bims-lypmec Biomed News
on Lysosomal positioning and metabolism in cardiomyocytes
Issue of 2023‒04‒23
seven papers selected by
Satoru Kobayashi
New York Institute of Technology
  1. Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.
  2. Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.
  3. Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis.
  4. An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.
  5. Rab GTPase-regulated phagosome-lysosome fusion is bypassed by micromolar calcium.
  6. Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1.
  7. The Lure of Cardiac Metabolism in the Diagnosis, Prevention, and Treatment of Heart Failure.
  1. FASEB J. 2023 May;37(5): e22930
    Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.
    Kyohei Ito, Miho Araki, Yuta Katai, Yuki Nishimura, Sota Imotani, Haruki Inoue, Genta Ito, Taisuke Tomita.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). Recent studies have shown that LRRK2 physiologically phosphorylates several Rab family proteins including Rab12 and that this phosphorylation is accelerated by the pathogenic mutations in LRRK2, although the significance in the PD pathogenesis remains unknown. Here we examined the effect of the overexpression of LRRK2 on the distribution of organelles in cultured cells and found that lysosomes become clustered in a perinuclear region upon the overexpression of pathogenic mutant LRRK2 in a manner dependent on its kinase activity. The perinuclear clustering of lysosomes was abolished by knocking out RAB12 as well as its effector protein RILPL1. Re-expression of Rab12 in RAB12 knockout cells suggested that the phosphorylation at Ser106 of Rab12 is required for the perinuclear clustering of lysosomes. Moreover, phosphorylated Rab12 was also accumulated on the clustered lysosomes, and the phosphorylation of Rab12 increased its interaction with RILPL1, leading us to conclude that the increase in the phosphorylation of Rab12 by pathogenic LRRK2 compromised intracellular lysosomal transport via the enhanced interaction of Rab12 with RILPL1. These data suggest the involvement of abnormal regulation of lysosomal transport in the LRRK2-mediated pathogenesis of PD.
    Keywords:  Rab-interacting lysosomal protein-like 1; Rab12; leucine-rich repeat kinase 2; lysosome
    DOI:  https://doi.org/10.1096/fj.202200780RR
  2. STAR Protoc. 2023 Apr 18. pii: S2666-1667(23)00194-6. [Epub ahead of print]4(2): 102236
    Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.
    Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.
      Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes. For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1.
    Keywords:  Cell Biology; Cell Culture; High-throughput Screening; Microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2023.102236
  3. Methods Mol Biol. 2023 ;2641 171-178
    Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis.
    Wendy P Loomis, Tessa Bergsbaken.
      Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1β and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis. This chapter outlines methods for measuring calcium flux, lysosome exocytosis, and membrane disruption after inflammatory caspase activation.
    Keywords:  Calcium; Gasdermin D; Inflammation; Inflammatory caspases; Lysosome exocytosis; Pyroptosis
    DOI:  https://doi.org/10.1007/978-1-0716-3040-2_14
  4. Sci Adv. 2023 04 21. 9(16): eadf8966
    An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.
    Samantha G Scharenberg, Wentao Dong, Ali Ghoochani, Kwamina Nyame, Roni Levin-Konigsberg, Aswini R Krishnan, Eshaan S Rawat, Kaitlyn Spees, Michael C Bassik, Monther Abu-Remaileh.
      Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.
    DOI:  https://doi.org/10.1126/sciadv.adf8966
  5. J Cell Sci. 2023 Apr 19. pii: jcs.260806. [Epub ahead of print]
    Rab GTPase-regulated phagosome-lysosome fusion is bypassed by micromolar calcium.
    Julia Becker, Ariane Schleinitz, Christina Hermsen, Sabrina Rappold, Paul Saftig, Andreas Jeschke, Albert Haas.
      Several ATP- and cytosol-dependent fusion processes between membranes of the endocytic and exocytic pathways have been biochemically reconstituted. We here present a phagosome-lysosome-fusion reaction driven by micromolar Ca2+ in the absence of ATP and cytosol. Investigating classical and Ca2+-driven fusion (CaFu) side-by-side in vitro, using the same membrane preparations, we show that CaFu is faster than standard fusion (StaFu), leads to larger fusion products, and is not blocked by established inhibitors of StaFu. Approximately 120 µM Ca2+ supports maximal membrane attachment and 15 µM Ca2+ maximal membrane fusion, assigning Ca2+ both a membrane binding and a fusion-promoting activity. StaFu and CaFu are inhibited by a mutant form α-SNAP that does not support SNARE (SNAP receptor) activation, and both are inhibited by a mixture of the cytosolic domains of three cognate Q-SNARE proteins, demonstrating a role of SNAREs in Ca2+-driven membrane merger. CaFu is independent of the calcium-regulated proteins synaptotagmin-7, calmodulin, and annexins A2 and A7. We propose that CaFu corresponds to the last step of phagosome-lysosome fusion, when a raised Ca2+ concentration from the compartment lumen activates SNAREs for fusion.
    Keywords:  Calcium; Lysosome; Membrane fusion; Pathogen; Phagosome; Reconstitution
    DOI:  https://doi.org/10.1242/jcs.260806
  6. Science. 2023 Apr 21. 380(6642): eabj5559
    Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1.
    Nazma Malik, Bibiana I Ferreira, Pablo E Hollstein, Stephanie D Curtis, Elijah Trefts, Sammy Weiser Novak, Jingting Yu, Rebecca Gilson, Kristina Hellberg, Lingjing Fang, Arlo Sheridan, Nasun Hah, Gerald S Shadel, Uri Manor, Reuben J Shaw.
      Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate-activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)-FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1-dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis.
    DOI:  https://doi.org/10.1126/science.abj5559
  7. JACC Heart Fail. 2023 Mar 17. pii: S2213-1779(23)00091-4. [Epub ahead of print]
    The Lure of Cardiac Metabolism in the Diagnosis, Prevention, and Treatment of Heart Failure.
    Daniele Rodolico, Gabriele G Schiattarella, Heinrich Taegtmeyer.
      Energy substrate metabolism and contractile function are tightly coupled in the heart. Within this framework, heart failure may be viewed as a state of impaired energy transfer. The metabolic changes in the failing heart are linked to functional and structural changes. A worthwhile goal is to measure metabolic flux and its regulation quantitatively, and to do this in a manner that leads to targeted interventions. For several good reasons, this goal has been elusive until now. The development of new analytical and imaging techniques offers the potential of exploring the landscape of metabolic changes across the different stages of heart failure. In this Review Topic of the Month, we focus on concepts and brevity to provide a strategic overview of cardiac metabolism in the diagnosis, prevention, and treatment of nonischemic heart failure.
    Keywords:  cardiac remodeling; flux-based analysis; imaging; metabolic unloading
    DOI:  https://doi.org/10.1016/j.jchf.2023.02.007
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