VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans

VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans Xiaomeng Yin , Ruoxi Wang , Andrea Thackeray , Eric H. Baehrecke , View ORCID Profile Mark J. Alkema doi: https://doi.org/10.1101/2025.01.22.634397 Xiaomeng Yin 1 Department of Neurobiology, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA 2 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School , Worcester, MA 01605 USA 3 School of Life Sciences, Southern University of Science and Technology , Shenzhen, 518055, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ruoxi Wang 2 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School , Worcester, MA 01605 USA 3 School of Life Sciences, Southern University of Science and Technology , Shenzhen, 518055, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andrea Thackeray 1 Department of Neurobiology, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eric H. Baehrecke 2 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School , Worcester, MA 01605 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: eric.baehrecke{at}umassmed.edu mark.alkema{at}umassmed.edu Mark J. Alkema 1 Department of Neurobiology, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mark J. Alkema For correspondence: eric.baehrecke{at}umassmed.edu mark.alkema{at}umassmed.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Mitochondria control cellular metabolism, serve as hubs for signaling and organelle communication, and are important for the health and survival of cells. VPS13D encodes a cytoplasmic lipid transfer protein that regulates mitochondrial morphology, mitochondria and endoplasmic reticulum (ER) contact, quality control of mitochondria. VPS13D mutations have been reported in patients displaying ataxic and spastic gait disorders with variable age of onset. Here we used CRISPR/Cas9 gene editing to create VPS13D related-spinocerebellar ataxia-4 (SCAR4) missense mutations and C-terminal deletion in VPS13D ’s orthologue vps-13D in C. elegans . Consistent with SCAR4 patient movement disorders and mitochondrial dysfunction, vps-13D mutant worms exhibit locomotion defects and abnormal mitochondrial morphology. Importantly, animals with a vps-13D deletion or a N3017I missense mutation exhibited an increase in mitochondrial unfolded protein response (UPR mt ). The cellular and behavioral changes caused by VPS13D mutations in C. elegans advance the development of animal models that are needed to study SCAR4 pathogenesis. Introduction Mitochondria are dynamic organelles that play an important role in cellular bioenergetics and viability. The mitochondrial network undergoes continuous fusion and fission to maintain its health. In addition, mitochondria form dynamic contacts with other organelles, especially the endoplasmic reticulum, to participate in various biological processes. Damaged mitochondria are selectively cleared by autophagy, and alterations in mitochondrial removal by autophagy have been associated with neurological disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis ( Collier et al. 2023 ). Vacuolar protein sorting-associated protein 13D, encoded by the VPS13D gene, is a key regulator of mitochondrial processes, including mitochondrial clearance, mitochondrial morphology, and inter-organelle contacts ( Anding et al. 2018 ; Shen et al. 2021b ; Guillen-Samander et al. 2021 ). VPS13D is a member of the conserved VPS13 protein family, which facilitates membrane contacts and transport lipids between organelles ( Leonzino et al. 2021 ). Unlike the other three human VPS13 proteins (VPS13A–C), VPS13D possesses a unique ubiquitin-associated (UBA) domain that has been shown to interact with K63 ubiquitin chains and participate in mitochondrial health ( Anding et al. 2018 ; Leonzino et al. 2021 ). Mutations in the VPS13A–C genes have been linked to specific neurological disorders, including chorea-acanthocytosis, Cohen syndrome, and early-onset Parkinson’s disease. Importantly, biallelic pathogenic variants in the VPS13D gene have also been associated with spastic ataxia and spastic paraplegia ( Seong et al. 2018 ; Gauthier et al. 2018 ). VPS13D movement disorder, also referred to as autosomal recessive spinocerebellar ataxia-4 (SCAR4; ( Swartz et al. 2002 ; Seong et al. 2018 )), manifests as a hyperkinetic movement disorder characterized by dystonia, chorea, and/or ataxia. This disorder is often accompanied with varying degrees of developmental delay and cognitive impairment (Meijer 2019). In the majority of the reported 42 cases, individuals have a combination of a loss-of-function variant and a seemingly milder variant, which can either be a missense mutation or a milder splicing variant ( Seong et al. 2018 ; Gauthier et al. 2018 ; Koh et al. 2020 ; Petry-Schmelzer et al. 2021 ; Wang Yu 2021; Huang and Fan 2022 ; Oztop-Cakmak et al. 2022 ; Durand et al. 2022 ; Pauly et al. 2023 ; Baker et al. 2023 ; Harada et al. 2024 ; Sultan et al. 2024 ; Nordli and Galan 2023 ; Kistol et al. 2023 ; Kistol et al. 2024 ; Algahtani et al. 2024 ; Lee et al. 2020 ). Both VPS13D -deficient human and Drosophila cells exhibit notable abnormalities in mitochondrial morphology and clearance ( Seong et al. 2018 ; Anding et al. 2018 ). In addition, a genetic screen for altered mitochondria in C. elegans identified C25H3.11 ( vps-13D ) ( Rolland and Conradt 2022 ). Recent studies have linked VPS13D to Leigh syndrome ( Kistol et al. 2023 ; Lee et al. 2020 ) and the Parkinson’s disease gene PINK1 ( Shen et al. 2021a ) suggesting that this gene has relevance to a broader spectrum of neurological diseases. Strong loss-of-function VPS13D alleles are lethal in Drosophila ( Anding et al. 2018 ) and mice ( Skarnes et al. 2011 ), thus presenting a challenge to model this disease in an intact animal. In this study, we used CRISPR/Cas9 gene editing techniques to create SCAR4 patient-specific mutations in the C. elegans orthologue of the human VPS13D gene: vps-13D . We analyzed the effects of these SCAR4 patient-specific mutations in vps-13D on worm fecundity, behavior, and mitochondrial morphology. We find that vps-13D deletion mutants display maternal effect sterility and locomotion defects. vps-13D deletions and the N3017I missense mutation displayed disrupted mitochondrial morphology and resulted in different degrees of activated mitochondrial unfolded protein response (UPR mt ). Furthermore, we identified a functional link between vps-13D and the mitochondria fusion regulator fzo-1 in the same pathway to regulate mitochondrial dynamics and homeostasis. The combined biological changes caused by VPS13D mutations in C. elegans advance the development of a new animal model to study SCAR4 pathogenesis. Materials and methods C. elegans strains and GFP fusion construction All C. elegans strains were maintained at 22°C on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 bacteria. The wild-type strain was Bristol N2. Sterile mutant vps-13D strains were balanced with the mIn1 balancer chromosome ( Edgley and Riddle 2001 ). The vps-13D mutant strains were crossed with the following transgenes: zcIs14 [ Pmyo-3::GFP ( mit )] ( Benedetti et al. 2006 ), zcIs13 [ Phsp-6::GFP + lin-15 ( + )] ( Yoneda et al. 2004 ; Yang et al. 2022 ). A full list of strains used in this study is shown in Supplementary Table S1. The Pvps-13D :: GFP transcriptional reporter was generated using the following primers: F(5’-ACAGGATCCTCGCATACAATCACATCGTC-3’) and R(5’-ACAGGTACCTGTCCAGGAATTGTGGTATC-3’). These primers were used to amplify a 3.5 kb fragment corresponding to the upstream promoter sequence (including exon 1 and part of exon 2) of vps-13D . The PCR product was digested by BamHI and KpnI restriction enzymes and inserted into pPD95.75 vector. The Pvps-13D :: GFP construct was microinjected at 50 ng/µl into temperature-sensitive lin-15 ( n765ts ) mutant animals along with the lin-15 ( + ) rescuing plasmid (pL15EK) at 80 ng/µl and pBSK DNA at 80 ng/µl. Transgenics were selected at 22°C based on a non-Muv (Multivulva) phenotype and GFP fluorescence. CRISPR/Cas9 design and gene editing The Bristol N2 strain was used as the wild-type strain for all CRISPR/Cas9 editing of vps-13D . Site-specific crRNAs and a repair template donor ssODNs were manually selected and generated by IDT (Integrated DNA Technologies, Inc.) who also provided the tracrRNA. Injection mixtures were prepared following established protocols ( Ghanta et al. 2021 ) and rol-6 was used as a co-CRISPR selection marker. All crRNAs, ssODNs, and PCR screening sequences are reported in Supplementary Table S2-S4. Correct substitution or deletion sequences were confirmed via Sanger sequencing. Sequencing of C-terminal deletion ΔC ( zf197 ) revealed a 1218-bp deletion (LGII 5670774–5671991) with a short 20-bp insertion positioned between the two breakpoints. The CRISPR-designed strains underwent four rounds of outcrossing to the N2 strain. Brood size assay Ten L4 animals of each genotype were picked and separated onto individual plates. Animals were transferred onto new plates every day until the cessation of egg-laying. Plates were counted for the total number of eggs after removal of the parent animal. Larval development analysis Five adult animals from each genotype were transferred to a new plate. After 2-hours, adult animals were removed, and the eggs were left to develop for over 96 hours. Two vps-13D deletion mutant strains were maintained as balanced heterozygotes using the mIn1 [ dpy-10 ( e128 ) mIs14 ] balancer. This balancer includes an integrated pharyngeal GFP reporter expressed in a semi-dominant manner. Consequently, the offspring segregate into wild type with a dim GFP signal, Dpy with a bright GFP signal ( mIn1 homozygotes), and non-GFP vps-13D deletion homozygotes. For the vps-13D deletion strains only non-GFP homozygotes were evaluated at each developmental stage. The developmental timing was calculated as the proportion of larvae among the total number of hatched embryos that reached each specific developmental stage. The developmental stage of each individual was determined based on body size and stage-specific morphological features. Multi-Worm Tracker assay 10 synchronized 1-day old or 3-day old (24 hours or three days post the L4 stage) worms were placed on a thin lawn E. coli OP50 on a medium (5 cm) NGM plate. The plate was then placed in the Multi-Worm Tracker (MWT) ( https://sourceforge.net/projects/mwt/ , ( Swierczek et al. 2011 )) and recorded for 10 minutes. The Multi-Worm Tracker package includes real-time image analysis software and behavioral parameter measurement software, Choreography. Tracking and analysis were conducted following previous studies ( Huang et al. 2019 ; Florman and Alkema 2022 ; Kang et al. 2024 ). The average speed of the population was measured over 5 minutes after a 5-minute acclimation period. Experiments were analyzed using custom MATLAB (MathWorks, Inc.) scripts to interface with Choreography analysis program. The MATLAB scripts used in this study are available at https://github.com/jeremyflorman/Tracker_GUI . Analysis was limited to objects that had been tracked for a minimum of 20 seconds and had moved a minimum of 5 body lengths. For the analysis of single worm tracks, a plate containing a single 3-day old animal was recorded for 10 minutes with images captured every 2 seconds. The sequential images from the final 5 minutes post-acclimation were stitched and processed in ImageJ to generate a continuous worm movie track. Thrashing assay 10 age-synchronized young adult animals were transferred to a fresh unseeded plate to remove residual bacteria. Individual animals were subsequently transferred to M9 buffer in a tiny plate. After 15 seconds acclimatization, the number of completed thrashes during 1 minute were counted per animal using a hand counter. Gene expression knockdown via RNAi treatment fzo-1 RNAi bacteria clones, obtained from the Ahringer library ( Kamath and Ahringer 2003 ), were selected by ampicillin (100 mg/ml) and tetracycline (12.5 mg/ml) and verified by DNA sequencing. The control L4440 or fzo-1 RNAi bacteria grown at 37°C overnight in LB with ampicillin (100 mg/ml), were concentrated (4X) and seeded on RNAi NGM plates that contain 6 mM IPTG and 100 mg/ml ampicillin. Young adult P0 animals were placed on the RNAi plates and allowed to produce offspring. L4 larvae of F1 progeny were transferred to a new RNAi plate. After two generations of RNAi-treatment, L4 larvae of F2 animals were analyzed. Imaging and fluorescence quantification To assess the sterile phenotype, adult wild-type animals and vps-13D deletion mutant animals were placed on 2% agarose pads containing 60 mM sodium azide. Adult sterile animals were identified by the absence of embryos or oocytes in the uterus through DIC imaging using a Zeiss LSM 700 microscope. To explore the expression pattern of vps-13D , adult animals expressing a Pvps-13D :: GFP reporter were mounted on 2% agarose pads containing 60 mM sodium azide. Images were captured using a Zeiss LSM 700 confocal microscope. To test the impact of vps-13D mutations on mitochondrial morphology, L4 animals expressing zcIs14 transgene were mounted on 2% agarose pads containing 10 mM levamisole. Images were recorded using a Zeiss LSM 700 confocal microscope. For each genotype mitochondrial morphology in body wall muscles in the middle of the worm body was scored for 10∼15 animals in total. To test the effects of vps-13D mutations on hsp-6 expression of C. elegans , six L4 animals expressing Phsp-6 :: GFP grown on E. coli OP50 were mounted together on 2% agarose pads containing 10 mM levamisole and 18∼36 animals in total. The GFP images were acquired with an Axioimager Z1 microscope (Zeiss) at 10x magnification, and the maximum fluorescence intensity was quantified using ImageJ FIJI software. Mitochondrial morphology analysis Mitochondrial images were segmented and quantified using the Mitochondrial Segmentation Network (MitoSegNet) toolbox, a deep learning-based tool that includes the MitoS segmentation tool and the MitoA analysis tool ( Fischer et al. 2020 ). Documentation for the MitoS and MitoA tools are available at https://github.com/mitosegnet , and the MitoSegNet segmentation model, along with the MitoA analysis and MitoS segmentation tools (GPU/CPU versions) for Linux and Windows, can be accessed at https://zenodo.org/search?page=1&size=20&q=mitosegnet . Initially, 8-bit raw images were segmented using the pretrained mitochondria-specific “basic mode” (MitoSegNet model) in the MitoS tool (CPU version). Upon completion, the program generated a prediction folder containing the segmented images. The segmented images and corresponding raw images were then subjected to quantitative measurement using the MitoA tool, which evaluates morphological features for each object and generates summary statistics for all object features in each image. The major axis lengths were determined by measuring the lengths of the line segment connecting the two vertices of an ellipse fitted around an object in the MitoA tool. Statistical analysis All statistical analysis and graph construction were performed using GraphPad Prism 8 software. Results are presented as standard error of the mean (SEM) from at least three independent experiments. Statistical comparisons were performed using ANOVA or Kruskal-Wallis H test with Dunnett’s correction for multiple samples. Significance was determined when P-value < 0.05. Results C. elegans vps-13D encodes an ortholog of VPS13D To explore the evolutionary relationships of the VPS13D proteins among various species, we first conducted a phylogenetic analysis. VPS13D proteins from different species formed a well-supported clade, indicating that they evolved from a common ancestor ( Fig. 1A ). The C. elegans C25H3.11 gene encodes a protein that shares the most significant similarity with VPS13D. The predicted Ricin B-type lectin domain-containing protein shares 36% similarity and 22% identity with the human VPS13D protein (Table S5). Furthermore, the highest sequence similarity between the two proteins was observed in the C-terminal region, which contains the VPS13 adaptor binding (VAB) domain (Bean et al. 2018) (also known as SHR binding domain or WD40-like region (Kumar et al. 2018)) and a Dbl homology (DH)-like domain (InterPro) ( Fig. 1B ). Given that C25H3.11 gene is the closest C. elegans orthologue of the human VPS13D gene, we hereafter refer to C25H3.11 as the C. elegans vps-13D gene. In humans, 14 of 30 reported families carry at least one missense mutation in the C-terminal region following the ubiquitin-associated (UBA) domain, and 5 of 8 reported homozygous missense mutations in patients are also located in this region ( Seong et al. 2018 ; Gauthier et al. 2018 ; Koh et al. 2020 ; Petry-Schmelzer et al. 2021 ; Wang Yu 2021; Huang and Fan 2022 ; Oztop-Cakmak et al. 2022 ; Durand et al. 2022 ; Pauly et al. 2023 ; Baker et al. 2023 ; Harada et al. 2024 ; Sultan et al. 2024 ; Nordli and Galan 2023 ; Kistol et al. 2023 ; Kistol et al. 2024 ; Algahtani et al. 2024 ; Lee et al. 2020 ). Several of these mutation sites associated with human pathogenicity are highly conserved in the C-terminal region in C. elegans VPS13D protein ( Fig. 1C ). Download figure Open in new tab Fig. 1. VPS13D orthologue vps-13D is conserved in C. elegans (A) Phylogenetic tree of VPS13D proteins. VPS13D protein sequences from various species were retrieved from UniProt. The evolutionary history was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Evolutionary analyses were conducted in MEGA11. The species depicted from top to bottom are Human (UniProt identifier: Q5THJ4), Bovine (UniProt identifier: E1BIF6), Mouse (UniProt identifier: B1ART2), Rat (UniProt identifier: A0A8I6G572), Zebrafish (UniProt identifier: A0A8M1QUR7), Drosophila (UniProt identifier: Q9VU08), C. elegans (labeled as brown diamond, UniProt identifier: A0A2K5ATR5) and Yeast (UniProt identifier: Q07878). (B) Human VPS13D protein topology (modified from ( Guillen-Samander et al. 2021 ) and predicted domains of the C. elegans VPS-13D protein with disease-related missense mutations labeled in dotted vertical lines and deletions annotated with horizontal lines. Domains and abbreviations are as follows: N, Chorein N-terminal domain; Chorein domain; Extended Chorein domain; UBA, Ubiquitin-associated domain; VAB, Vps13 Adaptor Binding/SHR-Binding/WD40-like domain; Ricin B-type lectin domain; DH, Dbl homology-like domain; and PH, Pleckstrin homology domain. (C) Sequence alignment of C terminal region between C. elegans VPS-13D, human VPS13D, and fly Vps13D protein by Clustal Omega. The three missense mutations enrolled in this study are highlighted with the red box, and the start residue of vps-13D ( ΔC ) deletion is labeled with the red asterisk. (D) Representative fluorescence image of adult hermaphrodites expressing Pvps-13D :: GFP under the 3570bp promoter (including exon 1 and part of exon 2). A, anterior; P, posterior; V, ventral side; D, dorsal side. The scale bar is 0.2 mm. We next focused on three conserved residues within the C-terminal region of VPS13D protein that are reported to be mutated in early onset SCAR4 patients. We used CRISPR/Cas9 gene editing to create these variants in vps-13D ( N2454S ( zf194 ), N3017I ( zf195 ) and R3144Q ( zf196 )) ( Table 1 ). In addition, we generated a C-terminal deletion ΔC ( zf197 ) that encompassed 1218 base pairs, resulting in a truncation of the VPS13D protein ( Fig. 1B ). We also obtained a vps-13D deletion allele, Δ ( ok2632 ), which contains a 1740 bp deletion removing part of exons 3 and 4, resulting in a premature stop and thus most likely represents a null allele. To analyze the expression of vps-13D , we investigated its expression pattern by generating a transcriptional fusion of GFP reporter with 3.5kb vps-13D upstream promoter fragment (including exon 1 and part of exon 2). View this table: View inline View popup Download powerpoint Table 1. VPS13D pathogenic missense mutations in this study. Fluorescence from the Pvps-13D :: GFP reporter was observed throughout the adult hermaphrodite, including intestine, hypodermis, muscle, glia and neurons ( Fig. 1D ). Our data indicate that vps-13D is expressed in most cell types consistently with RNA expression data sets ( Taylor et al. 2021 ). vps-13D mutants exhibit impaired locomotion Animals carrying either homozygous vps-13D deletion alleles ( Δ ( ok2632 ) or ΔC ( zf197 )) are viable. However, both homozygous vps-13D ( Δ ) and vps-13D ( ΔC ) animals display maternal effect sterility ( Fig. 2A ). Wild-type hermaphrodites produce oocytes that are fertilized in the spermatheca, resulting in the accumulation of developing embryos in the uterus. The germlines of either homozygous vps-13D ( Δ ) or vps-13D ( ΔC ) animals did not display visible oocytes or embryos ( Fig. 2B ). Due to their sterility, these two deletion strains were maintained as balanced heterozygotes but analyzed as homozygotes by selecting homozygous animals from a mixed population. In addition to fertility deficiencies, these two deletion mutants also exhibited a slower rate of development, taking 24∼48 hours longer than the wild type to reach the L4 stage ( Fig. 2C ). In contrast, three missense mutants vps-13D ( N2454S ), ( N3017I ), and ( R3144Q ) did not exhibit developmental arrest or delay and were able to produce a similar brood size compared to the wild type ( Fig. 2A ). Download figure Open in new tab Fig. 2. vps-13D deficiency affects worms’ fertility and locomotion. (A) Total brood size (n=30 broods) indicates the fertility ability of C. elegans to produce offspring in association with different vps-13D mutants. One-way ANOVA with Dunnett correction was used to compare the difference between wild-type and mutant animals. (B) DIC images of whole worm and gonad in hermaphrodites of the wild type and vps-13D ( Δ ) mutant strains. The scale bar in the whole worm image is 0.2 mm. The scale bar in the gonad image is 0.2 μm. Black arrows indicate oocyte nuclei. (C) The percentage of worms at the different developmental stages was determined for wild-type, vps-13D ( Δ ), and vps-13D ( ΔC ) mutant animals after 96 hours of growth from the embryonic stage at 22°C. (D) Individual animal tracks of 3-day-old wild type, vps-13D ( Δ ) and vps-13D ( ΔC ) mutant strains for the final 5 min recordings. The scale bar is 1 mm. (E) Comparison of average velocity for 3-day-old wild-type and vps-13D animals from the final 5 mins MWT recordings. The velocity has been normalized and calculated based on the worm body lengths of respective strains. The difference in average velocity between wild-type and mutants was analyzed by one-way ANOVA with Dunnett’s multiple comparison test. (F) Number of thrashes per minute in M9 liquid for 3-day-old wild type and vps-13D mutant animals (n=15-30). Differences between wild type and mutants were analyzed by One-way ANOVA with Dunnett correction for multiple comparisons. Next, we examined whether vps-13D mutant strains possess motor defects. We conducted a population-level motility assay using an automated multi-worm tracking system (MWT) ( Swierczek et al. 2011 ). We tracked wild type and vps-13D mutant strains for 5 minutes and extracted the behavioral dynamics of individual worms. One-day-old adult vps-13D mutants did not shown any difference compared to the wild-type (Fig. S1). However, vps-13D deletion animals displayed a noticeable movement impairment compared to wild-type animals. The average locomotion rate of 3-day-old wild-type animals was 0.101± 0.005 body lengths/sec, whereas either vps-13D ( Δ ) or vps-13D ( ΔC ) mutants displayed decreased locomotion rates of 0.064±0.010 and 0.065±0.012 body lengths/sec ( Fig. 2D-E ). The three vps-13D missense mutant strain locomotion rates were unaffected at day 3 of the adult stage (0.111±0.009, 0.106±0.010, 0.099±0.008 body lengths/sec, respectively, Fig. 2E ). Furthermore, we analyzed thrashing rates of single worm in liquid M9 medium. The average thrashing rate of 3-day-old wild-type animals was 113.2±6.9 thrashes/min. Significantly, vps-13D ( Δ ) and vps-13D ( ΔC ) mutant strains showed a 20∼26% decrease in thrashing rates compared to control wild-type animals (83.8 ± 11.0 thrashes/min, 89.8±8.7 thrashes/min, respectively, Fig. 2F ). Importantly, the thrashing rate of vps-13D ( N3017I ) mutant worms was 92.7±7.1 thrashes/min, showing an 18% reduction compared to control animals ( Fig. 2F ), where vps-13D ( N2454S ) and vps-13D ( R3144Q ) mutants exhibited similar thrashing rates to the wild type (110.5±11.0 thrashes/min, 108.3±9.5 thrashes/min, respectively). Combined, these results indicate that the C. elegans vps-13D mutations can result in locomotion defects. vps-13D mutants possess altered mitochondrial morphology Dynamic changes in mitochondrial morphology are essential for mitochondria health and homeostasis. Mitochondrial fusion and fission not only affect mitochondria morphology but also regulate multiple mitochondria biological processes, including mitochondria quality control and clearance by mitophagy ( Pickles et al. 2018 ). Previous studies indicate that VPS13D affects mitochondrial morphology and mitophagy in both human and Drosophila cells ( Anding et al. 2018 ). To test if vps-13D mutants exhibit a similar function to maintain mitochondrial morphology, we used a transgenic line zcIs14 ([ myo-3::GFP ( mit )]) that labels mitochondria in the body wall muscles ( Benedetti et al. 2006 ) and quantified the mitochondrial morphology using MitoSegNet ( Fischer et al. 2020 ). Although morphologies between different mutants were variable and possibly reflected allele strength, mutant strains of vps-13D displayed changes in mitochondrial morphology at the L4 larval stage compared to the wild type ( Fig. 3A ). Specifically, all homozygous vps-13D mutant worms possessed shorter mitochondria compared to control wild-type worms, as determined by the major axis length ( Fig. 3A and 3B ). In wild-type worms, the major axis length was 31.4±11.6 pixels. By contrast, vps-13D ( Δ ) and vps-13D ( ΔC ) mutant strains displayed significantly reduced major axis lengths of 18.1±3.7 pixels and 19.0±5.4 pixels ( Fig. 3B ). Similarly, the major axis length was also reduced in the three vps-13D missense mutant strains, measuring 19.7±5.7, 18.0±3.9, 22.3±9.5 pixels, respectively ( Fig. 3B ). These data are consistent with a recent report ( Rolland and Conradt 2022 ). In addition, both vps-13D ( Δ ) and vps-13D ( ΔC ) mutant strains demonstrated a notable increase in mitochondrial area measuring 154.0±32.6 pixels and 144.7±30.2 pixels, respectively, compared to 113.5±19.6 pixels in wild-type worms. Download figure Open in new tab Fig. 3. vps-13D regulates mitochondrial morphology in C. elegans (A) Mitochondrial morphology of the wild type and vps-13D mutant animals. Original images are at the top and MitoSegNet model segmentations are at the bottom. The scale bar is 20 μm. (B, C) Statistical analysis of mitochondrial morphology parameters. Major axis length (see materials and methods) and average area were measured in segmented images of mitochondria from wild-type and vps-13D mutant animals (n=15-25). One-way ANOVA with Dunnett correction was used to compare the difference between wild-type and mutant animals. Data is presented as mean ± S.E.M. *p <0.05, **p <0.01, *** p <0.001, **** p <0.0001, n.s. = not significant. By contrast, vps-13D ( N2454S ) and vps-13D ( N3017I ) mutant strains display slightly smaller mitochondrial areas of 86.5±20.9 pixels and 87.6±26.5 pixels, respectively, where vps-13D ( R3144Q ) mutant is similar to the wild type (102.3±49.4 pixels) ( Fig. 3A and 3C ). These results indicate that SCAR4 mutations in the C. elegans VPS13D ortholog vps-13D lead to alterations in mitochondrial morphology, supporting VPS-13D’s role in maintaining mitochondrial structure. vps-13D disruption induces mitochondrial unfolded protein response Although previous assays have examined the effects of VPS13D mutations on mitochondrial morphology, their impact on mitochondrial homeostasis remains unclear ( Anding et al. 2018 ). The mitochondrial unfolded protein response (UPR mt ) is a crucial cellular pathway that protects mitochondria from multiple forms of cell stress ( Shpilka and Haynes 2018 ). To test whether vps-13D mutants affect UPR mt , we used a transgene of the transcriptional reporter Phsp-6::mtHSP70::GFP to monitor UPR mt in C. elegans ( Yoneda et al. 2004 ; Yang et al. 2022 ). Interestingly, vps-13D ( Δ ) and vps-13D ( ΔC ) mutant strains exhibit a significant induction of the UPR mt with high levels of Phsp-6::mtHSP70::GFP intensity compared to wild-type animals at L4 larval age ( Fig. 4A and 4B ). While the relative fluorescence intensity of vps-13D ( N2454S ) and vps-13D ( R3144Q ) missense mutants were not significantly different from wild type, homozygous vps-13D ( N3017I ) mutant worms exhibited a slight increase in UPR mt reporter expression compared to control worms ( Fig. 4A and 4B ). Combined, these results indicate that VPS13D plays a crucial role in maintaining mitochondrial homeostasis. Since the large deletion Δ ( ok2632 ) allele, and the C-terminal deletion allele ΔC ( zf197 ) causes similar deficiencies in fertility, development, locomotion, as well as mitochondrial morphology, we cannot exclude that frameshift mutations near the C-terminus could compromise protein stability. Download figure Open in new tab Fig. 4. vps-13D dysfunction induces mitochondrial UPR (A) Phsp-6 :: GFP expression of wild type and vps-13D mutant worms. The scale bar is 0.2 mm. (B) Quantification of Phsp-6 :: GFP relative fluorescence intensity (n=3-5 groups*6 animals). Kruskal-Wallis H test with Dunnett correction was used to compare the difference between wild-type and mutant animals. Data is presented as mean ± S.E.M. *p <0.05, **p <0.01, *** p <0.001, **** p <0.0001, n.s. = not significant. vps-13D and fzo-1/MFN2 function in a pathway to regulate mitochondrial homeostasis In Drosophila , the orthologue of MFN2 , Marf acts downstream of Vps13D to regulate mitochondrial fusion ( Shen et al. 2021b ). The C. elegans MFN2 orthologue FZO-1 is also involved in mitochondrial fusion ( Rolland et al. 2009 ). Since our results indicate that vps-13D loss promotes alteration of mitochondrial morphology ( Fig. 3 ), we examined the potential relationship between VPS-13D and FZO-1 in the regulation of mitochondrial morphology. We performed RNA-mediated interference (RNAi) targeting fzo-1 either in vps-13D ( Δ ) mutant background or in control worms. fzo-1 knockdown worms exhibited a decrease in average major axis length (17.7±2.5 pixels) and mitochondria area (77.8± 8.5 pixels) compared to control animals, which exhibited a major axis length of 28.4± 6.7 pixels and a mitochondrial area of 119.2 ± 9.8 pixels ( Fig. 5A , 5B , and 5C). Knockdown of fzo-1 failed to suppress the abnormal mitochondria morphology observed in vps-13D ( Δ ) deficient animals ( Fig. 5A , 5B , and 5C ). The major axis length (20.6±6.5 pixels) and mitochondria area (146.7±57.4 pixels) of vps-13D ( Δ ); fzo-1 (RNAi) worms were similar to those observed in vps-13D ( Δ ) control animals (19.4±4.2 pixels in major axis length and 157.4±40.1 pixels in area). We also examined whether fzo-1 affects mitochondria unfolded protein response (UPR mt ). Consistent with previous studies ( Haeussler et al. 2020 ), the knockdown of fzo-1 in wild-type worms led to a significant increase in the level of the UPR mt reporter zcIs13 ( Fig. 5D and 5E ). However, fzo-1 inactivation in vps-13D ( Δ ) mutant worms did not result in a significant change in UPR mt compared to the vps-13D ( Δ ) single mutant strain ( Fig. 5D and 5E ). Collectively, these data suggest that vps-13D and fzo-1 function together to regulate mitochondria homeostasis. Download figure Open in new tab Fig. 5. vps-13D and fzo-1 regulate mitochondrial homeostasis in similar pathways (A) Mitochondrial morphology changes of wild type and vps-13D ( Δ ) animals grown on either control mock or fzo-1 ( RNAi ). Original images are at the top and MitoSegNet model segmentations are at the bottom. The scale bar is 20 μm. (B, C) Statistical analysis of mitochondrial morphology parameters. Major axis length (see materials and methods) and average area were measured in segmented images of mitochondria from wild type and vps-13D ( Δ ) animals grown on either control mock or fzo-1 ( RNAi ) (n=10-15). (D, E) Phsp-6 :: GFP expression of wild type and vps-13D ( Δ ) animals grown on either control mock or fzo-1 ( RNAi ) (n=3-15 groups*6 animals). The scale bar is 0.2 mm. Kruskal-Wallis H test with Dunnett correction was used to compare the difference between wild-type and mutant animals. Data is presented as mean ± S.E.M. *p <0.05, **p <0.01, *** p <0.001, **** p <0.0001, n.s.= not significant. Discussion We developed a new model to study VPS13D related SCAR4 disease in the nematode C. elegans . Our results indicate that C. elegans carrying the VPS13D deletions and disease-associated missense mutations, in the worm orthologue vps-13D, are viable. However, vps-13D null deletion mutations that remove the C-terminal display maternal effect sterility. In addition, vps-13D deletions and N3017I missense mutant C. elegans exhibit impaired locomotion, thus reflecting a similar role for this gene because human SCAR4 patients exhibit movement deficiencies. Importantly, vps-13D mutant C. elegans displayed abnormal mitochondrial morphology, and vps-13D deletions and vps-13D ( N3017I ) mutants displayed varying levels increased mitochondrial UPR. The key clinical features of SCAR4 patients include progressive development of hyperkinetic movement disorder (dystonia, chorea, and/or ataxia). SCAR4 patient muscle biopsies exhibited mitochondrial accumulation and mild lipidosis ( Gauthier et al. 2018 ). Similarly, altered mitochondrial morphology and mitochondria clearance is decreased in cultured VPS13D patient-derived fibroblasts, VPS13D mutant human HeLa cells, and Drosophila ( Seong et al. 2018 ; Anding et al. 2018 ; Shen et al. 2021b ). Although mitochondrial respiratory chain function was normal in the muscle biopsy of one SCAR4 patient, a decrease in energy production and complex (I, III, and IV) protein levels has been detected in some patient fibroblasts ( Gauthier et al. 2018 ; Seong et al. 2018 ; Durand et al. 2022 ). Consistent with the movement defect of SCAR4 patients, worms carrying either deletions or N3017I missense mutations in vps-13D exhibited defects in crawling and/or swimming behavior. Interestingly, the abnormal swimming ability of vps-13D mutant worms is suggestive of mitochondrial dysfunction since swimming has been shown to be more energetically demanding than crawling for C. elegans ( Laranjeiro et al. 2017 ; Kang et al. 2024 ). The similarities between SCAR4 patients and vps-13D mutant worms, including locomotion defects, mitochondrial morphology, and mitochondrial homeostasis changes, suggest that C. elegans will likely be a useful model to reveal other genes in this pathway because of the strength of this genetic model. Mitochondrial homeostasis is controlled by mitophagy machinery, including the PINK1-Parkin axis ( Pickrell and Youle 2015 ). VPS13D functions downstream of PINK1, but bifurcates with Parkin to regulate mitochondrial clearance ( Shen et al. 2021a ). Specifically, VPS13D binds to ubiquitin and affects both the PINK1 substrate ubiquitinphospho-serine 65 and localization of the mitophagy receptor Ref2p/p62 on mitochondria ( Shen et al. 2021a ). Previous studies suggest mitophagy and mitochondrial UPR are both required to maintain mitochondrial health ( Pellegrino and Haynes 2015 ). Mitophagy and mitochondrial UPR function in a complementary manner to either recycle the damaged mitochondria by autophagy or recover the less damaged mitochondria. Additionally, mitophagy inhibition also causes damaged mitochondrial DNA accumulation that triggers mitochondrial UPR. Recently, it has been shown that non-mitochondrial proteins, including VPS13D, also play a role in maintaining mitochondrial homeostasis ( Rolland and Conradt 2022 ). Although it is unclear if VPS13D regulates mitochondria clearance in worms, evidence of VPS13D regulating mitophagy in different animals and human cells suggest this possibility ( Anding et al. 2018 ; Shen et al. 2021a ). Therefore, vps-13D mutant worms may potentially accumulate damaged mitochondria to activate mitochondrial UPR. Mitochondria and ER contact dictates inter-organelle lipid transfer sites and mitochondrial membrane lipid composition ( Gatta and Levine 2017 ). The lipid composition of mitochondrial membranes, in turn, can influence crucial processes within mitochondria ( Bockler and Westermann 2014 ; Lahiri et al. 2015 ). Previous studies indicate Vmp1 and Vps13D regulate mitochondria-ER contact and mitophagy ( Shen et al. 2021b ). VPS13D binds the outer mitochondrial membrane GTPase-Miro (both Miro1 and Miro2), likely via the WD40-like/VAB domain) ( Guillen-Samander et al. 2021 ), and the conserved C-terminal regions have been shown to work synergistically with the VAB domain to facilitate the membrane targeting ( Dziurdzik and Conibear 2021 ). Moreover, the VPS13D C-terminus is similar to the lipid transfer protein ATG2 ( Oztop-Cakmak et al. 2022 ). Consistent with the importance of these domains, our data indicate that vps-13D C-terminus is important for mitochondrial homeostasis. The mitochondrial protein mitofusin-2 (MFN2) facilitates mitochondrial fusion, as well as mitochondria and ER contact ( Filadi et al. 2018 ). Mutations in MFN2 are associated with Charcot-Marie-Tooth disease type 2A (CMT2A), in which mitochondrial morphology is abnormal in nerve tissue and fibroblasts from patients ( Verhoeven et al. 2006 ; Amiott et al. 2008 ). Similarly, loss of fzo-1 , the worm MFN2 homolog, resulted in progressive movement deficits, fragmented mitochondria, and mitochondrial UPR activation in C.elegans ( Byrne et al. 2019 ; Haeussler et al. 2021 ). Consistent with these studies, our results indicate fzo-1 knockdown worms displayed altered mitochondrial morphology and induced mitochondrial UPR. UPR mt activation requires the key transcription factor ATFS-1, which regulates the hsp-6 reporter expression ( Lin et al. 2016 ; Xin et al. 2022 ; Shpilka et al. 2021 ; Nargund et al. 2015 ). Notably, knockdown of atfs-1 significantly suppressed UPR mt in the fzo-1 mutant worms, confirming that UPR mt induction by loss of fzo-1 is ATFS-1 dependent ( Chen et al. 2021 ). Unlike Marf/MFN2 function in the Vps13D pathway regulating mitochondrial morphology in Drosophila ( Shen et al. 2021b ), our results suggest that fzo-1 and vps-13D could function in a related pathway to affect mitochondrial UPR in C. elegans . In summary, this study provides evidence supporting the conserved role of VPS13D in regulating mitochondrial morphology and function in C. elegans and provides insights how disease mutations affect mitochondrial homeostasis and behavior. Funding This work was supported by National Institutes of Health grants R01 GM140480 (MJA) and R35 GM131689 (EHB). Conflicts of interest The authors declare no conflict of interest. Data availability All data files from this study have been uploaded to Mendeley Data (DOI: 10.17632/3mwgvd2dbx.1). Figure legends Fig. S1. Day 1 vps-13D mutant worms did not show a significant deficiency in locomotion. (A) Comparison of average velocity for 1-day-old young adult wild type and vps-13D mutant animals from the final 5 mins MWT recordings. The velocity has been normalized and calculated based on the worm body lengths of respective strains. The difference in average velocity between wild-type and mutants was analyzed by one-way ANOVA with Dunnett’s multiple comparison test. (B) Number of thrashes per minute in M9 liquid for 1-day-old young adult wild type and vps-13D mutant animals (n=10-15). Differences between wild type and mutants were analyzed by One-way ANOVA with Dunnett correction for multiple comparisons. Acknowledgments We thank the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for some worm strains. We thank W.K. Kang, J.T. Florman, and M. Gorczyca for technical support and helpful discussions. Literature Cited ↵ Algahtani , H. , B. Shirah , and M.I. Naseer , 2024 Autosomal recessive spinocerebellar ataxia type 4 due to a novel homozygous mutation in the VPS13D gene in a Saudi family . Clin Neurol Neurosurg 240 : 108271 . OpenUrl CrossRef PubMed ↵ Amiott , E.A. , P. Lott , J. Soto , P.B. Kang , J.M. McCaffery et al. , 2008 Mitochondrial fusion and function in Charcot-Marie-Tooth type 2A patient fibroblasts with mitofusin 2 mutations . Exp Neurol 211 ( 1 ): 115 – 127 . OpenUrl CrossRef PubMed Web of Science ↵ Anding , A.L. , C. Wang , T.K. Chang , D.A. Sliter , C.M. Powers et al. , 2018 Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance . Curr Biol 28 ( 2 ): 287 – 295 e286. OpenUrl CrossRef PubMed ↵ Baker , E.K. , J. Han , W.A. Langley , M.A. Reott , Jr. , B.E. Hallinan et al. , 2023 RNA sequencing reveals a complete picture of a homozygous missense variant in a patient with VPS13D movement disorder: a case report and review of the literature . Mol Genet Genomics 298 ( 5 ): 1185 – 1199 . OpenUrl CrossRef PubMed ↵ Benedetti , C. , C.M. Haynes , Y. Yang , H.P. Harding , and D. Ron , 2006 Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response . Genetics 174 ( 1 ): 229 – 239 . OpenUrl Abstract / FREE Full Text ↵ Bockler , S. , and B. Westermann , 2014 Mitochondrial ER contacts are crucial for mitophagy in yeast . Dev Cell 28 ( 4 ): 450 – 458 . OpenUrl CrossRef PubMed ↵ Byrne , J.J. , M.S. Soh , G. Chandhok , T. Vijayaraghavan , J.S. Teoh et al. , 2019 Disruption of mitochondrial dynamics affects behaviour and lifespan in Caenorhabditis elegans . Cell Mol Life Sci 76 ( 10 ): 1967 – 1985 . OpenUrl CrossRef PubMed ↵ Chen , L.T. , C.T. Lin , L.Y. Lin , J.M. Hsu , Y.C. Wu et al. , 2021 Neuronal mitochondrial dynamics coordinate systemic mitochondrial morphology and stress response to confer pathogen resistance in C. elegans . Dev Cell 56 ( 12 ): 1770 – 1785 e1712. OpenUrl CrossRef PubMed ↵ Collier , J.J. , M. Olahova , T.G. McWilliams , and R.W. Taylor , 2023 Mitochondrial signalling and homeostasis: from cell biology to neurological disease . Trends Neurosci 46 ( 2 ): 137 – 152 . OpenUrl CrossRef PubMed ↵ Durand , C.M. , C. Angelini , V. Michaud , C. Delleci , I. Coupry et al. , 2022 Whole-exome sequencing confirms implication of VPS13D as a potential cause of progressive spastic ataxia . BMC Neurol 22 ( 1 ): 53 . OpenUrl CrossRef PubMed ↵ Dziurdzik , S.K. , and E. Conibear , 2021 The Vps13 Family of Lipid Transporters and Its Role at Membrane Contact Sites . Int J Mol Sci 22 ( 6 ). OpenUrl CrossRef PubMed ↵ Edgley , M.L. , and D.L. Riddle , 2001 LG II balancer chromosomes in Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and recessively marked inversions . Mol Genet Genomics 266 ( 3 ): 385 – 395 . OpenUrl CrossRef PubMed Web of Science ↵ Filadi , R. , D. Pendin , and P. Pizzo , 2018 Mitofusin 2: from functions to disease . Cell Death Dis 9 ( 3 ): 330 . OpenUrl CrossRef PubMed ↵ Fischer , C.A. , L. Besora-Casals , S.G. Rolland , S. Haeussler , K. Singh et al. , 2020 MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology . iScience 23 ( 10 ): 101601 . OpenUrl CrossRef PubMed ↵ Florman , J.T. , and M.J. Alkema , 2022 Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response . PLoS Genet 18 ( 3 ): e1010091 . OpenUrl CrossRef PubMed ↵ Gatta , A.T. , and T.P. Levine , 2017 Piecing Together the Patchwork of Contact Sites . Trends Cell Biol 27 ( 3 ): 214 – 229 . OpenUrl CrossRef PubMed ↵ Gauthier , J. , I.A. Meijer , D. Lessel , N.E. Mencacci , D. Krainc et al. , 2018 Recessive mutations in VPS13D cause childhood onset movement disorders . Ann Neurol 83 ( 6 ): 1089 – 1095 . OpenUrl CrossRef PubMed ↵ Ghanta , K.S. , T. Ishidate , and C.C. Mello , 2021 Microinjection for precision genome editing in Caenorhabditis elegans . STAR Protoc 2 ( 3 ): 100748 . OpenUrl CrossRef PubMed ↵ Guillen-Samander , A. , M. Leonzino , M.G. Hanna , N. Tang , H. Shen et al. , 2021 VPS13D bridges the ER to mitochondria and peroxisomes via Miro . J Cell Biol 220 ( 5 ). OpenUrl CrossRef ↵ Haeussler , S. , F. Kohler , M. Witting , M.F. Premm , S.G. Rolland et al. , 2020 Autophagy compensates for defects in mitochondrial dynamics . PLoS Genet 16 ( 3 ): e1008638 . OpenUrl CrossRef PubMed ↵ Haeussler , S. , A. Yeroslaviz , S.G. Rolland , S. Luehr , E.J. Lambie et al. , 2021 Genome-wide RNAi screen for regulators of UPRmt in Caenorhabditis elegans mutants with defects in mitochondrial fusion . G3 (Bethesda) 11 ( 7 ). ↵ Harada , S. , Y. Azuma , Y. Misumi , H. Hayashi , S. Matsubara et al. , 2024 A Novel Mutation of VPS13D-related Disorders with Parkinsonism . Intern Med . ↵ Huang , X. , and D.S. Fan , 2022 Autosomal recessive spinocerebellar ataxia type 4 with a VPS13D mutation: A case report . World J Clin Cases 10 ( 2 ): 703 – 708 . OpenUrl CrossRef PubMed ↵ Huang , Y.C. , J.K. Pirri , D. Rayes , S. Gao , B. Mulcahy et al. , 2019 Gain-of-function mutations in the UNC-2/CaV2alpha channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans . Elife 8 . ↵ Kamath , R.S. , and J. Ahringer , 2003 Genome-wide RNAi screening in Caenorhabditis elegans . Methods 30 ( 4 ): 313 – 321 . OpenUrl CrossRef PubMed Web of Science ↵ Kang , W.K. , J.T. Florman , A. Araya , B.W. Fox , A. Thackeray et al. , 2024 Vitamin B(12) produced by gut bacteria modulates cholinergic signalling . Nat Cell Biol 26 ( 1 ): 72 – 85 . OpenUrl CrossRef PubMed ↵ Kistol , D. , P. Tsygankova , F. Bostanova , M. Orlova , and E. Zakharova , 2024 New Case of Spinocerebellar Ataxia, Autosomal Recessive 4, Due to VPS13D Variants . Int J Mol Sci 25 ( 10 ). ↵ Kistol , D. , P. Tsygankova , T. Krylova , I. Bychkov , Y. Itkis et al. , 2023 Leigh Syndrome: Spectrum of Molecular Defects and Clinical Features in Russia . Int J Mol Sci 24 ( 2 ). OpenUrl ↵ Koh , K. , H. Ishiura , H. Shimazaki , M. Tsutsumiuchi , Y. Ichinose et al. , 2020 VPS13D-related disorders presenting as a pure and complicated form of hereditary spastic paraplegia . Mol Genet Genomic Med 8 ( 3 ): e1108 . OpenUrl CrossRef ↵ Lahiri , S. , A. Toulmay , and W.A. Prinz , 2015 Membrane contact sites, gateways for lipid homeostasis . Curr Opin Cell Biol 33 : 82 – 87 . OpenUrl CrossRef PubMed ↵ Laranjeiro , R. , G. Harinath , D. Burke , B.P. Braeckman , and M. Driscoll , 2017 Single swim sessions in C. elegans induce key features of mammalian exercise . BMC Biol 15 ( 1 ): 30 . OpenUrl CrossRef PubMed ↵ Lee , J.S. , T. Yoo , M. Lee , Y. Lee , E. Jeon et al. , 2020 Genetic heterogeneity in Leigh syndrome: Highlighting treatable and novel genetic causes . Clin Genet 97 ( 4 ): 586 – 594 . OpenUrl CrossRef PubMed ↵ Leonzino , M. , K.M. Reinisch , and P. De Camilli , 2021 Insights into VPS13 properties and function reveal a new mechanism of eukaryotic lipid transport . Biochim Biophys Acta Mol Cell Biol Lipids 1866 ( 10 ): 159003 . OpenUrl PubMed ↵ Lin , Y.F. , A.M. Schulz , M.W. Pellegrino , Y. Lu , S. Shaham et al. , 2016 Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response . Nature 533 ( 7603 ): 416 – 419 . OpenUrl CrossRef PubMed Meijer , I.A. , 2019 VPS13D Movement Disorder in GeneReviews(®) , edited by M.P. Adam , G.M. Mirzaa , R.A. Pagon , S.E. Wallace , L.J.H. Bean et al. University of Washington, Seattle Copyright © 1993-2023, University of Washington, Seattle . GeneReviews is a registered trademark of the University of Washington, Seattle . All rights reserved., Seattle (WA) . ↵ Nargund , A.M. , C.J. Fiorese , M.W. Pellegrino , P. Deng , and C.M. Haynes , 2015 Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt) . Mol Cell 58 ( 1 ): 123 – 133 . OpenUrl CrossRef PubMed ↵ Nordli , D.R. , 3rd . , and F.N. Galan , 2023 Progressive basal ganglia damage and EE-SWAS in a patient with a VPS13D pathogenic variant . Epileptic Disord 25 ( 1 ): 117 – 119 . OpenUrl CrossRef PubMed ↵ Oztop-Cakmak , O. , G. Simsir , S. Tekgul , M.S. Aygun , O. Gokler et al. , 2022 VPS13D-based disease: Expansion of the clinical phenotype in two brothers and mutation diversity in the Turkish population . Rev Neurol (Paris) 178 ( 9 ): 907 – 913 . OpenUrl CrossRef PubMed ↵ Pauly , M.G. , N. Bruggemann , S. Efthymiou , A. Grozinger , S.H. Diaw et al. , 2023 Not to Miss: Intronic Variants, Treatment, and Review of the Phenotypic Spectrum in VPS13D-Related Disorder . Int J Mol Sci 24 ( 3 ). ↵ Pellegrino , M.W. , and C.M. Haynes , 2015 Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial infection . BMC Biol 13 : 22 . OpenUrl CrossRef PubMed ↵ Petry-Schmelzer , J.N. , N. Keller , M. Karakaya , B. Wirth , G.R. Fink et al. , 2021 VPS13D: One Family, Same Mutations, Two Phenotypes . Mov Disord Clin Pract 8 ( 5 ): 803 – 806 . OpenUrl CrossRef PubMed ↵ Pickles , S. , P. Vigie , and R.J. Youle , 2018 Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance . Curr Biol 28 ( 4 ): R170 – R185 . OpenUrl CrossRef PubMed ↵ Pickrell , A.M. , and R.J. Youle , 2015 The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease . Neuron 85 ( 2 ): 257 – 273 . OpenUrl CrossRef PubMed ↵ Rolland , S. , and B. Conradt , 2022 Genetic screen identifies non-mitochondrial proteins involved in the maintenance of mitochondrial homeostasis . MicroPubl Biol 2022 . ↵ Rolland , S.G. , Y. Lu , C.N. David , and B. Conradt , 2009 The BCL-2-like protein CED-9 of C. elegans promotes FZO-1/Mfn1,2- and EAT-3/Opa1-dependent mitochondrial fusion . J Cell Biol 186 ( 4 ): 525 – 540 . OpenUrl Abstract / FREE Full Text ↵ Seong , E. , R. Insolera , M. Dulovic , E.J. Kamsteeg , J. Trinh et al. , 2018 Mutations in VPS13D lead to a new recessive ataxia with spasticity and mitochondrial defects . Ann Neurol 83 ( 6 ): 1075 – 1088 . OpenUrl CrossRef PubMed ↵ Shen , J.L. , T.M. Fortier , R. Wang , and E.H. Baehrecke , 2021a Vps13D functions in a Pink1-dependent and Parkin-independent mitophagy pathway . J Cell Biol 220 ( 11 ). OpenUrl CrossRef ↵ Shen , J.L. , T.M. Fortier , Y.G. Zhao , R. Wang , M. Burmeister et al. , 2021b Vmp1, Vps13D, and Marf/Mfn2 function in a conserved pathway to regulate mitochondria and ER contact in development and disease . Curr Biol 31 ( 14 ): 3028 – 3039 e3027. OpenUrl CrossRef PubMed ↵ Shpilka , T. , Y. Du , Q. Yang , A. Melber , N. Uma Naresh et al. , 2021 UPR(mt) scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans . Nat Commun 12 ( 1 ): 479 . OpenUrl CrossRef PubMed ↵ Shpilka , T. , and C.M. Haynes , 2018 The mitochondrial UPR: mechanisms, physiological functions and implications in ageing . Nat Rev Mol Cell Biol 19 ( 2 ): 109 – 120 . OpenUrl CrossRef PubMed ↵ Skarnes , W.C. , B. Rosen , A.P. West , M. Koutsourakis , W. Bushell et al. , 2011 A conditional knockout resource for the genome-wide study of mouse gene function . Nature 474 ( 7351 ): 337 – 342 . OpenUrl CrossRef PubMed Web of Science ↵ Sultan , T. , G. Scorrano , M. Panciroli , M. Christoforou , J. Raza Alvi et al. , 2024 Clinical and molecular heterogeneity of VPS13D-related neurodevelopmental and movement disorders . Gene 899 : 148119 . OpenUrl CrossRef PubMed ↵ Swartz , B.E. , M. Burmeister , J.T. Somers , K.G. Rottach , I.N. Bespalova et al. , 2002 A form of inherited cerebellar ataxia with saccadic intrusions, increased saccadic speed, sensory neuropathy, and myoclonus . Ann N Y Acad Sci 956 : 441 – 444 . OpenUrl CrossRef PubMed Web of Science ↵ Swierczek , N.A. , A.C. Giles , C.H. Rankin , and R.A. Kerr , 2011 High-throughput behavioral analysis in C. elegans . Nat Methods 8 ( 7 ): 592 – 598 . OpenUrl CrossRef PubMed Web of Science ↵ Taylor , S.R. , G. Santpere , A. Weinreb , A. Barrett , M.B. Reilly et al. , 2021 Molecular topography of an entire nervous system . Cell 184 ( 16 ): 4329 – 4347 e4323. OpenUrl CrossRef PubMed ↵ Verhoeven , K. , K.G. Claeys , S. Zuchner , J.M. Schroder , J. Weis et al. , 2006 MFN2 mutation distribution and genotype/phenotype correlation in Charcot-Marie-Tooth type 2 . Brain 129 ( Pt 8 ): 2093 – 2102 . OpenUrl CrossRef PubMed Web of Science Wang Yu , W.S. , Cheng Yun , Song Bin , Jin Ping , Zhu Yulong , Sun Dandan , Ai Wenlong , Fu Xiaoming , Ye Qunrong , Li Kai , Wang Xun , 2021 A case report of complex heterozygous VPS13D mutation with spinocerebellar ataxia recessive type 4 . Chinese medical case repository 03 ( 01 ): E211 – E211 . OpenUrl ↵ Xin , N. , J. Durieux , C. Yang , S. Wolff , H.E. Kim et al. , 2022 The UPRmt preserves mitochondrial import to extend lifespan . J Cell Biol 221 ( 7 ). OpenUrl CrossRef ↵ Yang , Q. , P. Liu , N.S. Anderson , T. Shpilka , Y. Du et al. , 2022 LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria . Nat Cell Biol 24 ( 2 ): 181 – 193 . OpenUrl CrossRef PubMed ↵ Yoneda , T. , C. Benedetti , F. Urano , S.G. Clark , H.P. Harding et al. , 2004 Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones . J Cell Sci 117 ( Pt 18 ): 4055 – 4066 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted January 25, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans Xiaomeng Yin , Ruoxi Wang , Andrea Thackeray , Eric H. Baehrecke , Mark J. Alkema bioRxiv 2025.01.22.634397; doi: https://doi.org/10.1101/2025.01.22.634397 Share This Article: Copy Citation Tools VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans Xiaomeng Yin , Ruoxi Wang , Andrea Thackeray , Eric H. Baehrecke , Mark J. Alkema bioRxiv 2025.01.22.634397; doi: https://doi.org/10.1101/2025.01.22.634397 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18558) Cell Biology (25460) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15589) Genomics (22475) Immunology (17711) Microbiology (40327) Molecular Biology (17145) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)