Biology subjects

Iwatsubo, T.

Publications and source records attributed to Iwatsubo, T..

7 recordsLinked to original sources

bioRxiv2026

STING activation drives pericentrosomal positioning of acidic organelles via the VAIL-LRRK2-Rab35 pathway

The cGAS-STING pathway is an innate immune system that responds to cytoplasmic double-stranded DNA, but recent studies have highlighted its noncanonical role in facilitating lysosomal stress responses. Specifically, STING activation has been shown to trigger V-ATPase-ATG16L1-induced LC3 lipidation (VAIL) and to activate LRRK2, a Parkinson disease-associated kinase, but the detailed molecular mechanism and the physiological roles of the activation have remained unclear. Here, we found that STING activation in microglia induces the translocation of LRRK2 to the pericentrosomal area, where lysosomes and recycling endosomes (REs) concentrate. This process was VAIL-dependent, and LRRK2 was found to be localized to the organelles. The translocation of LRRK2 required intact microtubule structures and was mediated by LRRK2 kinase activity, its substrate Rab35 and the motor adaptor proteins including JIP4. All these proteins were concentrated at the pericentrosomal area upon STING stimulation. The hyperactivating G2019S mutation of LRRK2 promoted the pericentrosomal translocation of these proteins. Overall, we propose a novel STING-induced noncanonical pathway that involves LRRK2 to facilitate pericentrosomal organelle positioning.

cell biology↗

bioRxiv2026

Subtype-specific secretion of extracellular vesicles by LRRK2 and Rab GTPases under lysosomal stress

LRRK2, the Parkinsons disease-associated kinase, phosphorylates a subset of Rab GTPases and regulates membrane dynamics. We previously reported that lysosomal stress activates LRRK2 and thereby induces the exocytic secretion of lysosomal contents, but the detailed secretion mechanism remained unclear. Here we found that, under lysosomal stress, endolysosomal luminal and membrane components were secreted with extracellular vesicles (EVs) via LRRK2. Bis(monoacylglycerol)phosphate, an endolysosomal lipid and a urinary marker of LRRK2 activity, was similarly secreted via LRRK2, whereas CD9-positive EVs were not involved. Further dissection of the secreted EVs revealed that Alix-positive EVs were secreted via Rab8a as well as the ESCRT component VPS4, whereas LAMP1/cathepsin B-positive EVs were secreted via Rab10/Rab35, and SNARE proteins syntaxin 2 and VAMP8 regulated the secretion of both EV subtypes. These findings suggest a distinctive stress-induced secretory mechanism whereby LRRK2 facilitates the secretion of multiple EV subtypes by controlling Rab GTPases involved in each pathway.

cell biology↗

bioRxiv2025

LRRK2 is activated by phosphatidylinositol 3-phosphate in conjunction with CASM

LRRK2 is a Parkinsons disease (PD)-associated kinase that phosphorylates Rab GTPases. Hyperactivation of LRRK2, which is thought to cause PD, occurs on stressed endolysosomal membranes via CASM (conjugation of ATG8 to single membranes) and Rab, whereas the involvement of specific membrane lipids remained unclear. Here, we found that LRRK2 was potently activated upon treatment with PIKfyve inhibitors that increase cellular phosphatidylinositol 3-phosphate (PI3P) levels, and that additional treatment with the class III phosphoinositide 3-kinase (PI3K) inhibitors blocking PI3P generation suppressed LRRK2 activation. Cellular PI3P levels were indeed correlated with the LRRK2 kinase activity. CASM-induced LRRK2 activation, but not CASM itself, was suppressed by PI3K inhibitors, whereas inhibition of CASM or Rab12/Rab29 suppressed LRRK2 activation induced by PIKfyve inhibitors. LRRK2 and phospho-Rab10 were co-localized with PI3P on LAMP1-positive enlarged vacuoles, and LRRK2 directly bound PI3P in vitro via its N-terminal positively charged amino acids. Together, we propose an updated mechanism of LRRK2 activation that requires CASM, Rab and PI3P-rich membranes.

cell biology↗

bioRxiv2023

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate lysosomal stress responses

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinsons disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

molecular biology↗

bioRxiv2022

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinsons disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKC was deemed to be responsible for this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKC in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

molecular biology↗

bioRxiv2021

Compositional analysis of ALS-linked stress granule-like structures reveals factors and cellular pathways dysregulated by mutant FUS under stress

Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, global structural differences between mFAs and physiological SGs remain largely unknown, therefore it is unclear whether and how mFAs may affect cellular stress responses. Here we used affinity purification to characterise the protein and RNA composition of normal SGs and mFAs purified from stressed cells. Comparison of the SG and mFA proteomes revealed that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a hit from our analysis, a splicing factor hnRNPA3, confirmed its RNA-dependent sequestration into mFAs in cells and into pathological FUS inclusions in a FUS transgenic mouse model. Furthermore, silencing of the Drosophila hnRNPA3 ortholog dramatically enhanced FUS toxicity in transgenic flies. Comparative transcriptomic analysis of SGs and mFAs revealed that mFAs recruit a significantly less diverse spectrum of RNAs, including reduced recruitment of transcripts encoding proteins involved in protein translation, DNA damage response, and apoptotic signalling. However mFAs abnormally sequester certain mRNAs encoding proteins involved in stress signalling cascades. Overall, our study establishes molecular differences between physiological SGs and mFAs and identifies the spectrum of proteins, RNAs and respective cellular pathways affected by mFAs in stressed cells. In conclusion, we show that mFAs are compositionally distinct from SGs and that they cannot fully substitute for SG functions while gaining novel, potentially toxic functions in cellular stress response. Results of our study support a pathogenic role for stress-induced cytoplasmic FUS assemblies in ALS-FUS.

cell biology↗

bioRxiv2020

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinsons disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

molecular biology↗