Abstract

The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Highlights

  • IntroductionLate endosomes and other closely related acidic organelles (hereafter together referred to as lysosomes) are the major recycling stations of the cell

  • Lysosomes, late endosomes and other closely related acidic organelles are the major recycling stations of the cell

  • We selected U2OS cells due to their high proliferative rate and reduction in adherence when rounding up during mitosis. The former allowed for efficient synchronization by chemically arresting the progression of cell cycle and the latter enabled the efficient separation of the detached mitotic cells from those in the interphase that were adhered to the surface of cell culture dishes

Read more

Summary

Introduction

Late endosomes and other closely related acidic organelles (hereafter together referred to as lysosomes) are the major recycling stations of the cell. Instead of the glycosylated limiting membrane, luminal lipases are recruited to intraluminal vesicles (ILVs) through interactions with bis(monoacylglycero)phosphate (BMP) and other negatively charged phospholipids that are found on the surface of these vesicles [17,18]. At this site, lysosomal lipases degrade all sorts of intra- and extracellularly derived lipids, whose degradation products are exported to the cytosol by luminal, transmembrane, and cytosolic lipid transporters, such as Niemann-Pick type C1 (NPC1/NPC1), Niemann-Pick disease type C2

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call