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.

No abstract

Efficient synthesis of pyrano[3,2-c]pyranones motif as fused carbohybrids and their anticancer activities reported.

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved amongst yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63–79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452–480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc-phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.

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