Nafiseh Sabri

The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) mutants, whereas the level of CRM1-dependent EGFP-nuclear export signal (EGFP-NES) export is increased. We show that the nuclear accumulation of the Drosophila Rel protein Dorsal requires CRM1. DNup88 binds to DNup214 and DCRM1 in vitro, and both proteins become mislocalized from the nuclear rim into the nucleus of mbo mutants. Overexpression of DNup88 is sufficient to relocalize DNup214 and CRM1 on the nuclear envelope and revert the mutant phenotypes. We propose that a major function of DNup88 is to anchor DNup214 and CRM1 on the nuclear envelope and thereby attenuate NES-mediated nuclear export.

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)–rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin α/β−mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events.

The nucleoporin Nup214 sequesters CRM1 at the nuclear rim and modulates NFκB activation in Drosophila

CRM1-mediated protein export is an important determinant of the nuclear accumulation of many gene regulators. Here, we show that the NFκB transcription factor Dorsal is a substrate of CRM1 and requires the nucleoporin Nup214 for its nuclear translocation upon signaling. Nup214 bound to CRM1 directly and anchored it to the nuclear envelope. In nup214 mutants CRM1 accumulated in the nucleus and NES-protein export was enhanced. Nup214 formed complexes with Nup88 and CRM1 in vivo and Nup214 protected Nup88 from degradation at the nuclear rim. In turn, Nup88 was sufficient for targeting the complex to the nuclear pores. Overexpression experiments indicated that Nup214 alone attracts a fraction of CRM1 to the nuclear envelope but does not interfere with NES-GFP export. By contrast, overexpression of the Nup214-Nup88 complex trapped CRM1 and Dorsal to cytoplasmic foci and inhibited protein export and immune response activation. We hypothesize that variation in levels of the Nup214-Nup88 complex at the pore changes the amount of NPC-bound CRM1 and influences the relative strength and duration of NFκB signaling responses.

The Ct-RAE1 protein interacts with Balbiani ring RNP particles at the nuclear pore

RAE1 is an evolutionarily conserved protein that associates with both mRNPs and nucleoporins, and may bridge the interaction between mRNP export cargoes and the nuclear pore complex (NPC). However, the mechanism by which RAE1 functions in mRNA export is still unknown and the time point at which RAE1 interacts with the exported RNP has not been directly investigated. Here we have addressed this question in the Balbiani ring (BR) system of Chironomus tentans using immunoelectron microscopy. The RAE1 protein of C. tentans, Ct-RAE1, is 70% identical to human RAE1/mrnp41 (hRAE1) and is recognized by antibodies raised against hRAE1. As in vertebrate cells, Ct-RAE1 is concentrated at the nuclear envelope and also dispersed throughout the nuclear interior. Here we show that Ct-RAE1 does not bind to the BR particle either cotranscriptionally or in the nucleoplasm. Instead, the interaction between Ct-RAE1 and the exported BR particle occurs at the NPC. Moreover, the localization of Ct-RAE1 at the NPC is correlated with the presence of an exported RNP in the NPC. Finally, the anti-RAE1 antibody does not label the cytoplasmic side of BR particles in transit through the central channel, which indicates that Ct-RAE1 either remains anchored at the nuclear side of the NPC during translocation of the RNP through the central channel or becomes transiently associated with the RNP but is rapidly released into the cytoplasm.

The Nup155-mediated organisation of inner nuclear membrane proteins is independent of Nup155 anchoring to the metazoan nuclear pore complex

Summary The nuclear envelope (NE), an important barrier between the nucleus and the cytoplasm, is composed of three structures: the outer nuclear membrane, which is continuous with the ER, the inner nuclear membrane (INM), which interfaces with chromatin, and nuclear pore complexes (NPCs), which are essential for the exchange of macromolecules between the two compartments. The NPC protein Nup155 has an evolutionarily conserved role in the metazoan NE formation; but the in vivo analysis of Nup155 has been severely hampered by the essential function of this protein in cell viability. Here, we take advantage of the hypomorphicity of RNAi systems and use a combination of protein binding and rescue assays to map the interaction sites of two neighbouring NPC proteins Nup93 and Nup53 on Nup155, and to define the requirements of these interactions in INM protein organization. We show that different parts of Drosophila Nup155 have distinct functions: the Nup155 &bgr;-propeller anchors the protein to the NPC, whereas the &agr;-solenoid part of Nup155 is essential for the correct localisation of INM proteins lamin-B receptor (LBR) and otefin. Using chromatin extracts from semi-synchronized cells, we also provide evidence that the Nup155 &agr;-solenoid has a chromatin-binding activity that is stronger at the end of mitosis. Our results argue that the role of Nup155 in INM protein localisation is not mediated through the NPC anchoring activity of the protein and suggest that regions other than Nup155 &bgr;-propeller are necessary for the targeting of proteins to the INM.