished in NPC1 -, C18orf8 -, Ccz1- and Mon1A/B -deficient cells. ( A ) Wild-type, NPC1-, C18orf8-, Ccz1 and Mon1A/B-deficient cells were treated for 24 hours with the NPC1 inhibitor U18666A to increase lysosomal cholesterol (pulse, top panels), followed by a 24 hours chase in the presence of LPDS and mevastatin (lower panels). Lysosomal cholesterol accumulation was visualised using Filipin co-staining with the LE/Ly marker CD63. ( B ) Colocalisation of Filipin with CD63 was plotted as Pearson correlation, calculated from 3 independent experiments with 6 representative fields per experiment and >8 cells per field. ** p<0.01, *** p<0.001. ( C ) Cholesterol accumulation in C18orf8-deficient cells is abolished by overexpression of a hyperactive Rab7. C18orf8-deficient cells were transduced with a wild-type, dominant-negative (T22N) or hyperactive Rab7 (Q67L) or empty vector and co-stained with Filipin and anti-CD63. ( D ) Rab7 overexpression rescues lysosomal cholesterol accumulation in NPC patient fibroblasts. NPC1 I1061T/I1061T primary patient fibroblasts were transduced with a GFP-tagged wild-type Rab7 and analysed at day 7 for cholesterol accumulation using Filipin staining. Fibroblasts from a healthy individual were used as a control. Representative images are shown from 2 independent experiments. ( E ) Model for MCC and Rab7 function in lysosomal cholesterol export. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF activates mammalian Rab7, which then binds to the NPC1 cholesterol transporter and either a ) directly activates NPC1 cholesterol export function; or b ) assembles a down-stream membrane contact site (MCS) at which a yet-uncharacterised lipid transfer protein (LTP) mediates cholesterol transfer to the ER and/or plasma membrane. A combined Rab7 function in NPC1 activation and MCS formation would assure lysosomal cholesterol is only exported once a down-stream lipid transfer module is assembled.

Article Snippet: 2xHA-Rab7 constructs were cloned from GFP-HA-Rab7 wild-type, T22N and Q67L constructs (Addgene #28047, #28048, #28049, created by Prof. Qing Zhong, University of California Berkeley, USA ( )) and inserted into pHRSIN.pCMV pGK Puro.

Techniques: Staining, Marker, Over Expression, Transduction, Dominant Negative Mutation, Plasmid Preparation, Control, Membrane, Clinical Proteomics, Activation Assay