Biology subjects

Fajardo-Callejon, S.

Publications and source records attributed to Fajardo-Callejon, S..

3 recordsLinked to original sources

bioRxiv2026

Miniaturized Lysosome Enrichment through Selective Plasma Membrane Destabilization

Organelle enrichment presents a prerequisite for the unbiased biochemical analysis of cellular subcellular compartments. The performance of this step is decisive for the experiments success, and despite the high sensitivity of downstream analytical strategies, such as enzymatic assays, western blotting, or mass spectrometry (MS)-based OMICs approaches, typically tens of millions of cells are required, standing in stark contrast. Here, we demonstrate that selective rupture of the plasma membrane constitutes a limiting factor for the reduction of cell numbers and present an approach to overcome this limitation through detergent-based plasma membrane destabilization, followed by mechanical homogenization. By combination of this strategy with two common methods for lysosome enrichment, namely superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation via 3xHA-tagged TMEM192 (TMEM IP), we scale down lysosome enrichment to only half a million cells and demonstrate that reduced input cell numbers yield superior results with respect to sample purity and organelle proteome characterization. HighlightsO_LIRelease of intact lysosomes negatively correlates with cell concentration. C_LIO_LICombination of detergent-based plasma membrane destabilization and mechanical homogenization increases lysosomal intactness from low cell numbers. C_LIO_LIEnrichment columns require a minimum sample input. C_LIO_LIImmunoprecipitation of intact lysosomes enables enrichment from lower cell numbers. C_LIO_LIProteomics of low input lysosome enriched fractions identifies superior performance. C_LI MotivationModern mass spectrometry (MS)-based proteomics strategies facilitate the detection and quantification of peptides and proteins with unprecedented sensitivity, enabling the analysis of low input samples down to single-cells. However, subcellular fractionation experiments typically require tens of millions of cells to achieve sufficient yield and purity, presenting a strong contrast. This restricts the application of organelle profiling to cell lines that can be grown in sufficient amounts, excluding many physiologically relevant species which are only available in small quantities. To be able to miniaturize subcellular fractionation experiments, it is of crucial importance to overcome this limitation and to miniaturize sample preparation strategies.

cell biology↗

bioRxiv2026

ER-Lysosome Cholesterol Exchange Regulates Lysosomal Motility Through mTOR-Dependent LAMTOR1 Phosphorylation

The subcellular distribution of lysosomes, the main degradative organelles of mammalian cells, responds to metabolic cues in a highly dynamic way. While lysosomal positioning due to amino acid levels is well-characterized, cholesterol-dependent regulation of lysosomal motility is incompletely understood. We explored impaired lysosomal cholesterol export using a mass spectrometry-based multi-OMICs approach, identifying widespread reallocation of resources and signaling pathway modulation. We identified increased phosphorylation at LAMTOR1 serine 56 in response to cholesterol level perturbations. We demonstrate that this phosphorylation site is sufficient to disrupt Rag GTPases/SLC38A9 binding to the Ragulator complex, inhibiting canonical mTORC1 and facilitating binding of BORC, therefore promoting lysosomal retrograde movement. LAMTOR1 S56 phosphorylation responds exclusively to depletion of lysosomal limiting membrane cholesterol, is facilitated by mTOR, and presents a negative feedback loop for amino acid independent displacement of Ragulator bound Rag GTPases, limiting canonical mTORC1 activity. Mass spectrometry data are available via ProteomeXchange with identifier PXD073489. HighlightsO_LIPerturbation of lysosomal cholesterol homeostasis results in adaptation of cellular protein and lipid biosynthesis C_LIO_LILAMTOR1 is phosphorylated at serine 56 via mTORC1 C_LIO_LILAMTOR1 S56 phosphorylation is lysosomal membrane cholesterol dependent C_LIO_LILAMTOR1 S56 phosphorylation disrupts binding of Rag GTPases to the Ragulator complex C_LIO_LILAMTOR1 S56 phosphorylation promotes binding of Ragulator to BORC, facilitating lysosomal retrograde transport C_LI

molecular biology↗

bioRxiv2025

Absolute Quantification of Lysosomal Proteins by Multiple Reaction Monitoring Mass Spectrometry and QconCAT Protein Standards

ABSTRACTLysosomes are membrane-enclosed organelles that play a crucial role in the degradation of intra- and extracellular substrates and the regulation of metabolic signaling. These functions are carried out by a variety of proteins, of which > 150 are currently known to be located in the lysosomal lumen or to be embedded in its membrane. These proteins are typically low abundant, necessitating organelle enrichment experiments to enable their investigation by unbiased mass spectrometry-based proteomics analyses. Enrichment strategies have been applied in a plethora of studies to gain a deeper understanding of lysosomal composition and relative changes of lysosomal proteome abundance in a variety of pathological conditions. Such experiments are restricted, however, to selected cell lines and tissues and do not allow a direct analysis of the lysosomal proteome from whole cell or tissue lysates. Furthermore, they do not provide absolute quantities. We developed a multiple reaction monitoring mass spectrometry-based assay for the highly sensitive and reproducible absolute quantification of 143 mouse lysosomal proteins from any type of sample following the QconCAT strategy. We applied our approach to the investigation of mouse embryonic fibroblast whole cell lysates and lysosome-enriched fractions, providing absolute copy numbers per cell/lysosome for lysosomal hydrolases and membrane proteins. These data reveal a dynamic range of more than three orders of magnitude in protein expression and strong differences in the subcellular distribution of individual lysosomal proteins. Furthermore, we applied our strategy to the investigation of four primary cell types (macrophages, lung fibroblasts, osteoblasts, and osteoclasts), demonstrating pathway-specific heterogeneity of individual lysosomal protein classes and indicating protein-specific post-transcriptional regulation of expression levels.

molecular biology↗