bims-lypmec 2024-03-10 papers

Mol Biol Cell. 2024 Mar 06. mbcE23080322

Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Ioana Sava, Luther J Davis, Sally R Gray, Nicholas A Bright, J Paul Luzio.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/re-feeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E23-08-0322

Mol Cell. 2024 Feb 28. pii: S1097-2765(24)00125-4. [Epub ahead of print]

Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.

Kwamina Nyame, Andy Hims, Aya Aburous, Nouf N Laqtom, Wentao Dong, Uche N Medoh, Julia C Heiby, Jian Xiong, Alessandro Ori, Monther Abu-Remaileh.

Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.

Keywords: Batten disease; CLN3; GPDs; PLA2G15; PLBD2; glycerophosphodiesters; lysosomal storage disease; lysosome; neurodegeneration; phospholipase; phospholipid metabolism

DOI: https://doi.org/10.1016/j.molcel.2024.02.006

Anal Chim Acta. 2024 Apr 08. pii: S0003-2670(24)00104-1. [Epub ahead of print]1297 342303

A novel ratiometric fluorescent probe with high selectivity for lysosomal nitric oxide imaging.

Zhiling Xu, Songtao Liu, Liren Xu, Zichun Li, Xiaoli Zhang, Hao Kang, Yifan Liu, Jin Yu, Jing Jing, Guangle Niu, Xiaoling Zhang.

Nitric oxide (NO) plays critical roles in both physiology and pathology, serving as a significant signaling molecule. Recent investigations have uncovered the pivotal role of lysosome as a critical organelle where intracellular NO exists and takes function. In this study, we developed a novel ratiometric fluorescent probe called XL-NO and modified it with a morpholine unit, which followed the intramolecular charge transfer (ICT) mechanism. The probe could detect lysosomal nitric oxide with high selectivity and sensitivity. The probe XL-NO contained a secondary amine moiety that could readily react with NO in lysosomes, leading to the formation of the N-nitrosation product. The N-nitroso structure enhanced the capability in push-pull electron, which obviously led to the change of fluorescence from 621 nm to 521 nm. In addition, XL-NO was discovered to have some evident advantages, such as significant ratiometric signal (I521/I621) change, strong anti-interference ability, good biocompatibility, and a low detection limit (LOD = 44.3 nM), which were crucial for the detection of lysosomal NO. To evaluate the practical application of XL-NO, NO imaging experiments were performed in both living cells and zebrafish. The results from these experiments confirmed the feasibility and reliability of XL-NO for exogenous/endogenous NO imaging and lysosome targeting.

Keywords: Biological imaging; Lysosomes-targeted; Nitric oxide; Ratiometric fluorescent probe; Zebrafish

DOI: https://doi.org/10.1016/j.aca.2024.342303

Nat Commun. 2024 Mar 02. 15(1): 1948

A structural and dynamic visualization of the interaction between MAP7 and microtubules.

Agnes Adler, Mamata Bangera, J Wouter Beugelink, Salima Bahri, Hugo van Ingen, Carolyn A Moores, Marc Baldus.

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex.

DOI: https://doi.org/10.1038/s41467-024-46260-5

Nat Commun. 2024 Mar 06. 15(1): 2029

Microtubule damage shapes the acetylation gradient.

Mireia Andreu-Carbó, Cornelia Egoldt, Marie-Claire Velluz, Charlotte Aumeier.

The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.

DOI: https://doi.org/10.1038/s41467-024-46379-5