A novel live-cell imaging assay reveals regulation of endosome maturation
- Biozentrum, University of Basel, Switzerland
Figures
Figure 1 with 6 supplements
Rab conversion and completion of endolysosomal stages of endosome maturation can be observed in enlarged endosomes, induced with short nigericin treatment.
Nigericin was added to HeLa cells at 10 μM for 20 min and washed away, and cells were imaged by time-lapse microscopy. Recovery times are specified relative to removal of nigericin. (A,C,D) Cells …
Figure 1—figure supplement 1
Enlarged compartments interact with early endosomes and acquire intraluminal content from the endolysosomal pathway.
Nigericin was added to HeLa cells, stably expressing mApple-Rab5 and GFP-Rab7 (A,B) or mApple-Rab5 alone (C), for 20 min and washed away, and cells were imaged by time-lapse microscopy. Recovery …
Figure 1—figure supplement 2
Enlarged endosomes that undergo Rab conversion can be induced by different treatments.
HeLa cells stably expressing mApple-Rab5 and GFP-Rab7 were treated for 4 hr with ammonium chloride (A) or for 20 min with monensin (B), washed and imaged at specified times after the wash. …
Figure 1—figure supplement 3
Enlarged endosomes that undergo Rab conversion can be induced in different cell types.
Cells were transiently transfected with mApple-Rab5 and GFP-Rab7, and treated 20 min with nigericin followed by recovery and time-lapse microscopy. (A) HEK293 cells, (B) Neuro2A cells, (C) Cos-1 …
Figure 1—video 1
Rab5 recruitment, Rab conversion and endolysosomal maturation.
Figure 1—video 2
Homotypic fusion of two Rab5-positive endosome and subsequent Rab5 removal/weak Rab7 recruitment.
Figure 1—video 3
An enlarged Rab7-positive endosome was selected to show accumulation of Lysotracker concomitant with the loss of spherical shape and a reduction in size of the maturing endolysosome.
Figure 2 with 1 supplement
Nigericin-induced enlarged compartments originate at the Golgi and contain trans-Golgi marker GalT, later found in ILVs, with most enlarged compartments resolved by 48 hr.
Nigericin was added to HeLa cells for 20 min and washed away, and cells were processed for electron microscopy (A–C), imaged by time-lapse microscopy (D) or harvested for counting (E) at specified …
Figure 2—figure supplement 1
Cell growth is unaffected by nigericin treatment, which induces reversible Golgi vesiculation HeLa cells stably expressing mApple-Rab5 (A,B) or GalT-GFP (C–E) were treated for 20 min with 10 μM nigericin or 5 μM monensin, or left untreated.
(A) Images of cells stained with LC3b antibody to show absence of autophagy induction or presence of LC3B at the enlarged endosomes of nigericin-treated cells. Bafilomycin-treated cells are shown …
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Figure 2—figure supplement 1—source data 1
Cell growth numbers and quantification post nigericin treatment.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 3 supplements
Short nigericin treatment leads to trans-Golgi vesiculation and subsequent acquisition of Rab5.
(A) Images of untreated HeLa cells stably expressing mApple-Rab5 and transiently expressing GalT-GFP. Arrows point to puncta positive for both markers. (B–D) Nigericin was added to HeLa cells for 20 …
Figure 3—figure supplement 1
Endocytic cargo contributes to enlarged early endosome composition.
(A) HeLa cells stably expressing mApple-Rab5 were treated with nigericin, washed and 60 min later treated with 2.5 mg/mL Dextran-AF488 for 20 min. Cells were imaged at indicated times after washing …
Figure 3—figure supplement 2
Endocytosed plasma membrane transferrin receptor contributes to enlarged early endosome composition.
HeLa cells transiently transfected with mTagBFP2-Rab5 and TfR-EGFP were treated with nigericin and washed, with mCherry- anti-GFP nanobody added 110 min later to label surface TfR. Times are …
Figure 3—video 1
Kinetic to show fusion of a Rab5 negative enlarged compartment devoid of Dextran with a large Rab5 positive endosome containing endocytosed Dextran.
Figure 4 with 1 supplement
Enlarged endosomes recruit Rab5 and undergo Rab conversion with anticipated kinetics.
(A) Scheme to show experimental flow, starting with transfection of cells of choice with selected markers, followed by short nigericin treatment, and time-lapse microscopy during the recovery phase, …
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Figure 4—source data 1
Quantification of Rab5 and Rab7 recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Enlarged endosomes occasionally display multiple Rab5 peaks and form Rab5 subdomains.
HeLa cells, stably expressing mApple-Rab5 and GFP-Rab7 were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A) Time-lapse images of an endosome displaying …
Figure 5 with 2 supplements
PI(3)P is recruited to endosomes concomitantly with Rab5.
HeLa cells, stably expressing mApple-Rab5 and transiently transfected with the PI(3)P marker, GFP-FYVE, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A.…
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Figure 5—source data 1
Quantification of Rab5 and PI(3)P recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
PI(3)P is recruited to endosomes concomitantly with Rab5.
HeLa cells, stably expressing mApple-Rab5 and transiently transfected with the PI(3)P marker, GFP-FYVE, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A.…
Figure 5—video 1
Representative endosome to show transient Rab5 recruitment accompanied by PI(3)P.
Figure 6 with 2 supplements
Snx1 subdomain formation at the endosomes initiates with Rab5 recruitment and peaks during Rab conversion stages.
HeLa cells, stably expressing mApple-Rab5 and transiently transfected with Snx1-GFP, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A) Time-lapse …
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Figure 6—source data 1
Quantification of Rab5 and Snx1 recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig6-data1-v1.xlsx
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Figure 6—source data 2
Quantification of Rab5 and Snx1 subdomains at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig6-data2-v1.xlsx
Figure 6—figure supplement 1
Dynamic Snx1 recruitment suggest active sorting at the enlarged endosome.
Nigericin was added to HeLa cells for 20 min and washed away, and cells were imaged by time-lapse microscopy, as described in Figure 4A. (A–E) Cells stably expressing mApple-Rab5 (A–C) or …
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Figure 6—figure supplement 1—source data 1
Quantification of Rab5 and Snx1 subdomains at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig6-figsupp1-data1-v1.xlsx
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Figure 6—figure supplement 1—source data 2
Quantification of Rab7 and Snx1 subdomains at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig6-figsupp1-data2-v1.xlsx
Figure 6—video 1
Snx1 subdomain formation relative to Rab5 recruitment.
Figure 7 with 1 supplement
Snx1 subdomain formation at the endosomes initiates with Rab5 recruitment, peaks during Rab conversion stages and continues in late endosomes.
HeLa cells, stably expressing mApple-Rab7 and transiently transfected with Snx1-GFP, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A) Time-lapse …
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Figure 7—source data 1
Quantification of Rab7 and Snx1 recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig7-data1-v1.xlsx
Figure 7—video 1
Snx1 subdomain formation relative to Rab7 recruitment.
Figure 8 with 5 supplements
Rab11 interacts with the maturing endosome independently of Rab5 or Rab7.
HeLa cells, stably expressing mApple-Rab5 (A–D) or mApple-Rab7 (E–H) and transiently transfected with GFP-Rab11, were treated for 20 min with nigericin, washed and imaged over 3 h, as described in Fi…
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Figure 8—source data 1
Quantification of Rab5 and Rab11 recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-data1-v1.xlsx
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Figure 8—source data 2
Quantification of Rab5 and Rab11 subdomains at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-data2-v1.xlsx
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Figure 8—source data 3
Quantification of Rab7 and Rab11 recruitment at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-data3-v1.xlsx
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Figure 8—source data 4
Quantification of Rab7 and Rab11 subdomains at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-data4-v1.xlsx
Figure 8—figure supplement 1
Rab11 interacts with the maturing endosome independently of Rab5 or Rab7.
HeLa cells, stably expressing mApple-Rab5 (A,B) or mApple-Rab7 (C) and transiently transfected with GFP-Rab11, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Fig…
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Figure 8—figure supplement 1—source data 1
Quantification of Rab5 and Rab11 subdomains at endosomes in Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-figsupp1-data1-v1.xlsx
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Figure 8—figure supplement 1—source data 2
Numerical data for all analysed endosomes plotted in Figure 8—figure supplement 1A.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig8-figsupp1-data2-v1.xlsx
Figure 8—video 1
Rab11 interaction with the maturing endosome relative to Rab5.
Figure 8—video 2
Rab11 circling around the enlarged Rab5 positive compartments.
Figure 8—video 3
Rab11 circling around the enlarged Rab5 positive compartments.
Figure 8—video 4
Rab11 interaction with the maturing endosome relative to Rab7.
Figure 9 with 4 supplements
Selective cargo recycling takes place in enlarged Rab5-positive endosomes.
HeLa cells, stably expressing mApple-Rab5 and transiently transfected with the cargos TfR-GFP (A,D,G), GFP-CDMPR (B,E,H) or GalT-GFP (C,F,I), were treated for 20 min with nigericin, washed and …
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Figure 9—source data 1
Quantification of acquisition and removal of TfR to and from endosomes in relation to Rab5.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig9-data1-v1.xlsx
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Figure 9—source data 2
Quantification of acquisition and removal of CDMPR to and from endosomes in relation to Rab5.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig9-data2-v1.xlsx
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Figure 9—source data 3
Quantification of acquisition and removal of GalT to and from endosomes in relation to Rab5.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig9-data3-v1.xlsx
Figure 9—figure supplement 1
CDMPR can be acquired by Rab5-positive endosomes.
HeLa cells, stably expressing mApple-Rab5 and transiently transfected with CDMPR-GFP, were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. Time-lapse …
Figure 9—video 1
TfR acquisition and removal relative to Rab5 recruitment.
Figure 9—video 2
CDMPR acquisition and removal relative to Rab5 recruitment.
Figure 9—video 3
GalT acquisition and removal relative to Rab5 recruitment.
Figure 10 with 3 supplements
GalT-pHlemon sensor detects endosomal acidification.
HeLa cells were transiently transfected with the ratiometric pH sensor, GalT-pHlemon. (C,F) Cells were stably expressing mApple-Rab5. (B) Cells were incubated with Lysotracker Red (LTR) for 20 min …
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Figure 10—source data 1
Quantification of GalT-pHlemon signal in cells in calibration buffers of known pH.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig10-data1-v1.xlsx
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Figure 10—source data 2
Quantification of GalT-pHlemon signal in Golgi ribbon, endo-/lysosomal puncta, and enlarged endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig10-data2-v1.xlsx
Figure 10—figure supplement 1
GalT-pHlemon sensor responds to pH changes in a sigmoidal dose-response manner, and its puncta exhibit lysosome-like behavior.
HeLa cells were transiently transfected with the ratiometric pH sensor, GalT-pHlemon. (A) Graph to show response of GalT-pHlemon sensor to pH 4.0–9.0 range as displayed by YFP/CFP ratio measurements …
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Figure 10—figure supplement 1—source data 1
Sigmoidal dose-response curve to define relationship between GalT-pHlemon signal and pH.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig10-figsupp1-data1-v1.xlsx
Figure 10—video 1
GalT-pHlemon sensor localising to the enlarged transiently Rab5-positive endosome and changing YFP and CFP intensity consistent with endosomal acidification.
Figure 10—video 2
CFP-filled puncta fuse with the enlarged compartments, which shrink over time to re-obtain near-punctate morphology.
Figure 11 with 2 supplements
GalT-pHlemon sensor detects endosomal acidification, which correlates with Rab conversion.
HeLa cells, transiently expressing mApple-Rab5 (A–C) or stably expressing mApple-Rab7 (D–F) and transiently transfected with GalT-pHlemon, were treated for 20 min with nigericin, washed and imaged …
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Figure 11—source data 1
Quantification of Rab5 recruitment and GalT-pHlemon signal at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig11-data1-v1.xlsx
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Figure 11—source data 2
Quantification of Rab7 recruitment and GalT-pHlemon signal at endosomes.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig11-data2-v1.xlsx
Figure 11—video 1
Association of Rab conversion with acidification.
Figure 11—video 2
Association of Rab conversion with acidification.
Figure 12 with 5 supplements
Ccz1 KO disrupts Rab conversion and delays endosomal acidification.
HeLa cell lines with wild-type (WT) Ccz1 and knocked-out Ccz1 (KO) were transiently transfected with mApple-Rab5 and either GFP-Rab7 (A) or GalT-pHlemon (B–D). Ccz1 expression plasmid was …
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Figure 12—source data 1
Quantification of Rab5 recruitment and GalT-pHlemon signal at endosomes in Ccz1 WT, KO and rescue cells.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig12-data1-v1.xlsx
Figure 12—figure supplement 1
Validation and characterisation of Ccz1 knockout cell lines.
(A) Ccz1 mRNA expression levels in HeLa cell lines with wild-type (WT) Ccz1 and knocked-out Ccz1 (KO), three clones each, measured by qRT-PCR and normalised for actin. Raw RT-PCR data is available …
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Figure 12—figure supplement 1—source data 1
Raw RT-PCR data for Ccz1 expression levels in Ccz1 WT vs KO cells.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig12-figsupp1-data1-v1.xls
Figure 12—figure supplement 2
Validation and characterisation of Ccz1 the rescue construct.
(A) Western blot of the ccz1 rescue construct expressing NLS-mNeptune2-T2A-Ccz1-myc to show predominant production of two smaller products, NLS-mNeptune2 and ccz1-myc, as visualised by RFP and myc …
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Figure 12—figure supplement 2—source data 1
Original western blot images for NLS-mNeptune-T2A-myc expression.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig12-figsupp2-data1-v1.pdf
Figure 12—video 1
Rab conversion in Ccz1 WT cells.
Figure 12—video 2
Rab conversion in Ccz1 KO cells.
Figure 12—video 3
Rab conversion in Ccz1 KO cells expressing the Ccz1 rescue construct.
Figure 13 with 1 supplement
Lysosomes of Ccz1 knockout cells can acidify to the same extent as wild-type cells, with some delay.
HeLa cell lines with wild-type (WT) Ccz1 and knocked-out Ccz1 (KO) were transiently transfected with GalT-pHlemon. Ccz1 expression plasmid was co-transfected for 72 hr for rescue experiments. WT, KO …
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Figure 13—source data 1
Quantification of GalT-pHlemon signal in endo-/lysosomal puncta in Ccz1 WT, KO and rescue cells.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig13-data1-v1.xlsx
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Figure 13—source data 2
Quantification of GalT-pHlemon signal in endo-/lysosomal puncta in Ccz1 WT, KO and rescue cells post nigericin treatment.
- https://cdn.elifesciences.org/articles/70982/elife-70982-fig13-data2-v1.xlsx
Figure 13—figure supplement 1
Characterisation of Ccz1 knockout cell lines.
(A) Images of Ccz1 KO cells pre-treated with nigericin, recorded at 150 min recovery, showing Rab5-positive acidified hybrid compartments (arrows). Cells were transiently transfected with …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| cell line (Homo sapiens) | HeLa CCL2 | ATCC | RRID:CVCL_0030 | |
| cell line (Homo sapiens) | HeLa Kyoto | ATCC | RRID:CVCL_1922 | |
| cell line (Homo sapiens) | Neuro-2a | ATCC | RRID:CVCL_0470 | |
| cell line (Homo sapiens) | HEK293 | ATCC | RRID:CVCL_6910 | |
| cell line (monkey) | Cos-1 | ATCC | RRID:CVCL_023 | |
| antibody | anti-GFP (Rabbit polyclonal) | Abcam | RRID:AB_305564 | IEM(1:100) |
| antibody | anti-LC3b (Rabbit polyclonal) | Cell Signalling Technology | RRID:AB_2137707 | IF(1:400) |
| antibody | anti-GalT (Rabbit polyclonal) | Sigma | RRID:AB_1078254 | IF(1:50) |
| antibody | anti-mCherry (Goat polyclonal) | St John’s laboratory | STJ140001 | IEM(1:100) |
| antibody | anti-myc (Mouse monoclonal, 9E10) | Sigma | RRID:AB_2533008 | WB(1:2000) |
| antibody | anti-rabbit coupled to 10 nm Gold (Goat, IgG) | BB International | RRID:AB_2715527 | IEM (1:100) |
| antibody | anti-mouse coupled to 5 nm Gold (Goat, IgG) | BB International | RRID:AB_1769168 | IEM (1:100) |
| antibody | anti-rabbit coupled to 5 nm Gold (Donkey, IgG) | Jackson Immuno Research | RRID:AB_2340610 | IEM (1:100) |
| antibody | anti-goat (Mouse, IgG) | Jackson Immuno Research | RRID:AB_2339054 | IEM (1:100) |
| recombinant DNA reagent | mApple-Rab5a (Plasmid) | RRID:Addgene_54944 | ||
| recombinant DNA reagent | mApple-Rab7a (Plasmid) | RRID:Addgene_54945 | ||
| recombinant DNA reagent | GalT-mCherry (Plasmid) | RRID:Addgene_55052 | ||
| recombinant DNA reagent | mNeptune2-C1 (Plasmid) | RRID:Addgene_54836 | ||
| recombinant DNA reagent | mApple-Lamp1-pHluorin (Plasmid) | RRID:Addgene_54918 | ||
| recombinant DNA reagent | GFP-Rab11a (Plasmid) | RRID:Addgene_12674 | ||
| recombinant DNA reagent | EGFP-2xFYVE (Plasmid) | RRID:Addgene_140047 | ||
| recombinant DNA reagent | Lamp1-GFP (Plasmid) | RRID:Addgene_34831 | ||
| recombinant DNA reagent | pSpCas9(BB)–2A-GFP (pX458) (Plasmid) | RRID:Addgene_48138 | ||
| recombinant DNA reagent | pSpCas9(BB)–2A-puro (pX459) (Plasmid) | RRID:Addgene_48139 | ||
| recombinant DNA reagent | Snx1-turboGFP (Plasmid) | Origene | Origene # RG201844 | |
| recombinant DNA reagent | Ccz1-myc (Plasmid) | Origene | Origene # RC222195 | |
| recombinant DNA reagent | mTagBFP2-Rab5 (Plasmid) | RRID:Addgene_55322 | ||
| recombinant DNA reagent | mCherry-tagged anti-GFP (VHH) (Plasmid) | RRID:Addgene_109421 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70982/elife-70982-transrepform1-v1.docx
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Supplementary file 1
Oligonucleotide sequences used to generate specified constructs.
Table to specify oligonucleotide sequences and their description and purpose in generating constructs as outlined in Materials and methods.
- https://cdn.elifesciences.org/articles/70982/elife-70982-supp1-v1.docx
