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# Investigating the interplay between PIKfyve and CIC-7 in lysosomal acidification and trafficking.

Xavier Leray, Anowarul Amin, Mary Weston, Joseph Mindell

Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda

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PIKfyve is a lipid kinase found in the membrane of late endosomes and lysosomes. It is responsible for the synthesis of the phospholipid PI(3,5)P2. PIKfyve inhibition quickly and drastically impairs late endosomal/lysosomal formation, leading to the generation of big vacuoles. Recently, knocking out lysosomal Cl/H+ exchanger CIC-7 has been proposed to provide substantial resistance against this vacuole phenotype. Given that CIC-7 is suggested to play a role in lysosomal acidification, it raises the possibility that PIKfyve may regulate CIC-7 activity, in turn modulating lysosomal pH, and that endosomal/lysosomal pH could tune endocytic trafficking.

In this new study, we are investigating the interplay between PIKfyve and CIC-7 in lysosomal acidification and vacuole formation.

## Open Questions

* Is lysosomal pH altered under PIKfyve inhibition?

* Is CIC-7 critical for this process?

* Is lysosomal pH critical for vacuole formation?

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## 1) PIKfyve inhibition hyperacidifies lysosomes

Apilimod is a PIKfyve inhibitor. OG is a pH-sensitive ratiometric dye.

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For method explanations, see the bottom center section of this poster.

First, Oregon Green 488 dextran (OG) is loaded into the endocytic pathway by fluid phase endocytosis. Then, OG is washed out from the media and a 4h incubation period let OG-containing endosomes to mature and eventually fuse with lysosomes.

Finally cells are incubated for 3h with the PIKfyve inhibitor apilimod before performing pH-ratiometric live cell imaging with an epifluorescence microscope.

<figure><img src="image_5.png" /><figcaption>A Images of live U2OS cells lysosome-loaded with OG (bright spots).</figcaption></figure>

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pH calibration curves for control and apilimod treated cells are similar.

This control experiment indicates that pH measurements is not altered when done on big vacuoles instead of small lysosomes. pH measured in both conditions can thus be compared without bias.

C

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Cumulative distribution of lysosomal pH population.

Each symbol represents the proportion of lysosomes having a pH value below the pH value represented in the abscissa axis. It confirms that OG-containing vesicles in control condition are acidic endolysosomal compartment. It also reveals that hyperacidification is general and not restricted to a subset of lysosomes, as shown by the global pH shift and shape conservation of the curve for apilimod condition compared to control.

D

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E

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(E) Lysosomal pH. Representative experiment. Each dot represents the averaged lysosomal pH of one cell (n=16-17).

(F) pH-shift. Each dot represents the apilimod-induced lysosomal pH shift measured in one experiment (n=10).

## 2) Does lysosomal pH alteration and increase in size correlate?

### Apilimod and WX8 are PIKfyve inhibitors.

<figure><img src="image_11.png" /><figcaption>B Evolution of lysosomal pH and size after PIKfyve removal.</figcaption></figure>

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### Evolutions of lysosomal pH and size show different kinetics.

## 3) Is CIC-7 involved in hyperacidification?

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CRISPR-Cas9 CIC-7 KO in U2OS cells

ATGGCCAACGTCTCTAAGAAGGTGTCTGGTCCGGCCGGGACCGGGACGAGGAGGCGGCGCCGCTGCTGCGGAGGACGGCGCGCCCGGCGGGGGGACGCCGCTGCTGAACGGGGCTGGGCCCGGGGCTGCGCGCCAG

CLCN7 EXON 1 sequence
Allele 1 deletion site (118bp with frameshift)
Allele 2 deletion site (64bp with frameshift)

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Chase
control/apilimod
3h

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(D) Lysosomal pH. Each dot represents average of cells lysosomal pH in one experiment (n=10 and n=6 for CIC-7 Wt and KO, respectively).

(E) pH-shift. Each dot represents the apilimod-induced lysosomal pH-shift measured in one experiment.

(F) Lysosomal size. Each dot represents average of cells lysosomal size in one experiment.

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CIC-7
- is required for
lysosomal
hyperacidification
- participates but is
not required for
vacuolization.

### How to “dye-load” lysosomes

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### How to measure lysosomal pH

Oregon Green 488 Dextran is a pH ratiometric fluorescent dye. The 488nm/445nm ratio will predictably be affected by pH.

Monensin and Nigericin are ionophores that equilibrate pH between media, cytoplasm and lumen of lysosomes. Measures of 488/445 lysosomal ratio in pH calibrated buffers in presence of Monensin and Nigericin can be plotted as a function of buffers pH, generating a pH calibration curve as depicted below.

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Each point correspond to one cell's lysosomes averaged values

pH calibration curve allows transformation of 488/445 ratio measures into pH values.

## 4) Is lysosomal hyperacidification involved in vacuole formation?

### Chloroquine is a lysosomotrophic agent. Bafilomycin-A1 is a v-ATPase inhibitor.

### Effect of lysosomes pre-alkilinization on apilimod treatment.

A

OG loading
overnight

4h

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control + apilimod

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chloroquine + apilimod

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bafilomycin + apilimod

C

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(C/D) Lysosomal pH/size. Each dot represents average of cells lysosomal pH/size in one experiment (n=4).

D

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### Lysosomal pH is not critical for
lysosomal swelling.

### Model supported by our results

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### hyperacidification

### Lysosomal pH

### Vacuole formation

### Conclusions

- PIKfyve might regulate lysosomal pH through inhibition of CIC-7 activity.

- CIC-7 activity, but not lysosomal pH, might participate in endolysosomal trafficking.