Figure 3. 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) and acceptor (A, cyan) channels and the corresponding pseudocolored D/A images. Scale bar, 5 μm (b) Representative [Cl-] maps of lysosomes acquired in these genetic backgrounds using Clensor. Images are acquired in the Alexa 647 (R) and BAC (G) channels and the corresponding pseudocolored R/G images are shown. Scale bar, 5 μm. (c) Quantification of lysosomal pH and lysosomal Cl- in C. elegans mutants or RNAi knockdowns of genes responsible for the indicated lysosomal storage diseases in humans. Mutants are grouped according to dysregulation only in lysosomal pH (purple box); only in lysosomal chloride (green box) and both lysosomal pH and chloride (pink box) for n = 10 worms (≥100 lysosomes) Error bars indicate s.e.m.
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 pH measurements in lysosomes as a function of area of each vesicle in the indicated genetic background. The linear regression is shown in red. The coefficient of regression (p = ‒0.14) shows no correlation (n > 100 lysosomes). (d) R/G values obtained for individual Cl- measurement in lysosomes as a function of area of each vesicle in the indicated genetic background. The linear regression line is shown in red. The coefficient of regression (p= ‒0.18) shows no correlation (n > 100 lysosomes). A key phenotype observed in cells derived from patients suffering from lysosomal storage disorders (LSD) is the presence of enlarged lysosomes (Filocamo and Morrone, 2011; Platt et al., 2012). LSD-related gene knockdowns in worms show subtle to no observable whole organismal phenotypes (de Voer et al., 2008). However, on knocking down various LSD-related genes in the background of LMP-1::GFP worms we observed that the morphology of LMP-1 positive vesicles were altered and that the coelomocytes contained enlarged lysosomes, some as large as eight microns in diameter (Figure 3—figure supplement 1). Figure 3—figure supplement 1b represents a plot of the diameter distribution for LMP-1 positive vesicles under each indicated genetic background. Worms with LSD-related gene knockdowns show a broader distribution of vesicle sizes compared to a more tightly regulated size distribution in wild type nematodes or non-LSD related mutants. example for when clh-4, a plasma membrane resident chloride channel was knocked down, lysosomal morphology is not affected. We carried out pH and chloride measurements for worms from various genetic backgrounds and checked for correlations between lysosomal D/A (or R/G) and lysosome size. On plotting D/A values obtained from pH measurements in ncr-1 RNAi worms, against the area of each vesicle; we observe no correlation between the two parameters (Figure 3—figure supplement 1c). A similar observation is seen in the case of chloride R/G measurements (Figure 3—figure supplement 1d). Thus assaying only for lysosome size shows no correlation with lysosome functionality. However lumenal chloride concentration is the best correlate of lysosome dysfunction, irrespective of size.
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 post-injection. Scale bar: 5 μm. (c) Quantification of colocalization between the ClensorA647 and GFP in arIs37 worms. Mean of n = 10 cells. To check whether our DNA nanodevices can mark the lysosomes of coelomocytes in wild type worms, we injected 500 nM of I4cLYA647 or ClensorA647 into worms containing LMP1::GFP marker (Figure 3—figure supplement 2a). The worms were imaged 1 hr post injection in the GFP channel (green) and in Alexa 647 channel (red) to visualize the lysosomal marker and DNA reporter respectively. Merged images show colocalization of DNA devices with the lysosomal marker, similar to previous studies (Surana et al., 2011). We then proceeded to validate the devices in the lysosomes in coelomocytes of various LSD-related genes knocked down worms. LMP1::GFP positive worms that were RNAi-ed for indicated genes were injected with ClensorA647 and imaged 1 hr post injection (Figure 3—figure supplement 2b). We observe that, in worms, Clensor reliably marks the lysosomes in all LSD- related gene knockdowns with over 74% colocalization (Figure 3—figure supplement 2c).
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).