High lumenal chloride in the lysosome is critical for lysosome function
- University of Chicago, United States
- Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, University of Chicago, United States
Figures
Figure 1 with 3 supplements
Clensor recapitulates its chloride sensing characteristics in vivo.
(a) Schematic of the ratiometric, fluorescent chloride (Clˉ) reporter Clensor. It bears a Clˉ sensitive fluorophore, BAC (green star) and a Clˉ insensitive fluorophore, Alexa 647 (red circle) (b) …
Figure 1—figure supplement 1
(a) Quantification of co-localization between DNA nanodevices and GFP in arIs37 worms.
Mean of n = 10 cells. (b) Coelomocyte labeling efficiency with I-switch (I4A647, grey) and ClensorA647 (red) in the absence (-mBSA) or presence of 150 or 300 equivalents excess of maleylated bovine …
Figure 1—figure supplement 2
(a) Schematic of a DNA nanodevice, I-switch, that functions as a fluorescent pH reporter based on a pH triggered conformational change that is transduced to photonic changes driven by differential fluorescent resonance energy transfer between donor (D, green) and acceptor (A, red) fluorophores (b) pH calibration curve of I4cLYA488/A647 in vivo (red) and in vitro (grey) showing normalized D/A ratios versus pH.
Error bars indicate s.e.m. (n = 15 cells, ≥60 endosomes) (c) in vitro (grey) and in vivo (red) fold change in D/A ratios of I4cLYA488/A647 from pH 4.0 to pH 7.5. (d) Representative images of …
Figure 1—figure supplement 3
Selectivity of Clensor (200 nM) in terms of its fold change in R/G from ~0 to 100 mM of each indicated anion unless otherwise indicated.
(** ~ 0 to 10 mM). Since Cl− channels are known to exhibit poor anion selectivity, we investigate the selectivity of Clensor to demonstrate the signal change in cell is attributed by the change of …
Figure 2 with 2 supplements
Dysregulation in lysosomal [Cl-] correlates with reduced lysosomal degradation.
(a) Schematic depicting protein players involved in autosomal recessive osteopetrosis. (b) Representative images of Clensor in lysosomes of coelomocytes, in the indicated genetic backgrounds …
Figure 2—figure supplement 1
(a) Representative images of coelomocyte lysosomes labeled with Clensor one hour post injection, in the indicated genetic backgrounds acquired in the Alexa 647 (R) and BAC (G) channels and the corresponding pseudocolored R/G images.
Scale bar, 5 μm. (b) Histograms comparing the spread of R/G in coelomocytes in different RNAi background. (n = 10 cells; >100 lysosomes). (c) Lysosomal pH measured using I4cLYA488/A647 in the …
Figure 2—figure supplement 2
(a) Plots showing mean whole cell intensity of I4A647 per coelomocyte, as a function of time, post-injection in indicated genetic backgrounds.
Error bars indicate s.e.m. (n ≥ 10 cells). (b) Schematic depicting protein players involved in autosomal recessive osteopetrosis. Also shown is clh-4, a plasma membrane resident chloride channel, …
Figure 3 with 3 supplements
Lysosomal chloride dysregulation is observed in nematode models in several pH-related lysosomal storage disorders.
(a) Representative pH maps of lysosomes in coelomocytes labelled with a DNA-based pH reporter, I4cLYA488/A647, in the indicated genetic backgrounds. Images were acquired in the donor (D, magenta) …
Figure 3—figure supplement 1
(a) Representative images of LMP-1::GFP marked coelomocytes in the background of indicated RNAi.
Scale bar: 5 μm. (b) Histograms comparing the spread in size of LMP-1::GFP positive vesicles in coelomocytes in the indicated RNAi background (n = 20 cells). (c) D/A values obtained for individual …
Figure 3—figure supplement 2
(a) Worms expressing LMP-1::GFP in coelomocytes were injected with I4cLYA647 or ClensorA647 (red) and show maximal colocalization with LMP-1::GFP vesicles (green) at 60 min.
Scale bar: 5 μm (b) Representative images of worms expressing LMP-1::GFP (green) in the background of various indicated RNAi's, which were injected with ClensorA647 (red) and imaged 60 mins …
Figure 3—figure supplement 3
(a) Histograms comparing the spread of D/A in coelomocytes in different RNAi background.
(n = 10 cells; >100 lysosomes). (b) Histograms comparing the spread of R/G in coelomocytes in different RNAi background. (n = 10 cells; >100 lysosomes).
Figure 4 with 5 supplements
Lysosomal chloride is substantially depleted in mammalian cell culture models of lysosomal storage diseases.
(a) Calibration profile of Clensor in cells (red) and in vitro (grey) showing normalized Alexa 647 (R) and BAC (G) intensity (R/G) ratios versus [Cl-]. Error bars indicate s.e.m. (n = 20 cells,≥100 …
Figure 4—figure supplement 1
(a) Structure of Oregon Green (OG) and schematic of ImLy (b) Fluorescence emission spectra of ImLy at the indicated pH obtained using λExOG = 494 nm (green) and λEx Atto 647N = 650 nm (red).
(c) Representative 12% denaturing PAGE stained with Ethidium bromide (EtBr) showing conjugation of OG (green) to thiol labelled DNA (purple arrow) imaged in the ETBr and OG channels respectively (d) …
Figure 4—figure supplement 2
Plots showing mean whole cell intensity (wci, black line) of ClensorA647 in cells imaged as a function of indicated times in (a) J774A.1 cells and (b) THP-1 cells.
Blue lines show wci traces of individual cells as a function of time. Error bars indicate s.e.m. (n ≥ 25 cells). Representative images of (c) J774A.1 cells and (d) THP-1 cells at indicated time …
Figure 4—figure supplement 3
(a) Representative images showing colocalization of ImLyAT647 with TMR-Dex in J774A.1 and THP-1 macrophages (b) Macrophage labeling efficiency with ClensorA647 (A647) in the absence or presence of 50 equivalents excess of maleylated bovine serum albumin (mBSA) in comparison to TMR Dextran.
Error bars indicate s.e.m. (n = 20 cells). To verify whether ImLy was endocytosized to lysosome by anionic ligand binding receptor (ALBR) pathyway, cells were first pre-labelled with TMR-Dextran a …
Figure 4—figure supplement 4
Co-localization of Clensor (red) with lysosomes labelled with TMR–dextran (green) in J774A.1 cells treated with the indicated lysosomal enzyme inhibitors.
(a) Representative images of lysosomes of J774A.1 cells labelled with TMR dextran (TMR; G) and Clensor (Alexa 647; R) in the absence of any inhibitor, 50 μM NPPB, 10 μM Amitryptiline hydrochloride …
Figure 4—figure supplement 5
Representative pH and [Cl-] maps of ImLy and Clensor in lysosomes of J774A.1 cells in the absence and presence of 10 μM U18666A that gives a cell culture model of NP-C.
(A) Representative images of lysosomes labeled with Clensor in the Alexa 647 (R) channel and pseudocolored R/G images are shown. Scale bar: 10 μm (B) Representative images of lysosomes labeled with …
Figure 5 with 2 supplements
(a) Schematic of potential roles for lysosomal chloride.
Cl- ions can regulate lysosomal Ca2+ and/or affect lysosomal enzyme function. (b) Representative traces of Glycyl-L-phenylalanine 2-naphthylamide (GPN) (400 μM) triggered lysosomal Ca2+ release in …
Figure 5—figure supplement 1
Representative fluorescence images of cleaved substrates of ARSB and cathepsin L (E), DAPI (D) merge of E and D channels and respective pseudocolour E/D maps of J774A.1 cells with and without 50 µM NPPB.
Scale bar: 5 μm. To demonstrate chloride ion levels directly influence lysosome function, 50 µM of chloride channel inhibitor – NPPB was employed to decrease lysosomal chloride without affecting …
Figure 5—figure supplement 2
(a) Lysosomal pH and (b) chloride levels measured by ImLy and Clensor in J774A.1 cells with increasing concentrations of NPPB.
Here we show that by varying doses of NPPB that at < 100 µM of NPPB, we can selectively reduce lysosomal Cl- without changing in lysosomal pH. Consequently all further studies that use NPPB do so at …
Additional files
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Supplementary file 1
Sequences used for Clensor and I4cLYA488/A647 assemblies.
Oligo P, Oligo D1 and Oligo D2 combine to form Clensor. Oligo I4cLYA488 and I4cLYA647 combine to form I4cLYA488/A647. The sequences in matching colors are complementary.
- https://doi.org/10.7554/eLife.28862.023
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Supplementary file 2
Lysosomal storage disorders investigated in this study, their corresponding human genes and the C.elegans homologues.
- https://doi.org/10.7554/eLife.28862.024
