Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2

Abstract

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

The Torsin/ NEP1R1-CTDNEP1/ Lipin axis regulates nuclear envelope lipid metabolism for nuclear pore complex insertion

“…4N). Similar herniations contain nucleoporins in mammals (Rampello et al, 2020), and are reminiscent of immature NPC (Otsuka et al, 2016). We also examined Torip -/animals.…”

“…We focused on the role of Torsin in the poorly understood process of interphase NPC insertion ( Fig. 4A & B; (Rampello et al, 2020)). Notably, fat body cells exit the cell cycle after embryogenesis (Zheng et al, 2016) and thus, are ideal to study interphase events without interference from mitotic NE breakdown.…”

“…Torsin loss impairs nuclear pore complex (NPC) biogenesis and levels in worm and mammals (Rampello et al, 2020;VanGompel et al, 2015). Torsin and LAP1 loss also produce abnormal vesicle-like membranes in the NE lumen (Goodchild et al, 2005;Kim et al, 2010) which potentially represent stalled nuclear egress vesicles (Jokhi et al, 2013), or intermediates of NPC insertion (Rampello et al, 2020). These phenomena underlie the theory that Torsin-LAP1 ATPase activity directly induces membrane scission (Jokhi et al, 2013).…”

Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

“…Such an idea fits well with work exploring the mechanism behind the biogenesis of similar herniations observed in cells with a loss of function of the AAA+ ATPase, TorsinA (Goodchild et al, 2005; Laudermilch et al, 2016). As data continues to grow linking these herniations to defective NPC assembly (Laudermilch et al, 2016; Pappas et al, 2018; Rampello et al, 2020), so does evidence that they could be a product of defective lipid metabolism; in fact recent work suggests that TorsinA may inhibit Lipin (Grillet et al, 2016; Cascalho et al, 2019). Second, and as follows, while ultimately too much PA may be deleterious (Oliveira et al, 2010; Adeyo et al, 2011; Park et al, 2015), it seems probable that a tightly controlled system of PA generation may actually promote INM-ONM fusion during interphase NPC assembly.…”

“…Genetic evidence is mounting, however, that local changes in lipid metabolism may contribute to the NPC biogenesis mechanism, likely at the step of INM-ONM fusion (Schneiter et al, 1996; Siniossoglou et al, 1998; Scarcelli et al, 2007; Hodge et al, 2010; Lone et al, 2015; Zhang et al, 2018). For example, the formation of nuclear envelope blebs or herniations that appear over malformed NPCs might occur due to an inhibition of INM-ONM fusion (Wente and Blobel, 1993; Scarcelli et al, 2007; Onischenko et al, 2017; Zhang et al, 2018; Thaller et al, 2019; Allegretti et al, 2020; Rampello et al, 2020). These structures have been observed in both yeast and metazoan systems (Thaller and Lusk, 2018) and have been associated with defects in lipid metabolism (Schneiter et al, 1996; Grillet et al, 2016).…”