The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamics
2022; Elsevier BV; Volume: 298; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2022.102320
ISSN1083-351X
AutoresVincent Francis, Walaa Alshafie, Rahul Kumar, Martine Girard, Bernard Brais, Peter S. McPherson,
Tópico(s)Microtubule and mitosis dynamics
ResumoAutosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes. Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes. Correction: The ARSACS disease protein sacsin controls lysosomal positioning and reformation by regulating microtubule dynamicsJournal of Biological ChemistryVol. 298Issue 12PreviewThe author list should read as follows: Full-Text PDF Open Access ARSACS (autosomal recessive spastic ataxia of Charlevoix-Saguenay) is a progressive neurodegenerative disorder characterized by loss of cerebellar Purkinje neurons. Initially identified in the Charlevoix and Saguenay regions of Quebec (1Bouchard J.P. Barbeau A. Bouchard R. Bouchard R.W. Autosomal recessive spastic ataxia of Charlevoix-Saguenay.Can. J. Neurol. Sci. 1978; 5: 61-69Crossref PubMed Scopus (222) Google Scholar, 2Engert J.C. Bérubé P. Mercier J. Doré C. Lepage P. Ge B. et al.ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF.Nat. Genet. 2000; 24: 120-125Crossref PubMed Scopus (354) Google Scholar), the disease is now recognized worldwide and is the second most common recessive form of ataxia (3Vermeer S. Meijer R.P. Pijl B.J. Timmermans J. Cruysberg J.R. Bos M.M. et al.ARSACS in the Dutch population: a frequent cause of early-onset cerebellar ataxia.Neurogenetics. 2008; 9: 207-214Crossref PubMed Scopus (109) Google Scholar). Clinical features vary depending on the patient population but in the Quebec population, ARSACS features spasticity, ataxia, polyneuropathy, and retinal thickening (1Bouchard J.P. Barbeau A. Bouchard R. Bouchard R.W. Autosomal recessive spastic ataxia of Charlevoix-Saguenay.Can. J. Neurol. Sci. 1978; 5: 61-69Crossref PubMed Scopus (222) Google Scholar). Patients display an unsteady gait, become wheelchair bound at an average age of 41, and have a reduced life expectancy. The SACS gene, mutations in which are responsible for ARSACS, encodes a massive 4579 amino acid (521 kDa) protein (2Engert J.C. Bérubé P. Mercier J. Doré C. Lepage P. Ge B. et al.ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF.Nat. Genet. 2000; 24: 120-125Crossref PubMed Scopus (354) Google Scholar). Since the initial discovery of the founder mutation (c.8844 delT) in French-Canadian ARSACS patients, approximately 200 mutations have been identified spanning the full length of the protein (4Synofzik M. Soehn A.S. Gburek-Augustat J. Schicks J. Karle K.N. Schüle R. et al.Autosomal recessive spastic ataxia of charlevoix saguenay (ARSACS): expanding the genetic, clinical and imaging spectrum.Orphanet J. Rare Dis. 2013; 8: 41Crossref PubMed Scopus (127) Google Scholar). Sacsin is a multi-modular protein consisting of an ubiquitin-like domain, which binds to the proteasome (5Parfitt D.A. Michael G.J. Vermeulen E.G. Prodromou N.V. Webb T.R. Gallo J.M. et al.The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.Hum. Mol. Genet. 2009; 18: 1556-1565Crossref PubMed Scopus (130) Google Scholar), three large sacsin repeat regions suggested to have Hsp90-like chaperone function (6Anderson J.F. Siller E. Barral J.M. The sacsin repeating region (SRR): a novel Hsp90-related supra-domain associated with neurodegeneration.J. Mol. Biol. 2010; 400: 665-674Crossref PubMed Scopus (51) Google Scholar), a potential XPCB domain that binds the Ube3A ubiquitin protein ligase (7Anderson J.F. Siller E. Barral J.M. The neurodegenerative-disease-related protein sacsin is a molecular chaperone.J. Mol. Biol. 2011; 411: 870-880Crossref PubMed Scopus (51) Google Scholar), a DnaJ domain that interacts with Hsc70 (5Parfitt D.A. Michael G.J. Vermeulen E.G. Prodromou N.V. Webb T.R. Gallo J.M. et al.The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.Hum. Mol. Genet. 2009; 18: 1556-1565Crossref PubMed Scopus (130) Google Scholar), and a HEPN domain mediating sacsin dimerization (8Kozlov G. Denisov A.Y. Girard M. Dicaire M.J. Hamlin J. McPherson P.S. et al.Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).J. Biol. Chem. 2011; 286: 20407-20412Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This domain organization suggests a functional role for the protein in proteostasis. However, the cellular function of sacsin remains largely unknown and there are no therapies available for ARSACS. We previously demonstrated that in primary neurons and nonneuronal cell lines, depletion of sacsin with inhibitory RNA results in a hyperfused mitochondrial network (9Girard M. Larivière R. Parfitt D.A. Deane E.C. Gaudet R. Nossova N. et al.Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 1661-1666Crossref PubMed Scopus (142) Google Scholar). Moreover, mitochondria accumulate in neuronal cell bodies suggesting a mitochondrial transport defect that could result from the altered mitochondrial network or from a primary defect in organelle transport (9Girard M. Larivière R. Parfitt D.A. Deane E.C. Gaudet R. Nossova N. et al.Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 1661-1666Crossref PubMed Scopus (142) Google Scholar). A subsequent study reported decreased mitochondrial motility in the axons of Sacs−/− motor neurons, arguing for a primary transport defect (10Larivière R. Gaudet R. Gentil B.J. Girard M. Conte T.C. Minotti S. et al.Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.Hum. Mol. Genet. 2015; 24: 727-739Crossref PubMed Scopus (61) Google Scholar). This study also reported an accumulation of abnormal, nonphosphorylated neurofilaments in the somatodendritic region of various neuronal population in Sacs−/− mice (10Larivière R. Gaudet R. Gentil B.J. Girard M. Conte T.C. Minotti S. et al.Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.Hum. Mol. Genet. 2015; 24: 727-739Crossref PubMed Scopus (61) Google Scholar). In fibroblasts from ARSACS patients, intermediate filaments are present as collapsed bundles close to the microtubule (MT) organizing center (11Duncan E.J. Larivière R. Bradshaw T.Y. Longo F. Sgarioto N. Hayes M.J. et al.Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin.Hum. Mol. Genet. 2017; 26: 3130-3143PubMed Google Scholar). These cellular phenotypes are consistent with a functional role for sacsin in regulating cytoskeleton dynamics. To further characterize the physiological function of sacsin and to test the hypothesis that sacsin regulates organelle positioning by regulating cytoskeletal dynamics, we used sacsin KO cells, primary neuronal cultures from sacs KO mice, and ARSACS patient fibroblasts. Our results reveal that lysosome positioning and motility are disrupted upon loss of sacsin. We demonstrate that sacsin binds to MTs and regulates MT dynamics. We also identify a novel interaction of sacsin with JIP3, an adapter between MTs and organelles, and we provide evidence that this interaction is crucial in controlling lysosome positioning. The regulation of MT dynamics provides a unifying hypothesis that can explain all known sacsin loss-of-function phenotypes. Reduction of sacsin in neurons using inhibitory RNA results in the accumulation of mitochondria in cell bodies and decreased rates of mitochondrial transport in axons, suggesting an organelle transport function for the protein (9Girard M. Larivière R. Parfitt D.A. Deane E.C. Gaudet R. Nossova N. et al.Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 1661-1666Crossref PubMed Scopus (142) Google Scholar, 10Larivière R. Gaudet R. Gentil B.J. Girard M. Conte T.C. Minotti S. et al.Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.Hum. Mol. Genet. 2015; 24: 727-739Crossref PubMed Scopus (61) Google Scholar). To further test for sacsin involvement in organelle transport or positioning, we generated sacsin KO HeLa cells using CRISPR/Cas9, leading to complete loss of sacsin protein in two independent lines (Fig. 1A). Similar to previous reports, sacsin KO cells display vimentin bundling (11Duncan E.J. Larivière R. Bradshaw T.Y. Longo F. Sgarioto N. Hayes M.J. et al.Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin.Hum. Mol. Genet. 2017; 26: 3130-3143PubMed Google Scholar) (Fig. S1A). We examined for changes in organelle positioning and discovered that the distribution of lysosomes is altered in sacsin KO cells compared to WT cells. Specifically, in KO cells under steady-state conditions, lysosomes stained with LAMP1 display a peripheral distribution with little juxtanuclear accumulation, whereas in WT cells, lysosomes have a more typical juxtanuclear pattern with seemingly less peripheral staining (unstarved, Fig. 1B). In contrast, there was no change in the distribution of early endosomes or peroxisomes (Fig. S1B). As expected, mitochondria were hyperfused in KO cells and fibroblasts from ARSACS patients (Fig. S2, A–C). Lysosomes are highly dynamic organelles, sometimes stationary, but often moving between a perinuclear pool and a more distributed peripheral pool (12Ballabio A. Bonifacino J.S. Lysosomes as dynamic regulators of cell and organismal homeostasis.Nat. Rev. Mol. Cell Biol. 2020; 21: 101-118Crossref PubMed Scopus (584) Google Scholar). The positioning of lysosomes controls their activity and contributions to cellular functions including responses to changing nutrient levels, autophagy, antigen presentation, cell adhesion, cell migration, and cancer cell invasion (13Pu J. Guardia C.M. Keren-Kaplan T. Bonifacino J.S. Mechanisms and functions of lysosome positioning.J. Cell Sci. 2016; 129: 4329-4339Crossref PubMed Scopus (305) Google Scholar). For example, starvation induces autophagy and stimulates repositioning of lysosomes from the periphery to the perinuclear pool to facilitate autophagosome/lysosome fusion (14Korolchuk V.I. Saiki S. Lichtenberg M. Siddiqi F.H. Roberts E.A. Imarisio S. et al.Lysosomal positioning coordinates cellular nutrient responses.Nat. Cell Biol. 2011; 13: 453-460Crossref PubMed Scopus (635) Google Scholar). We thus starved cells and as expected, in WT cells, starvation repositions lysosomes, which show an even more clustered juxtanuclear distribution than under steady-state conditions (Fig. 1B). In KO cells, lysosomes remain scattered with little evidence of juxtanuclear accumulation (Fig. 1B). To quantify alterations in lysosome positioning resulting from sacsin KO, we developed a lysosome distribution measure. Cells were outlined and concentric rings were drawn at 10% intervals based on the shape of the cell (Fig. 1C). The cumulative LAMP1 intensity was then plotted relative to the whole cell from cell center to periphery (Figs. 1D and S3, A–C). Shifting of the cumulative LAMP1 intensity curve toward the left demonstrates a perinuclear distribution whereas a shift toward the right indicates a peripheral distribution. Sacsin KO lines had a significant shift of lysosomal distribution to the periphery as revealed by rightward shifts in the distribution curves (Fig. 1D). To further validate our quantification assay, we manipulated lysosome distribution by overexpression of GFP-Arl8 (Fig. S3D), which is known to reposition lysosomes to the periphery (15Bagshaw R.D. Callahan J.W. Mahuran D.J. The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes.Biochem. Biophys. Res. Commun. 2006; 344: 1186-1191Crossref PubMed Scopus (75) Google Scholar) and plotted the LAMP1 distribution (Fig. S3E). Overexpression of GFP-Arl8 shifted the curve toward the right demonstrating a more peripheral distribution of lysosomes (Fig. S3E). The source code and the implementation of this algorithm are available as an open source code at GitHub (https://github.com/Vincent-Francis/Quantification-LysosomeDistribution). Since the position of lysosomes within cells helps determine their luminal pH, we sought to measure the acidity levels of lysosomes in WT and sacsin KO cells using LysoSensor DND-189. In acidic organelles, the fluorescence intensity of LysoSensor DND-189 depends on the acidity levels. We observed decreased levels of acidic lysosomes in sacsin KO cells compared to WT cells (Fig. 1, F and G). Since autophagic clearance of degradative cargo by lysosomes is dependent both on lysosome position and acidity, we performed immunofluorescence on WT and sacsin KO mice brains to see if there are any defects in autophagy using LC3, an autophagosome marker. We observed accumulation of LC3 in sacsin KO mice compared to WT controls (Fig. 1E). Since ARSACS is primarily a neuronal disease, we tested to see if lysosome positioning or transport is affected in neurons following loss of sacsin. Sacsin KO mice are a robust model of the disease revealing phenotypes that reflect symptoms seen in human patients (10Larivière R. Gaudet R. Gentil B.J. Girard M. Conte T.C. Minotti S. et al.Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.Hum. Mol. Genet. 2015; 24: 727-739Crossref PubMed Scopus (61) Google Scholar). We generated cultures of cortical neurons from WT and KO mice and stained lysosomes with lysotracker. In WT neurons, lysosomes are concentrated in cell bodies (arrows, Video S1) but are also detected throughout neuronal processes where many are motile. In sacsin KO neurons, the overall distribution of lysosomes between cell bodies and processes is similar to WT (arrows indicate multiple cell bodies in the image), but mobility is strongly reduced with a higher proportion of stationary lysosomes (Video S2). The altered motility in neuronal processes is most obvious when presented in the form of kymographs, revealing that many lysosomes are stationary in the sacsin KO neurons compared to WT neurons (Fig. 2, A and B). In addition, we used validated small inhibitory RNA sequences specific for rat sacsin, prepared in a miRNA backbone (shRNAmiR) and packaged into lentivirus to knockdown (KD) sacsin in cultured rat cortical neurons, as previously described (9Girard M. Larivière R. Parfitt D.A. Deane E.C. Gaudet R. Nossova N. et al.Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 1661-1666Crossref PubMed Scopus (142) Google Scholar). Reduced lysosomal mobility was also observed in sacsin KD neurons (Video S3 and Fig. 2C) as compared to control shRNAmiR-treated cells (Video S4). To further examine lysosome dynamics, we quantified the anterograde and retrograde trafficking of lysosomes in WT and sacsin KO neurons. To study this, DIV8 WT and sacsin KO neurons were labeled with lysotracker, and live cell imaging was performed. We observed that the number of lysosomes moving in both anterograde and retrograde directions were reduced in both axons and dendrites (Fig. S4, A and B). Together, these data indicate that sacsin plays a role in lysosomal trafficking and positioning in both neurons and nonneuronal cells. To further elucidate the molecular mechanisms involved in sacsin regulation of lysosomal positioning and transport, we performed mass spectrometry analysis of sacsin immunoprecipitates and identified JIP3 (C-jun-amino-terminal kinase-interacting protein 3) as a sacsin-interacting partner. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033823 (Supplementary Data File 1). This is particularly relevant as JIP3 functions as an adapter for MT-dependent organelle transport, including that of lysosomes (16Drerup C.M. Nechiporuk A.V. JNK-interacting protein 3 mediates the retrograde transport of activated c-Jun N-terminal kinase and lysosomes.PLoS Genet. 2013; 9e1003303Crossref PubMed Scopus (95) Google Scholar). The sacsin–JIP3 interaction was further validated through immunoprecipitation experiments. JIP3 belongs to the JIP family of proteins that comprises four isoforms (JIP1–4) (17Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. The JIP group of mitogen-activated protein kinase scaffold proteins.Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (410) Google Scholar). The four JIP isoforms, each with an N-terminal Flag tag, were immunoprecipitated with anti-Flag antibody following transfection in HEK-293 cells. The immunoprecipitates were then immunoblotted with antibodies recognizing Flag and sacsin (Fig. 3A). Endogenous sacsin coimmunoprecipitates with JIP3 but not with the other three JIP isoforms (Fig. 3A). We also detect interaction of sacsin with endogenous JIP3 (Fig. 3B). To further investigate the role of JIP3 in lysosome dynamics, we generated a JIP3 KO HeLa cell line using CRISPR/Cas9, leading to complete loss of protein in two independent KO lines (Fig. 3C). In WT cells, lysosomes are in both peripheral and juxtanuclear pools, and starvation causes a redistribution toward the juxtanuclear pool (Fig. 3D). In JIP3 KO cells, LAMP1-positive lysosomes have a more peripheral distribution and fail to redistribute toward the juxtanuclear area following nutrient deprivation (Fig. 3D), similar to what is seen in sacsin KO cells (Fig. 1). The cumulative LAMP1 intensity was plotted for WT and JIP3 KO cells under conditions of starvation and revealed a similar shift as for the sacsin KO cells (Figs. 1D and 3E). Since lysosomal positioning is important for facilitating the fusion of autophagosomes with lysosomes, the failure of lysosomes to reposition in JIP3 KO cells could impair autophagy. To test if autophagy is altered in JIP3 KO HeLa cells, both WT and KO cells were starved and treated with bafilomycin and LC3 levels were determined. We found that autophagy was impaired in JIP3 KO cells, as reduced levels of LC3-II were observed in JIP3 KO cells when compared to control (Fig. 3, F and G). We also tested the loss of JIP3 on lysosome dynamics in primary rat neuronal cultures. Neurons were transduced with a lentivirus driving JIP3-specific shRNAmiR and efficient KD was confirmed by qPCR (Fig. 2E). Compared to control shRNAmiR-treated neurons (Video S4), JIP3 KD neurons contain seemingly aggregated lysosomes in neuronal processes (Video S5). The control-treated neurons showed bidirectional lysosomal trafficking within neuronal processes, whereas JIP3 KD neurons displayed a significantly higher number of stationary lysosomes (Video S5 and Fig. 2D). These results are similar to those observed with sacsin KD and to an earlier study where disruption of JIP3 led to altered axonal transport of lysosomes and promoted amyloid plaque pathology (18Gowrishankar S. Wu Y. Ferguson S.M. Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology.J. Cell Biol. 2017; 216: 3291-3305Crossref PubMed Scopus (74) Google Scholar). Organelle transport is mediated primarily by MTs and their associated adapters (19Ross J.L. Ali M.Y. Warshaw D.M. Cargo transport: molecular motors navigate a complex cytoskeleton.Curr. Opin. Cell Biol. 2008; 20: 41-47Crossref PubMed Scopus (260) Google Scholar). We hypothesized that mispositioning of lysosomes upon loss of sacsin and JIP3 could be due to alterations in MT-based transport and/or MT dynamics. We thus investigated MT growth dynamics in WT and ARSACS fibroblasts. MTs were depolymerized using nocodazole and then allowed to regrow after drug washout. At 1 h following nocodazole wash out, cells were fixed and stained to reveal MT networks. At steady-state, MT networks appeared similar when examining control versus fibroblasts from ARSACS patients (Fig. 4A). However, at 1 h following nocodazole treatment, differences were observed in the organization of MTs (Fig. 4A). MTs in ARSACS fibroblasts were disorganized, clustered, and nonradial, compared to the radial and regular network of MTs in control fibroblasts. Since we observed altered MT organization in ARSACS fibroblast, we tested if sacsin has the ability to bind MTs and/or to regulate MT dynamics. Given the large size of the sacsin protein, we adapted a classical method of isolating MT-associated proteins (MAPs). Starting from brain lysates, cycles of temperature-dependent polymerization and depolymerization of MTs in the presence of GTP and glycerol, coupled to high speed centrifugation, significantly enriches MAPs (20Shelanski M.L. Gaskin F. Cantor C.R. Microtubule assembly in the absence of added nucleotides.Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 765-768Crossref PubMed Scopus (2046) Google Scholar). Using this method, we enriched sacsin and MAP1A (Fig. 4B). We also performed affinity-binding assays by incubating highly purified, Taxol-stabilized MTs with soluble rat brain lysate, followed by pelleting MTs and their MAPs by centrifugation. Using this approach, we could readily cosediment sacsin with MTs, with GAPDH as a control for a nonsedimenting protein (Fig. 4C). Since sacsin interacts with MTs, we tested to see if sacsin regulates MT dynamics. MT end-binding protein 3 (EB3) caps the plus ends of growing MTs. Monitoring the dynamics of fluorescently labeled EB3 is routinely used to measure MT dynamics (21Yang C. Wu J. de Heus C. Grigoriev I. Liv N. Yao Y. et al.EB1 and EB3 regulate microtubule minus end organization and Golgi morphology.J. Cell Biol. 2017; 216: 3179-3198Crossref PubMed Scopus (56) Google Scholar). EB3-GFP was transfected into WT and sacsin KO HeLa cells, and live cell imaging was performed to quantify the growth rate of MTs (Fig. 4, D and E). The TrackMate plugin of FIJI software was used to plot the tracks of EB3-GFP as indicated in Figure 4D. By applying a robust MT tracking protocol, we observed that the growth rate of MTs in sacsin KO cells was slower than WT cells (Fig. 4E). The Rho family of GTPases are known to play an important role in the regulation of MT assembly status. For example, MT disassembly, such as that induced by nocodazole treatment, is known to lead to enhanced activation (GTP-bound form) of Rho (22Ren X.D. Kiosses W.B. Schwartz M.A. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1374) Google Scholar). There are numerous effectors of Rho including ROCK-I and ROCK-II that bind Rho selectively in the active, GTP-bound form (23Bagci H. Sriskandarajah N. Robert A. Boulais J. Elkholi I.E. Tran V. et al.Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.Nat. Cell Biol. 2020; 22: 120-134Crossref PubMed Scopus (89) Google Scholar). The Rho-binding domain of ROCK-1 expressed as a GST fusion protein (RBD-GST) was used to pull down GTP-Rho in control and ARSACS fibroblasts. Interestingly, ARSACS patient fibroblasts have relatively high levels of GTP-Rho compared to control fibroblasts (Fig. S5, A and B). These results suggest that sacsin binds to MTs and regulates their dynamics, which in turn controls organelle positioning. Following fusion of autophagosomes with lysosomes, the last step in autophagy, lysosome homeostasis is maintained through a process termed autolysosome reformation (ALR), which involves the formation of nascent lysosomes from pre-existing lysosomes (24Chen Y. Yu L. Development of research into autophagic lysosome reformation.Mol. Cell. 2018; 41: 45-49Google Scholar). The first step in ALR involves the formation of tubules from autolysosomes, which subsequently bud off into small protolysosomes. Protolysosomes contain autolysosome components that mature and acquire acidity to form fully functional lysosomes. ALR is a recently discovered cellular process and the mechanistic details are not clearly understood. Spinster, mTOR, clathrin, and the clathrin adapter AP2 have been shown to mediate ALR in a MT-dependent manner (24Chen Y. Yu L. Development of research into autophagic lysosome reformation.Mol. Cell. 2018; 41: 45-49Google Scholar). Since MT dynamics are altered in sacsin KO cells, loss of sacsin could also affect ALR. To investigate this hypothesis, ALR was initiated in sacsin KO and WT cells through nutrient deprivation (25Dai A. Yu L. Wang H.-W. WHAMM initiates autolysosome tubulation by promoting actin polymerization on autolysosomes.Nat. Commun. 2019; 10: 3699Crossref PubMed Scopus (35) Google Scholar). Loss of sacsin reduces formation of tubules budding from lysosomes (Fig. 5, A, B and D; Videos S6 and S7). Loss of LAMP1 tubules in sacsin KO cells are similar to cells treated with nocodazole (Video S9). Previous reports have demonstrated that clathrin and AP2 are important for ALR formation (26Rong Y. Liu M. Ma L. Du W. Zhang H. Tian Y. et al.Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation.Nat. Cell Biol. 2012; 14: 924-934Crossref PubMed Scopus (219) Google Scholar). In a mass spectrometry screen for binding partners of the DNAJ-HEPN domain of sacsin, we identified AP2 as an interacting partner. We confirmed the interaction using various GST-DNAJ-HEPN fusion proteins and mapped the AP2-binding site to a region between the DNAJ and HEPN domain (Fig. 5E). Kinesin motor protein (KIF5B) and Arl8 are important for ALR initiation. To further test the role of sacsin in ALR, we performed immunoprecipitation to test if it interacts with these proteins (27Du W. Su Q.P. Chen Y. Zhu Y. Jiang D. Rong Y. et al.Kinesin 1 drives autolysosome tubulation.Dev. Cell. 2016; 37: 326-336Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Immunoprecipitation reveals interaction of sacsin with KIF5B and ARL8 (Fig. 5F). Since ARL8 cycles between active and inactive forms, we tested to see if interaction with sacsin is nucleotide dependent (28Khatter D. Sindhwani A. Sharma M. Arf-like GTPase Arl8: moving from the periphery to the center of lysosomal biology.Cell Logist. 2015; 5e1086501Crossref PubMed Scopus (57) Google Scholar). Immunoprecipitation reveals ARL8 interaction with sacsin is nucleotide independent (Fig. S6). We also performed density gradient fractionation of 293T cell lysates under basal and ALR inducing starvation conditions and immunoblotted for clathrin and sacsin proteins. We observed greater cofractionation of sacsin with clathrin under starvation conditions (Fig. S7). We also tested to see if JIP3, which functions in MT dynamics, also regulates ALR. We initiated ALR with nutrient deprivation in JIP3 KO cells and observed no deficits in ALR (Fig. 5, C and D and Video S8). Since the initial discovery of ARSACS in the Charlevoix and Saguenay regions of Quebec, patients have been reported worldwide and over 200 mutations have been identified, which span the entire protein. Analysis of the sacsin protein domain structure suggests that sacsin may function as a molecular chaperone and regulate protein quality control mechanisms in the cell (29Romano A. Tessa A. Barca A. Fattori F. de Leva M.F. Terracciano A. et al.Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.Hum. Mutat. 2013; 34: 525-537Crossref PubMed Scopus (25) Google Scholar). However, the cellular phenotypes seen in cells with disrupted sacsin function, which includ
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