Scientists Uncover Major Pathway Cells Use to Mend Leaky Lysosomes

Lysosomes are tiny sacs of digestive enzymes that declutter cells by breaking down waste. But they can also be troublesome: When their outer surface is damaged, their destructive proteins begin to spill into the cytoplasm and harm the cell. Indeed, the frequency of this leakiness increases as a person ages and likely plays a role in aging-associated diseases such as neurodegenerative conditions. Now, a study published September 7 in Nature uncovers a previously unknown pathway that cells use to repair leaky lysosomes, which may have implications for treating these diseases.

It’s a “very complete and well-designed” study, and the first to link lipid transport to a nonmetabolic biological process, says Marja Jäättelä, a professor of cell death and metabolism at the Danish Cancer Society Research Centre, who was not involved in the work.

Research had already established one way that cells repair leaky lysosomes. Previously, a collection of proteins known as the ESCRT machinery was found to patch up holes in the organelles’ membranes. Yet “something was missing,” according to coauthor Jay Tan, a cell biologist at the University of Pittsburgh. Up to 90 percent of lysosomal damage is fixed even when the ESCRT complex is inhibited, suggesting a different pathway performs the bulk of repair, Tan tells The Scientist.

To pinpoint the individual components of the missing pathway, Tan used a lentivirus to express an enzyme called Turbo-ID in human cells, which sticks a biotin tag onto any protein within a ten-nanometer radius. Using a version of the enzyme which localizes to lysosomes and a chemical known to puncture the lysosomal membrane, he was able to biotinylate all the proteins surrounding the damaged organelle.

Tan isolated the biotin-labelled proteins and identified them using mass spectrometry. Among the purified molecules were the components of the ESCRT complex, but also proteins known to interact with phosphoinositides, a family of lipids known to regulate key cellular processes, including proliferation and migration.

Further experiments revealed that when the lysosome membrane is compromised, an enzyme called phosphatidylinositol-4-kinase type 2a, or PI4K2A, is recruited to the organelle’s surface, possibly in response to calcium ions leaking out of the lysosome, says Toren Finkel, a study coauthor and a professor of medicine at the University of Pittsburgh. PI4K2A generates a lipid called phosphatidylinositol-4-phosphate (PI4P), which acts as a danger signal and recruits several proteins known as ORPs (for oxysterol-binding protein-related proteins), which tether the endoplasmic reticulum to the lysosome.

These ORPs then swap PI4P with lipids from the endoplasmic reticulum, including phosphatidylserine, which recruits the lipid transporter ATG2—the final component of the pathway. “ATG2 is like a firehose” for lipids, says Finkel, pumping molecules into the membrane to plug the hole.

red lysosomes with bits of green endoplasmic reticulum enveloping them

Lipids are shuttled from the endoplasmic reticulum (green) to seal holes in damaged lysosomes (red). Cell nuclei are shown in blue.


The researchers then mutated the various subunits of ATG2 to confirm that the lipid transporter is an essential piece of the pathway. “Instead of a smooth tunnel, we put up a bunch of molecular toll booths by changing amino acids, making it harder for lipids to flow,” says Finkel. These changes indeed blocked lipid transport in cell culture and stalled lysosomal repair.

As an ode to the University of Pittsburgh, the two scientists christened the pathway phosphoinositide-initiated membrane tethering and lipid transport, or PITT for short.

Tan believes that the two mechanisms have evolved to repair different types of damage, with the ESCRT complex mending small pores while the PITT pathway repairs larger holes.

The new pathway may be performing most of the cell’s handywork. The researchers found that cells usually take around an hour to repair damaged lysosomes, but this healing requires up to 11 hours in cells lacking PI42KA. “It looks like quite a significant pathway, perhaps more so than the ESCRT pathway,” says Antony Galione, a pharmacologist at the University of Oxford in the UK who was not involved in the study.

Tan says the findings could point to drug targets for neurodegenerative diseases, such as Alzheimer’s, as protein aggregates like tau escape in the lysosome membrane, preventing their destruction and facilitating their spread between neurons, says Tan. Indeed, the researchers found that depletion of PI4K2A, the initiator of the PITT pathway, increased tau spreading in cell culture.

It’s “definitely an important process to further study in disease models of neurodegeneration,” says molecular biologist Caroline Mauvezin at the University of Barcelona, Spain, who was not involved in the study. But the pathway might function differently in other cells, including neurons, so more work is needed, she adds.

The researchers plan to screen drugs for their ability to activate the pathway, starting with currently available drugs that could be repurposed. One intriguing lead is ginseng, a plant used in traditional Chinese medicine, the components of which appear to activate PI42KA, says Tan.


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