Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

SummaryNuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation

Correction: Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila

International audienceNuclear shape is different in stem cells and differentiated cells and reflects important changes in the mechanics of the nuclear envelope (NE). The current framework emphasizes the key role of the nuclear lamina in nuclear mechanics and its alterations in disease. Whether active stress controls nuclear deformations and how this stress interplays with properties of the NE to control NE dynamics is unclear. We address this in the early Drosophila embryo, in which profound changes in NE shape parallel the transcriptional activation of the zygotic genome. We show that microtubule (MT) polymerization events produce the elementary forces necessary for NE dynamics. Moreover, large-scale NE deformations associated with groove formation require concentration of MT polymerization in bundles organized by Dynein. However, MT bundles cannot produce grooves when the farnesylated inner nuclear membrane protein Kugelkern (Kuk) is absent. Although it increases stiffness of the NE, Kuk also stabilizes NE deformations emerging from the collective effect of MT polymerization forces concentrated in bundles. Finally, we report that MT-induced NE deformations control the dynamics of chromatin and its organization at steady state. Thus, the NE is a dynamic organelle, fluctuations of which increase chromatin dynamics. We propose that such mechanical regulation of chromatin dynamics by MTs might be important for gene regulation

Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos

International audienceNuclear shape is different in stem cells and differentiated cells and reflects important changes in the mechanics of the nuclear envelope (NE). The current framework emphasizes the key role of the nuclear lamina in nuclear mechanics and its alterations in disease 1, 2 . Whether active stress controls nuclear deformations and how this stress interplays with properties of the NE to control NE dynamics is unclear. We address this in the early Drosophila embryo, where profound changes in NE shape parallel the transcriptional activation of the zygotic genome. We show that microtubule (MT) polymerization events produce the elementary forces necessary for NE dynamics. Moreover, large-scale NE-deformations associated with groove formation require concentration of microtubule polymerization in bundles organized by Dynein. However, MT bundles cannot produce grooves when the farnesylated inner nuclear membrane protein Charleston/Kugelkern (Char/Kuk) is absent 3, 4 . Although it increases stiffness of the NE, Char/Kuk also stabilizes NE deformations emerging from the collective effect of MT polymerization forces concentrated in bundles. Finally we report that MT induced NE deformations control the dynamics of the chromatin and its organization at steady state. Thus, the NE is a dynamic organelle, whose fluctuations increase chromatin dynamics. We propose that such mechanical regulation of chromatin dynamics by MT may be important for gene regulation

Microtubule-induced nuclear envelope fluctuations control chromatin dynamics in Drosophila embryos

International audienceNuclear shape is different in stem cells and differentiated cells and reflects important changes in the mechanics of the nuclear envelope (NE). The current framework emphasizes the key role of the nuclear lamina in nuclear mechanics and its alterations in disease. Whether active stress controls nuclear deformations and how this stress interplays with properties of the NE to control NE dynamics is unclear. We address this in the early Drosophila embryo, in which profound changes in NE shape parallel the transcriptional activation of the zygotic genome. We show that microtubule (MT) polymerization events produce the elementary forces necessary for NE dynamics. Moreover, large-scale NE deformations associated with groove formation require concentration of MT polymerization in bundles organized by Dynein. However, MT bundles cannot produce grooves when the farnesylated inner nuclear membrane protein Kugelkern (Kuk) is absent. Although it increases stiffness of the NE, Kuk also stabilizes NE deformations emerging from the collective effect of MT polymerization forces concentrated in bundles. Finally, we report that MT-induced NE deformations control the dynamics of chromatin and its organization at steady state. Thus, the NE is a dynamic organelle, fluctuations of which increase chromatin dynamics. We propose that such mechanical regulation of chromatin dynamics by MTs might be important for gene regulation

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes

The structures of the two leader protease (L(pro)) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 Å. This singular, pseudo-polymeric organization is present in a new L(pro) crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this L(pro) structure, refined at 1.9 Å resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of L(pro) activity on a fluorescent hexapeptide substrate showed that L(pro), in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the L(pro) structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties. (C) 2000 Academic Press.This work was supported by DGICYT (BIO99-0865 to I.F. and PB96-0271 to J.T.) and by the Austrian Science Foundation (P-11222 and P-13667 to T.S.). Data collection in Hamburg was supported by the Human Capital Mobility Project, contract CHGE-CT93-0040Peer Reviewe