Torsin ATPases are required to complete nuclear pore complex biogenesis in interphase

Abstract

32Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the 33 phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the 34 connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based 35 approach to identify MLF2 as a luminal component of the bleb. Using an MLF2-based live cell 36imaging platform, we demonstrate that NE blebbing occurs rapidly and synchronously 37 immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of 38 ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane 39 nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for 40 interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG 41 nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its 42 dependence on POM121, and a reduction of mature NPCs in Torsin deficient cells lead us to 43 conclude that the hallmark phenotype of Torsin manipulation represents the accumulation of 44 stalled NPC assembly intermediates. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 We previously presented a system that resolves both of these limitations by generating a 93 quadruple Torsin deletion HeLa cell line (designated 4TorKO) in which all four Torsin genes 94 (TorsinA, TorsinB, Torsin 2A and Torsin3A) have been deleted using CRISPR/Cas9 genome 95 engineering. This 4TorKO cell line abundantly exhibits the hallmark cellular phenotype of NE 96 blebbing in which the inner nuclear membrane (INM) bulges into the perinuclear space (PNS) to 97 form an omega-shaped herniation. Ubiquitin (Ub) conjugates of the K48 linkage type are 98 enriched in the lumen of the bleb in 4TorKO cells and in mouse models of Torsin dysfunction 99

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1

“…Based on studies in human cells, mice and Drosophila, diverse roles of Torsins have been suggested, including functions in ER homeostasis, secretion, lipid metabolism, NPC biogenesis, and the nuclear export of large particles like mRNPs or HSV1 capsids by a vesicular transport process across the NE (Chen et al, 2010, Grillet et al, 2016, Jokhi et al, 2013, Maric et al, 2011, Nery et al, 2011, Pappas et al, 2018, Rampello et al, 2019, Shin et al, 2019, Turner et al, 2015. However, while all these suggested functions are supported by the reported cellular phenotypes, biochemical and genetic data, it is currently unclear what are direct or indirect consequences of Torsin function since the molecular mechanism of Torsin action has remained elusive.…”

The Role of Torsin AAA+ Proteins in Preserving Nuclear Envelope Integrity and Safeguarding Against Disease

“…Upon Torsin manipulation, interphase assembly is stalled presumably prior to the fusion of the inner nuclear membrane (INM)/outer nuclear membrane (ONM). Note that cytoplasmic fibrils (CFs) containing NUP358 are only added to the nascent NPC after INM/ONM fusion during interphase assembly (i.e., the observed absence of NUP358 from blebs containing NPC components supports the idea of blebs representing stalled NPC assembly intermediates) [32,84].…”

“…While there is accumulating evidence indicating that Torsins contribute a critical function in NPC biogenesis, their precise role in this molecular pathway remains largely unknown. The recent finding that the loss of TorsinA and its three paralogs in HeLa cells results in a stalling of interphase assembly provides new insight into this mode of NPC assembly [32]. The formation of Nup-containing NE blebs follows a cell cycle-dependent growth and loss pattern, with an increase in the number during interphase and a substantial reduction following mitosis (Figure 3).…”

“…However, many distinct properties of Torsins produce a complicated system that has been reported to affect an ever-expanding number of cellular processes (Figure 1). Some of these diverse processes include lipid metabolism [21][22][23], nucleo-cytoskeleton coupling [24][25][26], membrane remodeling [13,27,28], ER redox monitoring [7,29], nuclear pore complex (NPC) biogenesis [30][31][32], and protein quality control [33][34][35][36][37] (Figure 1). In this review, we discuss recent findings that support roles for Torsins in NPC biogenesis and lipid metabolism and speculate how its AAA+ properties may relate to these processes.…”

“…The dynamics of bleb formation were also monitored through a live cell imaging platform utilizing lattice light sheet microscopy and a fluorescently tagged derivative of myeloid leukemia factor 2 (MLF2), a protein highly enriched in the bleb lumen [32]. These aberrant structures arise rapidly and are remarkably synchronous during early G1 phase at~700 s after anaphase onset.…”