5 research outputs found
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ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic
Funder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: Gates Cambridge Trust; doi: http://dx.doi.org/10.13039/501100005370Abstract: Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER–endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic
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Characterising the roles of mammalian atlastins in the endoplasmic reticulum and beyond
Hereditary spastic paraplegia (HSP) is a group of currently incurable disorders caused by the degeneration of corticospinal upper motor neuron axons, which are among the longest in the body. Recent research suggests that a few unifying pathways could link many genetically distinct HSP subtypes. Investigating these pathways and the pathological mechanisms affecting them is crucial to advance the development of effective treatments for HSP and related neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease, multiple sclerosis, peripheral neuropathies and various motor neuron diseases.
This project focuses on endoplasmic reticulum (ER) proteins atlastins (ATLs), particularly ATL1, as *ATL1* gene mutations cause the most frequent childhood-onset form of HSP. Using CRISPRi, I engineered ATL1 knock-down (KD) into the i3 human induced pluripotent stem cells (iPSCs) and utilised iPSC-derived neurons to investigate the roles of ATL1 in the ER and other cellular compartments and processes commonly affected in HSP. Preliminary findings showed fewer ER junctions in ATL1 KD neurons, consistent with known atlastin’s role in ER tubule fusion. Additionally, ER proteins were highly enriched in a protein list with altered abundance in ATL1 KD neurons determined through proteomics. Moreover, abnormalities in the endo-lysosomal system were identified in ATL1-depleted neurons. They had slightly fewer and larger lysosomes with reduced degradative capacity. Additionally, neuronal endosomal tubules were elongated, pointing towards a potential tubule fission defect which could lead to missorting of diverse cargo, such as lysosomal hydrolases. Lipidomics and neutral lipid staining revealed higher triglyceride content in neurons lacking ATL1. Moreover, blocking lipolysis degradation led to lipid droplet (LD) accumulation, with significantly more LDs in ATL1 KD neurons. The cellular content of other lipid classes was also altered, indicating potential lipid imbalance resulting from ATL1 loss. Finally, developing ATL1-depleted neurons exhibited altered morphology, with a significantly longer dominant neurite. Despite the detected phenotypes, the lack of ATL1 had no effect on neuronal viability.
These observations demonstrate that atlastin deficiency impacts cellular processes beyond its conventional role in ER shaping. In addition, the findings and tools developed in this study provide a foundation to investigate underlying mechanisms and pathways connecting the phenotypes resulting from ATL1 deficiency.Gates Cambridge
Medical Research Council (MRC)
Cambridge Institute for Medical Researc
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ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic
Funder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: Gates Cambridge Trust; doi: http://dx.doi.org/10.13039/501100005370Abstract: Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER–endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic
Whole-genome sequencing of patients with rare diseases in a national health system
Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare
Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort (Nature, (2020), 583, 7814, (90-95), 10.1038/s41586-020-2265-1)
An amendment to this paper has been published and can be accessed via a link at the top of the paper