Channel width modulates the permeability of DNA origami based nuclear pore mimics.

Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importing substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope.

Autophagic mechanisms that maintain nuclear envelope homeostasis are bulwarks to aging and disease. By leveraging 4D lattice light sheet microscopy and correlative light and electron tomography, we define a quantitative and ultrastructural timeline of a nuclear macroautophagy (nucleophagy) pathway in yeast. Nucleophagy initiates with a rapid local accumulation of the nuclear cargo adaptor Atg39 at the nuclear envelope adjacent to the nucleus-vacuole junction and is delivered to the vacuole in ~300 seconds through an autophagosome intermediate. Mechanistically, nucleophagy incorporates two consecutive and genetically defined membrane fission steps: inner nuclear membrane (INM) fission generates a lumenal vesicle in the perinuclear space followed by outer nuclear membrane (ONM) fission to liberate a double membraned vesicle to the cytosol. ONM fission occurs independently of phagophore engagement and instead relies surprisingly on dynamin-like protein1 (Dnm1), which is recruited to sites of Atg39 accumulation at the nuclear envelope. Loss of Dnm1 compromises nucleophagic flux by stalling nucleophagy after INM fission. Our findings reveal how nuclear and INM cargo are removed from an intact nucleus without compromising its integrity, achieved in part by a non-canonical role for Dnm1 in nuclear envelope remodeling.

An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

Changing the guard-nuclear pore complex quality control.

The integrity of the nuclear envelope depends on the function of nuclear pore complexes (NPCs), transport channels that control macromolecular traffic between the nucleus and cytosol. The central importance of NPCs suggests the existence of quality control (QC) mechanisms that oversee their assembly and function. In this perspective, we emphasize the challenges associated with NPC assembly and the need for QC mechanisms that operate at various stages of an NPC's life. This includes cytosolic preassembly QC that helps enforce key nucleoporin-nucleoporin interactions and their ultimate stoichiometry in the NPC in addition to mechanisms that monitor aberrant fusion of the inner and outer nuclear membranes. Furthermore, we discuss whether and how these QC mechanisms may operate to sense faulty mature NPCs to facilitate their repair or removal. The so far uncovered mechanisms for NPC QC provide fertile ground for future research that not only benefits a better understanding of the vital role that NPCs play in cellular physiology but also how loss of NPC function and/or these QC mechanisms might be an input to aging and disease.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity.

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

Kap-ß2/Transportin mediates ß-catenin nuclear transport in Wnt signaling.

Wnt signaling is essential for many aspects of embryonic development including the formation of the primary embryonic axis. In addition, excessive Wnt signaling drives multiple diseases including cancer, highlighting its importance for disease pathogenesis. ß-catenin is a key effector in this pathway that translocates into the nucleus and activates Wnt responsive genes. However, due to our lack of understanding of ß-catenin nuclear transport, therapeutic modulation of Wnt signaling has been challenging. Here, we took an unconventional approach to address this long-standing question by exploiting a heterologous model system, the budding yeast Saccharomyces cerevisiae, which contains a conserved nuclear transport machinery. In contrast to prior work, we demonstrate that ß-catenin accumulates in the nucleus in a Ran-dependent manner, suggesting the use of a nuclear transport receptor (NTR). Indeed, a systematic and conditional inhibition of NTRs revealed that only Kap104, the ortholog of Kap-ß2/Transportin-1 (TNPO1), was required for ß-catenin nuclear import. We further demonstrate direct binding between TNPO1 and ß-catenin that is mediated by a conserved PY-NLS. Finally, using Xenopus secondary axis and TCF/LEF (T Cell factor/lymphoid enhancer factor family) reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the nuclear localization signal in ß-catenin and its cognate NTR, our study suggests new therapeutic targets for a host of human diseases caused by excessive Wnt signaling. Indeed, we demonstrate that a small chimeric peptide designed to target TNPO1 can reduce Wnt signaling as a first step toward therapeutics.

Quality control mechanisms that protect nuclear envelope identity and function.

The nuclear envelope (NE) is a specialization of the endoplasmic reticulum with distinct biochemistry that defines inner and outer membranes connected at a pore membrane that houses nuclear pore complexes (NPCs). Quality control mechanisms that maintain the physical integrity and biochemical identity of these membranes are critical to ensure that the NE acts as a selective barrier that also contributes to genome stability and metabolism. As the proteome of the NE is highly integrated, it is challenging to turn over by conventional ubiquitin-proteasome and autophagy mechanisms. Further, removal of entire sections of the NE requires elaborate membrane remodeling that is poorly understood. Nonetheless, recent work has made inroads into discovering specializations of cellular degradative machineries tailored to meeting the unique challenges imposed by the NE. In addition, cells have evolved mechanisms to surveil and repair the NE barrier to protect against the deleterious effects of a breach in NE integrity, in the form of either a ruptured NE or a dysfunctional NPC. Here, we synthesize the most recent work exploring NE quality control mechanisms across eukaryotes.

Emerging Connections between Nuclear Pore Complex Homeostasis and ALS.

Developing effective treatments for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) requires understanding of the underlying pathomechanisms that contribute to the motor neuron loss that defines the disease. As it causes the largest fraction of familial ALS cases, considerable effort has focused on hexanucleotide repeat expansions in the C9ORF72 gene, which encode toxic repeat RNA and dipeptide repeat (DPR) proteins. Both the repeat RNA and DPRs interact with and perturb multiple elements of the nuclear transport machinery, including shuttling nuclear transport receptors, the Ran GTPase and the nucleoporin proteins (nups) that build the nuclear pore complex (NPC). Here, we consider recent work that describes changes to the molecular composition of the NPC in C9ORF72 model and patient neurons in the context of quality control mechanisms that function at the nuclear envelope (NE). For example, changes to NPC structure may be caused by the dysregulation of a conserved NE surveillance pathway mediated by the endosomal sorting complexes required for the transport protein, CHMP7. Thus, these studies are introducing NE and NPC quality control pathways as key elements in a pathological cascade that leads to C9ORF72 ALS, opening entirely new experimental avenues and possibilities for targeted therapeutic intervention.

One ring doesn't rule them all: Distinct nuclear pore complexes in a single cell.

Determining the structural plasticity and dynamics of nuclear pore complexes (NPCs) are major challenges. In this issue of Cell, Akey et al. present comprehensive NPC structures from in vitro and in vivo preparations, demonstrating multiple NPC forms in single cells while providing insight into the evolutionary origin of NPC architecture.

Nuclear pore complexes feel the strain.

A cryo-electron tomography structure of the human nuclear pore complex captured in cellulo by Schuller, Wojtynek et al. reveals that nuclear envelope tension expands the central transport channel and imposes asymmetry in the pore membrane.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Nuclear detox of unsaturated fat.

Fatty acid saturation in phospholipid bilayers alters their fluidity; whether saturation impacts inner nuclear membrane function has never been addressed. In this issue of Developmental Cell, Romanauska and Köhler (2021) show that the inner nuclear membrane detoxifies itself of unsaturated fatty acids by shunting them into cytosolic lipid droplets.

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS.

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)-derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43-associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

DNA-Origami NanoTrap for Studying the Selective Barriers Formed by Phenylalanine-Glycine-Rich Nucleoporins.

DNA nanotechnology provides a versatile and powerful tool to dissect the structure-function relationship of biomolecular machines like the nuclear pore complex (NPC), an enormous protein assembly that controls molecular traffic between the nucleus and cytoplasm. To understand how the intrinsically disordered, Phe-Gly-rich nucleoporins (FG-nups) within the NPC establish a selective barrier to macromolecules, we built a DNA-origami NanoTrap. The NanoTrap comprises precisely arranged FG-nups in an NPC-like channel, which sits on a baseplate that captures macromolecules that pass through the FG network. Using this biomimetic construct, we determined that the FG-motif type, grafting density, and spatial arrangement are critical determinants of an effective diffusion barrier. Further, we observed that diffusion barriers formed with cohesive FG interactions dominate in mixed-FG-nup scenarios. Finally, we demonstrated that the nuclear transport receptor, Ntf2, can selectively transport model cargo through NanoTraps composed of FxFG but not GLFG Nups. Our NanoTrap thus recapitulates the NPC's fundamental biological activities, providing a valuable tool for studying nuclear transport.

Heh2/Man1 may be an evolutionarily conserved sensor of NPC assembly state.

Integral membrane proteins of the Lap2-emerin-MAN1 (LEM) family have emerged as important components of the inner nuclear membrane (INM) required for the functional and physical integrity of the nuclear envelope. However, like many INM proteins, there is limited understanding of the biochemical interaction networks that enable LEM protein function. Here, we show that Heh2/Man1 can interact with major scaffold components of the nuclear pore complex (NPC), specifically the inner ring complex (IRC), in evolutionarily distant yeasts. Although an N-terminal domain is required for Heh2 targeting to the INM, we demonstrate that more stable interactions with the NPC are mediated by a C-terminal winged helix (WH) domain, thus decoupling INM targeting and NPC binding. Inhibiting Heh2's interactions with the NPC by deletion of the Heh2 WH domain leads to NPC clustering. Interestingly, Heh2's association with NPCs can also be disrupted by knocking out several outer ring nucleoporins. Thus, Heh2's interaction with NPCs depends on the structural integrity of both major NPC scaffold complexes. We propose a model in which Heh2 acts as a sensor of NPC assembly state, which may be important for NPC quality control mechanisms and the segregation of NPCs during cell division.

Direct binding of ESCRT protein Chm7 to phosphatidic acid-rich membranes at nuclear envelope herniations.

Mechanisms that control nuclear membrane remodeling are essential to maintain the integrity of the nucleus but remain to be fully defined. Here, we identify a phosphatidic acid (PA)-binding capacity in the nuclear envelope (NE)-specific ESCRT, Chm7, in budding yeast. Chm7's interaction with PA-rich membranes is mediated through a conserved hydrophobic stretch of amino acids, which confers recruitment to the NE in a manner that is independent of but required for Chm7's interaction with the LAP2-emerin-MAN1 (LEM) domain protein Heh1 (LEM2). Consistent with the functional importance of PA binding, mutation of this region abrogates recruitment of Chm7 to membranes and abolishes Chm7 function in the context of NE herniations that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that a PA sensor specifically accumulates within these NE herniations. We suggest that local control of PA metabolism is important for ensuring productive NE remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

Nucleoporin NUP205 plays a critical role in cilia and congenital disease.

Ciliopathies affect a variety of tissues during development including the heart, kidneys, respiratory tract, and retina. Though an increasing number of monogenic causes of ciliopathies have been described, many remain unexplained. Recently, recessive variants in NUP93 and NUP205 encoding two proteins of the inner ring of the nuclear pore complex were implicated as causes of steroid resistant nephrotic syndrome. In addition, we previously found that the inner ring nucleoporins NUP93 and NUP188 function in proper left-right patterning in developing embryos via a role at the cilium. Here, we describe the role of an additional inner ring nucleoporin NUP205 in cilia biology and establishment of normal organ situs. Using knockdown in Xenopus, we show that Nup205 depletion results in loss of cilia and abnormal cardiac morphology. Furthermore, by transmission electron microscopy, we observe a loss of cilia and mispositioning of intracellular ciliary structures such as basal bodies and rootlets upon depleting inner ring nucleoporins. We describe a model wherein NUP93 interacting with either NUP188 or NUP205 is necessary for cilia. We thus provide evidence that dysregulation of inner ring nucleoporin genes that have been identified in patients may contribute to pathogenesis through cilia dysfunction.