Lysosomal Changes in Mitosis

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.

Lysosome repair by ER-mediated cholesterol transfer

“…The purification yielded in total ∼1,500 picomoles (pmol) of these lipids from untreated (0 min) cells, in comparison to only ∼150 pmol in the negative control, in which the anti-LAMP1 antibody was replaced with a control IgG antibody (Figs 1A and EV1C). Further supporting its purity, the LAMP1+ fraction had in average ∼30 fold higher molar percentage (mol%, molar quantity relative to the total pmol of all lipids) values of bismonophosphatidic acid (BMP) species, the primarily polyunsaturated GPLs residing in the intraluminal vesicles of lysosomes (Nielsen et al , 2020; Stahl-Meyer et al ., 2022; van Meer et al , 2008), than the WCL (Bilgin et al ., 2017; Nielsen et al ., 2020) (Fig EV1D,E). The LAMP1+ fraction was abundant in STs and SLs (Fig 1B) but depleted of most other diacyl-GPL classes than BMP, including the major lipid class of HeLa ER membrane phosphatidylcholine (PC) (Scrima et al , 2022) and the fully depleted mitochondrial cardiolipin (CL) (Figs EV1D and EV2).…”

“…To elucidate the effects of membrane damage on the lipid compositions of lysosomes, we quantitatively profiled lipids in HeLa cells treated with a lysosomotropic compound, L-leucyl-L-leucine methyl ester (LLOMe) (Thiele & Lipsky, 1990) for 0, 10, or 45 min. We immuno-affinity purified lysosomes from the prepared post-nuclear fractions using a primary antibody against LAMP1 and a secondary antibody conjugated to magnetic microbeads (Bilgin et al , 2017; Stahl-Meyer et al , 2022) (Fig EV1A).…”

“…Lysosomes were purified and their lipid contents were analyzed with quantitative mass spectrometry-based shotgun lipidomics as described previously (Nielsen et al ., 2020; Stahl-Meyer et al ., 2022). In brief, the post-nuclear fractions prepared from the LLOMe-treated or untreated cells were incubated with anti-LAMP-1 primary antibody (Abcam, #ab24170) or IgG control (Invitrogen, Waltham, MA, USA, #31235) and then with anti-Rabbit IgG MicroBeads (Miltenyi Biotec, #130-048-602).…”

“…For shotgun lipidomics, a series of lipid internal standards in appropriate and known quantities (Stahl-Meyer et al ., 2022) were added to each sample prior to lipid extraction using a modified Bligh and Dyer protocol. The lipid extracts were mixed with positive (13.3 mM ammonium bicarbonate in 2-propanol) or negative (0.2% ( v / v ) tri-ethyl-amine in chloroform:methanol 1:5 ( v / v )) ionization solvents.…”

“…The copyright holder for this preprint this version posted September 28, 2022. ; https://doi.org/10.1101/2022.09.26.509457 doi: bioRxiv preprint 1990) for 0, 10, or 45 min. We immuno-affinity purified lysosomes from the prepared post-nuclear fractions using a primary antibody against LAMP1 and a secondary antibody conjugated to magnetic microbeads (Bilgin et al, 2017;Stahl-Meyer et al, 2022) (Fig EV1A).…”