OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP‐mediated microtubule anchoring

Wang, Biao; He, Wei; Prosseda, Philipp P.; Li, Liang; Kowal, Tia J.; Alvarado, Jorge A.; Wang, Qing; Hu, Yang; Sun, Yang

doi:10.15252/embr.202052173

Cited by 13 publications

(14 citation statements)

References 41 publications

(82 reference statements)

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“…We next applied the RAP uptake assay to an ocrl mutant zebrafish line to examine whether neuroepithelial RAP endocytosis and trafficking is impaired in the absence of Ocrl. Ocrl is a inositol-5-phosphatase that acts on pools of PI(4,5)P 2 coupled to membrane remodelling events at multiple stages of the endocytic pathway, which includes clathrin-mediated endocytosis (Choudhury et al, 2009; Erdmann et al, 2007; Nandez et al, 2014), receptor recycling from endosomes (Choudhury et al, 2005; van Rahden et al, 2012; Vicinanza et al, 2011) and lysosome fusion (De Leo et al, 2016; Wang et al, 2021; Zhang et al, 1998). Mutations in OCRL are responsible for Lowe syndrome and Dent 2 disease, X-linked multi-systemic disorders that predominantly affect the eyes, kidney and central nervous system (Bokenkamp and Ludwig, 2016).…”

Section: Resultsmentioning

confidence: 99%

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Williams

Güngördü

Jackson-Crawford

et al. 2022

Preprint

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Endocytosis is a vital process, required during development and for maintenance of tissue homeostasis, that allows cells to internalize a wide range of molecules from their environment as well maintain their plasma membrane composition. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualize endocytosis in the neuroepithelium of living zebrafish embryos. These assays rely on injection of fluorescent tracers into the brain ventricles followed by live imaging and can be used to study fluid-phase or receptor-mediated endocytosis, for which we use receptor-associated protein (RAP) as a ligand for LDL receptor-related protein (LRP) receptors expressed at the neuroepithelium. Using dual colour imaging combined with transient or stable expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also find that the RAP binding receptor Lrp2 appears to only partially contribute to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo, and support a role for Ocrl in this process.

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Section: Resultsmentioning

confidence: 99%

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Williams

Güngördü

Jackson-Crawford

et al. 2022

Preprint

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“…Moreover, the reduction in insulin signaling evidenced in our experiments at the cellular level, specifically in conditions of reduction of OCRL1 function, not only would reduce megalin recycling but also would potentially decrease megalin endocytosis as pAKT is required for the efficient megalin-mediated endocytosis of albumin, a physiologically relevant ligand of the receptor present in the proximal tubule ( Silva-Aguiar et al, 2022 ). Regarding AKT activity, it has been recently found that the mTORC1 complex is inactivated in OCRL1 deficient cells ( Madhivanan et al, 2020 ), a defect that triggers a lack of nutrient-sensing ( Wang et al, 2021 ) due to mTORC1 is required for proper insulin signaling ( Saltiel and Kahn, 2001 ). Insulin signaling is also highly dependent on the cell type, something we observed in our experiments, and is associated with insulin receptor trafficking ( Iraburu et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Participation of OCRL1, and APPL1, in the expression, proteolysis, phosphorylation and endosomal trafficking of megalin: Implications for Lowe Syndrome

Sandoval

Fuentealba²,

Marzolo³

2022

Front. Cell Dev. Biol.

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Megalin/LRP2 is the primary multiligand receptor for the re-absorption of low molecular weight proteins in the proximal renal tubule. Its function is significantly dependent on its endosomal trafficking. Megalin recycling from endosomal compartments is altered in an X-linked disease called Lowe Syndrome (LS), caused by mutations in the gene encoding for the phosphatidylinositol 5-phosphatase OCRL1. LS patients show increased low-molecular-weight proteins with reduced levels of megalin ectodomain in the urine and accumulation of the receptor in endosomal compartments of the proximal tubule cells. To gain insight into the deregulation of megalin in the LS condition, we silenced OCRL1 in different cell lines to evaluate megalin expression finding that it is post-transcriptionally regulated. As an indication of megalin proteolysis, we detect the ectodomain of the receptor in the culture media. Remarkably, in OCRL1 silenced cells, megalin ectodomain secretion appeared significantly reduced, according to the observation in the urine of LS patients. Besides, the silencing of APPL1, a Rab5 effector associated with OCRL1 in endocytic vesicles, also reduced the presence of megalin’s ectodomain in the culture media. In both silencing conditions, megalin cell surface levels were significantly decreased. Considering that GSK3ß-mediated megalin phosphorylation reduces receptor recycling, we determined that the endosomal distribution of megalin depends on its phosphorylation status and OCRL1 function. As a physiologic regulator of GSK3ß, we focused on insulin signaling that reduces kinase activity. Accordingly, megalin phosphorylation was significantly reduced by insulin in wild-type cells. Moreover, even though in cells with low activity of OCRL1 the insulin response was reduced, the phosphorylation of megalin was significantly decreased and the receptor at the cell surface increased, suggesting a protective role of insulin in a LS cellular model.

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“…It is interesting to note that loss of Lrp2 does not affect neural development in zebrafish, whereas in mammals it causes forebrain malformation, due to defects in SHH endocytosis and morphogen gradient formation (Christ et al, 2012;Kur et al, 2011). This difference may reflect different requirements for SHH signalling between mammals and teleosts, or perhaps differential requirements for LRP family receptors in endocytosis and developmental signalling between the two classes of vertebrates (Willnow and Christ, 2017). Uncovering the complement of receptors that mediate zebrafish neuroepithelial RAP endocytosis may therefore provide further insights into whether and how LRP family members contribute to neural development in zebrafish and possibly in mammals too (Christ et al, 2012;Kur et al, 2011).…”

Section: Journal Of Cell Science • Accepted Manuscriptmentioning

confidence: 99%

“…For genotyping of ocrl mutant adult zebrafish a PCR fragment spanning the SpeI restriction site in the ocrl gene was amplified from genomic DNA prepared from adult fish as previously described (Wilkinson et al, 2013). The resulting 720 bp fragment was digested using SpeI restriction enzyme (New England Biolabs) and the digested product resolved by gel electrophoresis.…”

Section: Genotyping Of Ocrl Mutant Adult Zebrafishmentioning

confidence: 99%

“…Sensing of signals that influence neuroepithelial cell behaviour is in part mediated by receptors present at the apical surface, with cell surface receptor levels often maintained by receptor recycling from endosomes. As an example, apical enrichment of the recycling receptor LRP2 (Nagai et al, 2003) in mammalian neuroepithelial cells shapes tissue patterning within the forebrain through endocytosis of the secreted morphogen Sonic Hedgehog (SHH) with the absence of LRP2 leading to an impairment of SHH signalling during early neurogenesis in mice (Christ et al, 2012;Willnow and Christ, 2017). Bound ligands internalised into endosomes from the neuroepithelial cell surface can also influence neuroepithelial cell behaviour.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Williams

Güngördü

Jackson-Crawford

et al. 2022

Journal of Cell Science

View full text Add to dashboard Cite

Endocytosis allows cells to internalize a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualize endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we use receptor-associated protein (RAP) as a ligand for LDL receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also find that the RAP binding receptor Lrp2 appears to only partially contribute to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo, and support a role for Ocrl in this process.

show abstract

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP‐mediated microtubule anchoring

Cited by 13 publications

References 41 publications

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Participation of OCRL1, and APPL1, in the expression, proteolysis, phosphorylation and endosomal trafficking of megalin: Implications for Lowe Syndrome

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

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