# Investigating the interplay between PIKfyve/PI(3,5)P2 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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NATIONAL INSTITUTE OF
NEUROLOGICAL
DISORDERS AND STROKE

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National Institutes
of Health

PI(3,5)P2 is a low abundant phospholipid synthesized by the lipid kinase PIKfyve and found in the membrane of late endosomes and lysosomes. Following pharmacological inhibition of PIKfyve, PI(3,5)P2 depletion quickly and drastically impairs late endosomal/lysosomal formation, leading to the generation of big vacuoles. In a recent study, Gayle et al. observed that the deletion of CIC-7, a lysosomal transporter thought to facilitate lysosomal acidification, abolished the effects of PIKfyve inhibition, including vacuole formation (Gayle et al., Blood, 2017).

In this new study, we are investigating the interplay between PIKfyve/PI(3,5)P2 and CIC-7 in lysosomal acidification and vacuole formation.

## Open Questions

**Does PI(3,5)P2 level impact lysosomal/vacuole pH?**

Is CIC-7 a target of PI(3,5)P2?

Is the lysosomal pH essential for the vacuole formation?

## Main Results

PI(3,5)P2 depletion leads to lysosomal/vacuole hyperacidification.

Knocking out CIC-7 protects against lysosomal hyperacidification, but not vacuolization.

Lysosomal alkalanizing agent (chloroquine) does not prevent vacuolization.

## Conclusions

PI(3,5)P2/PIKfyve activity inhibits lysosomal acidification through CIC-7.

Vacuolization is not link to lysosomal hyperacidification.

## How to "dye-load" lysosomes

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## How to extract lysosomal fluorescence

We use the software Slicer2D to detect fluorescent lysosomes and extract through image processing their size and fluorescent intensity at 488nm and 445nm.

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For each cell, the 488/445 ratio of each individual fluorescent lysosome is measured.

## 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 equilibrates the pH between media, cytoplasm and lysosomes. Measuring 488/445 lysosomal ratio in pH calibrated buffers in presence of Monensin and Nigericin generates a pH calibration curves.

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## 1) Does PI(3,5)P2 level impact lysosomal pH?

**Apilimod is a PIKfyve inhibitor.**

Timecourse effect of apilimod 100nM on lysosomal pH of U2OS cells.

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PI(3,5)P2 depletion leads to hyperacidic lysosomes/vacuoles.

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

**A** Construction of a U2OS CIC7-KO clone through CRISPR/Cas9 approach (LentiCRISPRv2-GeCKO)

Because no antibody tagerting CIC-7 are available, we selected and validated the CIC7-KO clone at the DNA/RNA level

genomique level:
PCR using primer set F2R2
on U2OS cell extracted DNA

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transcript level:
PCR on cDNA obtain from U2OS cell extracted mRNA

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transcript level:
qPCR on cDNA obtain from RNA

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Model of the two DEL-CICN7 alleles present in the CIC7-KO clone created after sequencing:

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We have generated
a CIC7-KO U2OS
cell-line.

**B** Effect of apilimod (100nM, 3h) on lysosomal pH in CIC-7-KO U2OS cells.

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Representative data. Data replicated in two independent experiments.

CIC-7 is implicated in lysosomal hyperacidification, but not vacuolization.

## 3) Is lysosomal hyperacidification linked to vacuole formation?

Effect of chloroquine (5µM, overnight) on apilimod effect (100nM, 3h) in U2OS cells.

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Lysosomal hyperacidification is not essential for vacuolization.

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## Results interpretation

Model supported by our results:

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hyperacidification

Lysosomal pH

Endolysosomal
trafficking

Vacuole formation

PIKfyve/PI(3,5)P2 regulation
of lysosomal pH and endolysosomal
trafficking are not connected.

## Perspectives

Is the kinase activity of PIKfyve or its product, PI(3,5)P2, that regulates CIC-7?

Transfection of PI(3,5)P2; Inhibition of PI(3)P production (the substrate required to produce PI(3,5)P2).

Direct evidence for PI(3,5)P2 regulation/inhibition of CIC-7 activity?

Patch clamp of HEK cells expressing CIC-7 at the plasma membrane.

Is CIC-7 important for lysosomes reformation after releasing of PIKfyve inhibition?

PIKfyve inhibition by apilimod is reversible, and is associated with reformation of normal sized lysosomes in a timescale of hours.