ARTICLE PREPRINT 1 TorsinA is essential for the timing and localization of neuronal nuclear pore complex biogenesis Sumin Kim1,2, Sébastien Phan3, Thomas R. Shaw4, Mark H. Ellisman3, Sarah L. Veatch4, Sami J. Barmada1,2,*, Samuel S. Pappas5,6,*, and William T. Dauer5,6,7,* 1Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI;2Department of Neurology, University of Michigan, Ann Arbor, MI;3National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA;4Department of Biophysics, University of Michigan, Ann Arbor, MI;5Peter O9Donnell Jr. Brain Institute, UT Southwestern, Dallas, TX;6Department of Neurology, UT Southwestern, Dallas, TX;7Department of Neuroscience, UT Southwestern, Dallas, TX; *To whom correspondence should be addressed:sbarmada@med.umich.edu;Samuel.Pappas@utsouthwestern.edu;William.Dauer@utsouthwestern.edu Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss offunctioncausestheneurodevelopmentalmovementdisorderDYT-TOR1A(DYT1)dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs. INTRODUCTION Nuclear pore complexes (NPCs) are large, evolutionarily conserved structures that serve as the gateway between the nucleus and cytoplasm, allowing passive diffusion of small moleculesandfacilitatingnucleocytosolictransportof proteins and RNA1,2. Regulation of NPC biogenesis and functioniscriticalforcoordinatinginformationtransfer betweenthenucleusandcytoplasm,allowingcellsto dynamically respond to internal and external cues. Neurons are heavily dependent on NPC function; neuronal plasticity requires transport of signaling molecules and transcription factors336and mRNA export for local translation is essential for several neurodevelopmental processes7311. Accordingly, NPCdefectsarepresentinseveralnervoussystem diseases12316and mutations in nucleoporins cause early- onsetneurologicalillness17,18.Indeed,neuronsfacea considerablechallengeinmaintainingpropernuclear functionthroughouttheirlifetime.Whereasmitoticcells disassemble and recreate the nucleus with each division, neurons must manage nuclear pore number and localization within a closed interphase nucleus.Neuronal NPCs exhibit minimal turnover and nucleoporins (Nups) that constitute the NPC are among the longest-lived proteins19,20, underscoring theuniquechallengeneuronsfaceinregulatingNPC formation and function. Despite the biological and clinical importanceoftheseevents,littleisunderstoodabout neuronalNPCbiogenesis.Furthermore,mechanisms underlying NPC number, organization, and dynamics in both neuronal and non-neuronal systems remain elusive. TorsinA appears to lie at the intersection of neuronal NPC biogenesis and neurodevelopmental disease. TorsinA is a AAA+ protein that resides in the endoplasmic reticulum (ER)/nuclearenvelope(NE)lumen21329.The neurodevelopmentalmovementdisorderDYT-TOR1A (DYT1) dystonia is caused by an in-frame 3-bp deletion in theTOR1AgenethatencodesaE-torsinAmutant protein30,31. Several observations demonstrate that the NE is an active site of torsinA activity. A <substrate trap= mutant of torsinAprimarilylocalizestotheNE24,25andperturbing torsinAlevelscauseschangesintheLINC(linkerof nucleoskeletonandcytoskeleton)complex32,33. Mislocalizationofnuclearmembraneproteinsisalso observed inC. elegansgerm cells lacking the torsin homolog OOC-5, which additionally exhibit asymmetric plaques of Nups34. TorsinA-knockout (KO) or homozygous E mutant mice develop abnormal NE evaginations or <blebs= exclusively in post-migratory maturing neurons35, establishing E as a loss-of-function (LOF)mutation.Biochemicalstudies are consistent with a LOF effect of the E mutation36,37. NE blebs are inner nuclear membrane (INM) outpouchings that project into the NE lumen. These blebs occur transiently, emerging .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 27, 2023.;https://doi.org/10.1101/2023.04.26.538491doi:bioRxiv preprint
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