Nuclear Envelope Budding Research Articles

Nuclear envelope budding inhibition slows down progerin-induced aging process

Progerin causes Hutchinson-Gilford progeria syndrome (HGPS), but how progerin accelerates aging is still an interesting question. Here, we provide evidence linking nuclear envelope (NE) budding and accelerated aging. Mechanistically, progerin disrupts nuclear lamina to induce NE budding in concert with lamin A/C, resulting in transport of chromatin into the cytoplasm where it is removed via autophagy, whereas emerin antagonizes this process. Primary cells from both HGPS patients and mouse models express progerin and display NE budding and chromatin loss, and ectopically expressing progerin in cells can mimic this process. More excitingly, we screen a NE budding inhibitor chaetocin by high-throughput screening, which can dramatically sequester progerin from the NE and prevent this NE budding through sustaining ERK1/2 activation. Chaetocin alleviates NE budding-induced chromatin loss and ameliorates HGPS defects in cells and mice and significantly extends lifespan of HGPS mice. Collectively, we propose that progerin-induced NE budding participates in the induction of progeria, highlight the roles of chaetocin and sustained ERK1/2 activation in anti-aging, and provide a distinct avenue for treating HGPS.

Vesicular translocation of PARP-1 to cytoplasm causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

ADP-ribosylation plays a significant role in various biological processes including genomic stability maintenance, transcriptional regulation, energy metabolism, and cell death. Using macrodomain pull-down assay with microglia lysates and MALDI-TOF-MS analysis, we identified vimentin as a major protein highly ADP-ribosylated by the poly(ADP-ribose) polymerases-1 (PARP-1) in response to LPS. ABT-888, a potent inhibitor of PARP-1/2 blocks the disassembly and ADP-ribosylation of vimentin. PARP-1 is a highly abundant nuclear protein. Its nuclear functions in repairing DNA damages induced by various stress signals, such as inflammatory stresses, have been well studied. In contrast, limited studies have been done on the cytoplasmic role(s) of PARP-1. Our study focuses on the cytoplasmic role of PARP-1 during microglia activation. Using immunofluorescence microscopy and Western blotting, we showed that a significant amount of PARP-1 is present in the cytosol of microglia cells stimulated and activated by LPS. Live cell imaging showed the translocation of nuclear PARP-1-EGFP to the cytoplasm in vesicular structures upon LPS stimulation. ABT-888 and U0126 can block this translocation. Immunofluorescence staining with various organelle marker antibodies revealed that PARP-1 vesicles show colocalization with Lamin A/C, suggesting they might be derived from the nuclear envelope through nuclear envelope budding. In conclusion, we demonstrated that PARP-1 is translocated from the nucleus to cytoplasm via vesicles upon LPS stimulation and that cytoplasmic PARP-1 causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

Nuclear envelope budding: Getting large macromolecular complexes out of the nucleus.

Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.

Centralspindlin proteins Pavarotti and Tumbleweed along with WASH regulate nuclear envelope budding.

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.

Ceramide-rich microdomains facilitate nuclear envelope budding for non-conventional exosome formation.

Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B4 (LTB4) refs. 1,2. We previously demonstrated that LTB4 and its synthesizing enzymes, 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and leukotriene A4 hydrolase, are packaged and released in exosomes3. Here we report that the biogenesis of the LTB4-containing exosomes originates at the nuclear envelope (NE) of activated neutrophils. We show that the neutral sphingomyelinase 1 (nSMase1)-mediated generation of ceramide-enriched lipid-ordered microdomains initiates the clustering of the LTB4-synthesizing enzymes on the NE. We isolated and analysed exosomes from activated neutrophils and established that the FLAP/5-LO-positive exosome population is distinct from that of the CD63-positive exosome population. Furthermore, we observed a strong co-localization between ALIX and FLAP at the periphery of nuclei and within cytosolic vesicles. We propose that the initiation of NE curvature and bud formation is mediated by nSMase1-dependent ceramide generation, which leads to FLAP and ALIX recruitment. Together, these observations elucidate the mechanism for LTB4 secretion and identify a non-conventional pathway for exosome generation.

Nuclear envelope budding is a response to cellular stress

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.

Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding.

Nuclear envelope (NE) budding is a recently described phenomenon wherein large macromolecular complexes are packaged inside the nucleus and extruded through the nuclear membranes. Although 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 in a Drosophila model system. 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 by 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.

Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding

Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marfwas insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis ofNE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

Torsin Mediates Primary Envelopment of Large Ribonucleoprotein Granules at the Nuclear Envelope

A previously unrecognized mechanism through which large ribonucleoprotein (megaRNP) granules exit the nucleus is by budding through the nuclear envelope (NE). This mechanism is akin to the nuclear egress of herpes-type viruses and is essential for proper synapse development. However, the molecular machinery required to remodel the NE during this process is unknown. Here, we identify Torsin, an AAA-ATPase that in humans is linked to dystonia, as a major mediator of primary megaRNP envelopment during NE budding. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the messenger RNAs contained within fail to reach synaptic sites, preventing normal synaptic protein synthesis and thus proper synaptic bouton development. These studies begin to establish the cellular machinery underlying the exit of megaRNPs via budding, offer an explanation for the "nuclear blebbing" phenotype found in dystonia models, and provide an important link between Torsin and the synaptic phenotypes observed in dystonia.

Nuclear Envelope Budding Enables Large Ribonucleoprotein Particle Export during Synaptic Wnt Signaling

Localized protein synthesis requires assembly and transport of translationally silenced ribonucleoprotein particles (RNPs), some of which are exceptionally large. Where in the cell such large RNP granules first assemble was heretofore unknown. We previously reported that during synapse development, a fragment of the Wnt-1 receptor, DFrizzled2, enters postsynaptic nuclei where it forms prominent foci. Here we show that these foci constitute large RNP granules harboring synaptic protein transcripts. These granules exit the nucleus by budding through the inner and the outer nuclear membranes in a nuclear egress mechanism akin to that of herpes viruses. This budding involves phosphorylation of A-type lamin, a protein linked to muscular dystrophies. Thus nuclear envelope budding is an endogenous nuclear export pathway for large RNP granules.