Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.

Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy.

Centriolar satellites are high-order assemblies, scaffolded by the protein PCM1, that gravitate as particles around the centrosome and play pivotal roles in fundamental cellular processes notably ciliogenesis and autophagy. Despite stringent control mechanisms involving phosphorylation and ubiquitination, the landscape of post-translational modifications shaping these structures remains elusive. Here, we report that necrosulfonamide (NSA), a small molecule known for binding and inactivating the pivotal effector of cell death by necroptosis MLKL, intersects with centriolar satellites, ciliogenesis, and autophagy independently of MLKL. NSA functions as a potent redox cycler and triggers the oxidation and aggregation of PCM1 alongside select partners, while minimally impacting the overall distribution of centriolar satellites. Additionally, NSA-mediated ROS production disrupts ciliogenesis and leads to the accumulation of autophagy markers, partially alleviated by PCM1 deletion. Together, these results identify PCM1 as a redox sensor protein and provide new insights into the interplay between centriolar satellites and autophagy.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis.

Centriolar satellites are multiprotein aggregates that orbit the centrosome and govern centrosome homeostasis and primary cilia formation. In contrast to the scaffold PCM1, which nucleates centriolar satellites and has been linked to microtubule dynamics, autophagy, and intracellular trafficking, the functions of its interactant CEP131 beyond ciliogenesis remain unclear. Using a knockout strategy in a non-ciliary T-cell line, we report that, although dispensable for centriolar satellite assembly, CEP131 participates in optimal tubulin glycylation and polyglutamylation, and microtubule regrowth. Our unsupervised label-free proteomic analysis by quantitative mass spectrometry further uncovered mitochondrial and apoptotic signatures. CEP131-deficient cells showed an elongated mitochondrial network. Upon cell death inducers targeting mitochondria, knockout cells displayed delayed cytochrome c release from mitochondria, subsequent caspase activation, and apoptosis. This mitochondrial permeabilization defect was intrinsic, and replicable in vitro with isolated organelles. These findings extend CEP131 functions to life-and-death decisions and propose ways to interfere with mitochondrial apoptosis.