Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. While a general outline of the cellular events occurring during NE-budding is now in place, little is yet known about the molecular machinery and mechanisms underlying the physical aspects of NE-bud formation. Using a multidisciplinary approach, we identify Wash, its regulatory complex (SHRC), capping protein, and Arp2/3 as new molecular components involved in the physical aspects of NE-bud formation. Interestingly, Wash affects NE-budding in two ways: indirectly through general nuclear lamina disruption via an SHRC-independent interaction with Lamin B leading to inefficient NE-bud formation, and directly blocking NE-bud formation along with its SHRC, capping protein, and Arp2/3. In addition to NE-budding emerging as an important cellular process, it shares many similarities with herpesvirus nuclear egress mechanisms suggesting new avenues for exploration in both normal and disease biology.

Nuclear envelope (NE) budding is a nuclear pore–independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development, and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott–Aldrich Syndrome protein, Wash, its regulatory complex (SHRC), and Arp2/3, and requires Wash’s actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin-bundling activity of Pav is required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development.

Nuclear envelope (NE) budding is a nuclear pore independent nuclear export pathway, analogous to the egress of herpesviruses, and required for protein quality control, synapse development and mitochondrial integrity. The physical formation of NE buds is dependent on the Wiskott-Aldrich Syndrome protein Wash, its regulatory complex (SHRC), and Arp2/3, and requires Wash actin nucleation activity. However, the machinery governing cargo recruitment and organization within the NE bud remains unknown. Here, we identify Pavarotti (Pav) and Tumbleweed (Tum) as new molecular components of NE budding. Pav and Tum interact directly with Wash and define a second nuclear Wash-containing complex required for NE budding. Interestingly, we find that the actin bundling activities of Wash and Pav are required, suggesting a structural role in the physical and/or organizational aspects of NE buds. Thus, Pav and Tum are providing exciting new entry points into the physical machineries of this alternative nuclear export pathway for large cargos during cell differentiation and development.

A 74-year-old woman was urgently referred to haematology. She had initially presented to her general practitioner with breathlessness and weight loss. She was a long-term smoker. A chest X-ray showed a widened mediastinum, and a follow up computerized tomography (CT) scan (left) demonstrated marked thoracic lymphadenopathy and splenomegaly of 19 cm. There were no palpable peripheral lymph nodes. The clinical suspicion was of lymphoma.Her full blood count showed a haemoglobin concentration of 134 g/l, platelet count of 139 · 10 9 /l and normal white count and differential. A blood film demonstrated numerous spherocytes and polychromasia. Lactate dehydrogenase was 212 iu/l (normal: 140-240), bilirubin 99 lmol/l (normal: 2-20) and Coombs test negative. She had a past medical history and family history of hereditary spherocytosis (HS). This was later confirmed by a positive eosin-5maleimide binding test.A bone marrow aspirate and trephine biopsy demonstrated erythroid hyperplasia only. A CT guided biopsy of the mediastinal mass demonstrated extramedullary haematopoiesis (EMH), with no evidence of lymphoma (right). A videoassisted thoracoscopic biopsy similarly showed only EMH.She was negative for the JAK2 V617F mutation. High performance liquid chromatography, for variant haemoglobins and thalassaemia, was normal. A subsequent splenectomy demonstrated a disrupted splenic parenchyma with an increase in the red pulp and no evidence of neoplasia, which would be consistent with splenomegaly secondary to HS.The patient is now symptomatically better, having had a splenectomy to reduce the haemolysis, the presumed driving force for EMH, and also having had her chronic obstructive airways disease managed optimally. A follow-up CT scan 4 months post-splenectomy showed some reduction in the thoracic paravertebral soft tissue mass with increased fat content, suggesting conversion of haematopoietic marrow to fat.This case illustrates how significant posterior mediastinal widening can be due to EMH, which is a rare complication of chronic haemolysis. EMH has been reported in congenital haemolytic anaemias, such as HS, and in thalassaemias, as well as in malignant and myeloproliferative disorders, such as primary myelofibrosis.Case reports of treatment for symptomatic EMH have demonstrated varying success. Treating the underlying cause can reduce the driving force for EMH, resulting in regression of the mass. More immediate benefit can be seen with radiotherapy or surgical resection; however the latter can result in significant bleeding. It must be born in mind that EMH is usually a compensatory mechanism for haemolysis.

Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes can be packaged inside the nucleus and be extruded through the nuclear membranes, completely bypassing nuclear pores. While factors have been identified both as cargos or actively involved in this process, much remains unknown about the molecules that generate the forces and membrane deformations which appear inherent. Using fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model, we identify Wash, its regulatory complex, and Arp2/3 as novel players in NE-budding. Surprisingly, Wash's role in this process is bipotent and, independent of Recently, Nuclear Envelope (NE-) budding was identified as an alternative pathway for nuclear exit, particularly for large developmentally-required ribonucleoprotein (megaRNP) complexes that would otherwise need to unfold/remodel to fit through the NPCs (Fradkin and Budnik, 2016;Hatch and Hetzer, 2014;Hatch and Hetzer, 2012;Jokhi et al., 2013;Li et al., 2016;Parchure et al., 2017;Speese et al., 2012). In this pathway, large macromolecule complexes, such as megaRNPs, are encircled by the nuclear lamina (type-A and type-B lamins) and inner nuclear membrane, are pinched off from the inner nuclear membrane, cross the perinuclear space, fuse with the outer nuclear membrane, and release the megaRNPs into the cytoplasm (Figure 1A-C).Strikingly, NE-budding shares many features with the nuclear egress mechanism used by herpesviruses, common pathogens that cause and/or contribute to a diverse array of human diseases (Bigalke and Heldwein, 2016;Hagen et al., 2015;Lye et al., 2017;Mettenleiter et al., 2013;Parchure et al., 2017;Roller and Baines, 2017). As viruses often take advantage of preexisting host pathways for their livelihoods, the parallel between nuclear exit of herpesvirus nucleocapsids and that of megaRNPs suggests that NE-budding may be a general cellular mechanism that elegantly allows for the nuclear export of endogenous megaRNPs and/or other large cargos (cf. (Fradkin and Budnik, 2016;Mettenleiter et al., 2013;Parchure et al., 2017;Roller and Baines, 2017). Indeed, this pathway has also been implicated in the removal of obsolete macromolecular complexes or other material (i.e., large protein aggregates, polyubiquitylated proteins) from the nucleus (Jokhi et al., 2013;Ramaswami et al., 2013;Rose and Schlieker, 2012).The NE-budding pathway was first demonstrated in Drosophila synapse development, proving to be essential for neuromuscular junction integrity. Here, a C-terminal fragment (dFz2C) of the fly Wingless receptor, dFz2C, was shown to associate with megaRNPs that formed foci at the nuclear periphery and exited the nucleus by budding through the nuclear envelope ( Figure 1B-C) (Speese et al., 2012). Failure of this process resulted in aberrant