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: Nature Communications

Article Title: Endosomal trafficking participates in lipid droplet catabolism to maintain lipid homeostasis

doi: 10.1038/s41467-025-57038-8

Figure Lengend Snippet: A The knockdown efficiency of Rab5a in A549 cells was assessed by western blot analysis ( n = 3). B Control or Rab5a-knockdown A549 cells were fixed by 4% PFA, and stained with Bodipy 493/503 (1 μM) and DAPI (1 μg/mL) for 30 min. The number, size and area of LDs, as well as the fluorescence intensity of Bodipy 493/503 were quantified by ImageJ ( n = 30 for all figures; p < 0.0001 for all figures). C The triglyceride level in control or Rab5a-knockdown A549 cells was determined ( p < 0.0029). D A549 cells were transiently transfected with mc-Rab5a or mc-Rab5a-S23N, and then stained with Bodipy 493/503 (1 μM) for 30 min. The number and size of LDs, and fluorescence intensity of Bodipy 493/503 were quantified by ImageJ ( n = 13 for all figures; p = 0.0080, 0.0303 and 0.0015 for number of LDs, average size of LDs and relative Bodipy fluorescence, respectively). Images were captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The graphs represented data from three independent experiments. The statistical significance of differences was determined by using the unpaired two-tailed student’s t test, and data quantifications were expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: no significance.

Article Snippet: pEGFP-C1-ADRP (addgene, #87161), mcherry-ACSL3 (addgene, #87158), GFP-Rab5 (addgene, #174454), mcherry-Rab5 (addgene, #55126), mcherry-Rab5 S23N, GFP-Rab7 (addgene, #61803), mcherry-Rab7 (addgene, #55127), GFP-Rab7 T22N (addgene, #28048), GFP-Rab7 Q67L (addgene, #28049), pCDNA3.1-Twinstrep Rab5 (Elife.

Techniques: Knockdown, Western Blot, Control, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test