bims-lycede 2025-10-19 papers

bims-lycede

Biomed News

on Lysosome-dependent cell death

Issue of 2025–10–19
eight papers selected by
Sofía Peralta, Universidad Nacional de Cuyo

Proteomics Insights Into Lysosome Biogenesis and Maturation.
Visualization of lysosomal membrane proteins by cryo electron tomography.
Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.
Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.
Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
Assessment of Lysosome Trafficking in iPSC-Derived Neurons Utilizing Time-Lapse Videos and TrackMate ImageJ Plugin Software.
Protocol for monitoring the stability of transcription factor EB using global protein stability assay.
Lysosomal pH Measurements via Dual-Imaging Microscopy.

Proteomics. 2025 Oct 15. e70058

Proteomics Insights Into Lysosome Biogenesis and Maturation.

Katharina Hirn, Sofía Fajardo-Callejón, Dominic Winter.

  Lysosomes constitute the main degradative organelle of most eukaryotic cells and are capable of breaking down a wide spectrum of biomolecules, including proteins, lipids, glycans, and DNA/RNA. They play crucial roles in the regulation of cellular homeostasis, acting as metabolic signaling centers for the correlation of nutrient availability and biosynthetic processes. The lysosome's importance is highlighted by several human diseases associated with its dysfunction, including both early- and late-onset conditions, dependent on the level of functional impairment. Lysosomal biogenesis presents a multi-step process consisting of various delivery routes for its individual constituents, enabling strict activity control of the currently known ∼60 lysosomal hydrolases to prevent cellular self-digestion and proper assembly of the lysosomal membrane. In this review, we recapitulate the contribution of mass spectrometry (MS)-based proteomics to the characterization of lysosomal biogenesis in the last two decades. The enrichment and proteomic analysis of lysosomes and lysosomal proteins played an invaluable role for the investigation of lysosomes, encompassing the control of lysosomal gene expression, the characterization of sorting/trafficking processes, and the assignment of lysosomal proteins. This has resulted so far in the definition of ∼350 proteins which have been identified to be located in/at lysosomes or are of crucial importance for their function.

Keywords:  biogenesis; lysosomal proteins; lysosome; mass spectrometry; maturation; protein trafficking; proteomics; vesicular transport

DOI:  https://doi.org/10.1002/pmic.70058
Nat Commun. 2025 Oct 17. 16(1): 9234

Visualization of lysosomal membrane proteins by cryo electron tomography.

Bridget M McVeigh, José J De Jesús-Pérez, Dirk H Siepe, Prerana Gogoi, Shrawan Kumar Mageswaran, Marian Kalocsay, Elaine M Mihelc, Vera Y Moiseenkova-Bell.

Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.

DOI: https://doi.org/10.1038/s41467-025-64314-0
Methods Mol Biol. 2026 ;2976 189-207

Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.

William D Kim, Robert J Huber.

  Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.

Keywords:  Dictyostelium discoideum; Enzyme; Immunofluorescence; Lysosomes; Model organism; Neurodegeneration; Secretion; Trafficking; Western blotting

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_14
Methods Mol Biol. 2026 ;2976 25-34

Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.

Kirstin O McDonald, Stephanie M Hughes, Indranil Basak.

  Fluorescent molecular probes have frequently been used to monitor lysosomal health, localization, abundance, and movement through the detection of acidic organelles and lysosomal enzyme activity. Flow cytometry technology provides rapid and accurate analysis of single cells (neurons) or particles (lysosomes) in suspension through laser detection. Herein, we describe how to detect lysosomes via LysoTracker™ and Magic Red® Cathepsin Activity assays in iPSC-derived human neuron cultures by flow cytometry.

Keywords:  Flow cytometry; Fluorescent probes; Lysosome acidity; Lysosome function; iPSC-derived neuron cultures

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_3
Methods Mol Biol. 2026 ;2976 1-10

Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.

Etienne Sauvageau, Stephane Lefrancois.

  Lysosomes are responsible for a number of cellular functions, including the degradation of various biological molecules. Soluble enzymes within the lysosomal lumen are required to perform this function. Lysosomal activity can be disrupted in a variety of diseases, and measuring the activity of specific enzymes can be performed. In this chapter, we detail how lysosomal enzyme activity can be measured either in cell lysates or intact cells. This can be used to study fundamental cell biology or the effect of therapeutics targeting lysosomal function.

Keywords:  Batten disease; Fluorogenic substrates; Lysosomal enzyme activity; Lysosomes

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_1
Methods Mol Biol. 2026 ;2976 135-149

Assessment of Lysosome Trafficking in iPSC-Derived Neurons Utilizing Time-Lapse Videos and TrackMate ImageJ Plugin Software.

Oluwatobi N Eboda, Indranil Basak, Stephanie M Hughes.

  In order to maintain cell homeostasis, lysosomes must be trafficked throughout the cell and associated with different compartments for the delivery and breakdown of macromolecules. This process is especially important in polar cells, such as neurons, that require the trafficking of lysosomes to distal locations to perform their functions. Here, we employed LysoTracker DND-99, a live-cell fluorometric dye that stains acidic organelles, to label lysosomes prior to time-lapse imaging on a Nikon A1 confocal microscope and subsequent assessment of total distance travelled using the TrackMate ImageJ plugin.

Keywords:  ImageJ; LysoTracker; Lysosome trafficking; TrackMate

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_11
STAR Protoc. 2025 Oct 11. pii: S2666-1667(25)00547-7. [Epub ahead of print]6(4): 104141

Protocol for monitoring the stability of transcription factor EB using global protein stability assay.

Boju Wang, Yueling Zhao, Ruiyan Li, Xinying Li, Cheng Dong, Wenyi Mi, Xiaolu Wang.

  Transcription factor EB (TFEB), the master regulator of autophagy-lysosomal networks, undergoes multiple regulatory mechanisms to maintain proteostasis. Here, we present a protocol for quantifying TFEB stability following loss of function of its regulator (YEATS domain-containing protein 2 [YEATS2] knockdown), utilizing a dual-fluorescent reporter system based on flow cytometry. We describe strategies for vector construction, cell transfection, and fluorescence detection. We then detail procedures for analytical workflows. This protocol is designed to enhance sensitivity and reduce processing duration. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Keywords:  Cell-based Assays; Flow Cytometry; High Throughput Screening

DOI:  https://doi.org/10.1016/j.xpro.2025.104141
Methods Mol Biol. 2026 ;2976 11-24

Lysosomal pH Measurements via Dual-Imaging Microscopy.

Emily Kirkham, Sian Gardiner, Emyr Lloyd-Evans, Hannah Best, Helen Waller-Evans.

  Dual-wavelength ratiometric fluorescent probes are the current gold standard for generating a quantitative measure of subcellular compartment ion contents. This chapter describes the use of fluorescent probes to measure lysosomal pH, encompassing probe selection, calibration, instrumentation, and the miniaturization of assays to use for high-throughput readouts.

Keywords:  Dual-wavelength ratio imaging; Fluorescein isothiocyanate; Fluorescent microscopy; High-throughput; LysoSensor; Lysosome; pH

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_2