Biology subjects

Lusk, C. P.

Publications and source records attributed to Lusk, C. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Expandable DNA-origami rings as a nanomechanical platform for studying disordered nucleoporins

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

biophysics↗

bioRxiv2026

Mechanoresponsive modulation of nuclear pore complex structure and function by O-GlcNAc

Nuclear pore complexes (NPCs) control molecular exchange across the nuclear envelope, but how they tailor their selective permeability to meet the needs of specific cell types and/or environments remains poorly understood. We demonstrate that the strength of the NPC diffusion barrier differs across cell types, is particularly stringent in cultured neurons, and correlates with the O-linked N-acetylglucosamine (GlcNAc) modification of nucleoporins. Using conditional tools that specifically control nucleoporin GlcNAcylation, we show that GlcNAc modulates NPC permeability. Interestingly, nucleoporin GlcNAcylation is mechanosensitive, increasing in cells plated on stiff substrates, a condition where nuclear pores dilate. Indeed, we demonstrate that increasing or decreasing GlcNAcylation dilates and constricts NPCs, respectively. Further, O-linked N-acetylglucosamine transferase is recruited to modify NPCs upon their acute constriction during osmotic shock. Thus, cells employ GlcNAc to modulate steady-state NPC permeability in response to mechanical inputs and to counteract critical changes to their osmotic environment.

cell biology↗

bioRxiv2026

Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

Fibrosis and pathological stiffening of tissue are driven by mechanical and biochemical signaling pathways. Here, we find that Sun2, an integral inner nuclear membrane component of Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, is up-regulated in the lung of patients suffering from fibrotic conditions and in fibroblasts during an injury-induced mouse model of lung fibrosis. Sun2 protein levels also increase in primary lung fibroblasts in a substrate stiffness-dependent manner. Sun2-/- primary lung fibroblasts respond to TGF{beta}, become contractile, and express a key marker of extracellular matrix-producing fibroblasts, Cthrc1. Consistent with this, Sun2 is dispensable for myofibroblast formation and repairing the alveolar barrier after bleomycin injury. Remarkably, however, fibrosis does not develop in bleomycin-treated Sun2-/- mouse lungs. This is explained by the requirement for Sun2 to up-regulate genes encoding extracellular matrix proteins. We therefore suggest that Sun2-containing LINC complexes contribute to a mechanical coincidence detection mechanism that acts in concert with canonical TGF{beta} signaling necessary for pathologic extracellular matrix protein production, representing a nuclear mechanosensing node for intervention in fibrotic diseases of the lung.

cell biology↗

bioRxiv2026

Components of an ESCRT-independent nuclear envelope assembly pathway

In many cells, the nuclear envelope (NE) must be reassembled after mitosis and holes in the nuclear membrane must be "sealed." During NE assembly, the NE-specific adaptor, Cmp7, recruits/activates ESCRT-III proteins to mediate NE sealing. However, recent evidence suggests the presence of additional mechanisms. In a screen using the fission yeast, S. japonicus, we recently implicated the ESCRT adaptor, Alx1, and a conserved, but little studied protein, Vid27, in Cmp7-independent NE assembly. Here, we provide direct evidence that Alx1 functions in a Cmp7- and ESCRT-independent NE assembly pathway via positive regulation of Vid27. Consistent with a role in membrane remodeling, Vid27 localizes to sites of postmitotic NE sealing and is essential in S. japonicus. Alx1 and Vid27 interact and mutations disrupting the predicted interaction interface abolish Alx1s enhancement of Vid27 function at the NE. These findings define components of a new Cmp7- and ESCRT-independent NE assembly pathway, advancing our understanding of mechanisms that maintain the integrity of the nucleus.

cell biology↗

bioRxiv2025

Detecting Nuclear Pore Complex assembly in living cells

The formation of nuclear pore complexes (NPCs) -- vital gateways regulating nuclear-cytoplasmic transport -- is a highly orchestrated process requiring the integration of hundreds of nucleoporins into the nuclear envelope. A major challenge in studying this assembly process in living cells has been the difficulty to distinguish newly forming NPCs from their mature counterparts. Here, we present a powerful nanobody-based approach that overcomes this limitation. We demonstrate that a nanobody targeting the nucleoporin Nic96 from Saccharomyces cerevisiae selectively binds newly synthesized Nic96 subcomplexes prior to its incorporation into NPCs in vivo. Importantly, nanobody-bound Nic96 is incorporated in NPCs, and expression of the nanobody does not disrupt nuclear transport, cell growth, or lifespan, nor does it show genetic interactions with known NPC assembly surveillance pathways -- making it an ideal, non-perturbing tool to study NPC biogenesis in yeast. Illustrating the use of the Nic96 nanobody we report novel aspects of the early stages of assembly, including co-recruitment of Kap121 and VHH[Nic96] to putative assembly sites. In addition, we show local enrichment of newly synthesized nucleoporins on nuclear envelope proximal lipid droplets, including a specific subset of Pdr16- and Ldo16-positive lipid droplets near the nucleus-vacuole junction. When NPC assembly is delayed, association of Nic96 subcomplexes with lipid droplets increases. These findings illustrate how the strategy to pulse-label newly forming NPCs opens new avenues for dissecting the spatiotemporal regulation of NPC assembly and misassembly.

cell biology↗

bioRxiv2025

A yeast model reveals the SMAD3 nuclear import mechanism

TGF-{beta} signaling is important for patterning tissues during embryonic development and relies on the nuclear import of SMADs. Basal SMAD nuclear import mechanisms remain to be fully understood and could benefit from advances in our understanding of nuclear transport machinery. To assess the molecular determinants of SMAD nuclear transport, we take advantage of the unicellular S. cerevisiae model that lacks the TGF-{beta} signaling complexity of vertebrates but has conserved nuclear transport machinery. In this minimal system, we find that the steady-state distribution of SMAD2/3, SMAD4, and SMAD1/5 are Ran-dependent and either accumulate in, or are depleted from the nucleus. Conditionally inhibiting each of the karyopherin/importins demonstrates that SMAD3 is imported by orthologues of importin-{beta}, importin-7 and importin-8. The previously defined nuclear localization signal is insufficient to confer nuclear import. Instead, our data suggest the entire MH1 domain may act as an importin binding surface. Deleting a portion of this domain impairs SMAD3 function in Xenopus and SMAD3 nuclear enrichment in HEK293T cells. Thus, the yeast platform provides an efficient strategy to illuminate the nuclear transport mechanisms of embryonic signaling effectors.

developmental biology↗

bioRxiv2025

The LINC complex component Kms1 and CENP-B protein Cbp1 cooperate to enforce faithful homology-directed DNA repair at the nuclear periphery in S. pombe

While homologous recombination (HR) is often considered to be an error-free DNA repair mechanism, the fidelity of this pathway depends on the cells ability to engage the ideal template, the replicated sister chromatid. This is particularly challenging during repair of repetitive genome regions for which non-allelic sequences can errantly be used as templates. Here, we develop a model to study spontaneous DNA damage and repair that occurs at repetitive protein coding genes of the S. pombe flocculin family. We observe that genes encoding most members of this protein family constitutively reside at the nuclear periphery by virtue of their close proximity to binding sites for the CENP-B like protein, Cbp1. Tethering via Cbp1 to the nuclear periphery enhances the stability of the flocculin genes against intragenic recombination and restrains intergenic recombination between homoeologous repeat-encoding sequences. The LINC complex component Kms1 also antagonizes both intragenic and intergenic recombination at the flocculin genes as well as microhomology-mediated end-joining (MMEJ). Our observations suggest that S. pombe leverages nuclear compartmentalization to maintain the stability of repetitive genic regions at the nuclear periphery while association of DSBs with Kms1-containing LINC complexes enforces stringency to avoid mutagenic end-joining and use of the incorrect template during HR.

genetics↗

bioRxiv2024

Visualizing nuclear pore complex plasticity with Pan-Expansion Microscopy

The exploration of cell-type and environmentally-responsive nuclear pore complex (NPC) plasticity requires new, accessible tools. Using pan-Expansion Microscopy (pan-ExM), NPCs were identified by machine learning-facilitated segmentation with resolved cytoplasmic rings (CR), inner rings (IR) and nuclear rings (NR). They exhibited a large range of diameters with a bias for dilated NPCs at the basal nuclear surface in clusters suggestive of local islands of nuclear envelope (NE) tension. Whereas hyperosmotic shock constricted NPCs analogously to those found in annulate lamellae (AL), depletion of LINC complexes specifically eliminated the modest nuclear surface diameter biases. Therefore, LINC complexes may contribute locally to nuclear envelope tension to toggle NPC diameter between dilated, but not constricted, states. Lastly, POM121 shifts from the NR to the IR specifically in induced pluripotent stem cell derived neurons (iPSNs) from a patient with C9orf72 amyotrophic lateral sclerosis (ALS). Thus, pan-ExM is a powerful tool to visualize NPC plasticity in physiological and pathological contexts at single NPC resolution. SummaryMorgan et al. demonstrate that pan-Expansion Microscopy is an exceptional tool for probing the molecular composition and structural plasticity of individual NPCs: they reveal LINC-complex dependent NPC diameter biases across the nuclear surface and changing nucleoporin positions in an ALS model.

cell biology↗

bioRxiv2024

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, importin-{beta}1 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.

biophysics↗

bioRxiv2024

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 nuclear macroautophagy (nucleophagy) in yeast. Nucleophagy begins with a rapid accumulation of the selective autophagy receptor Atg39 at the nuclear envelope and finishes in [~]300 seconds with Atg39-cargo delivery to the vacuole. Although there are several routes to the vacuole, at least one pathway incorporates two consecutive membrane fission steps: inner nuclear membrane (INM) fission to generate an INM-derived vesicle in the perinuclear space and 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 1 (Dnm1), which is recruited to sites of Atg39 accumulation by Atg11. 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.

cell biology↗

bioRxiv2022

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. Here, we leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body (SPB) to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both depending on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, while replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization surprisingly remains intact despite NE discontinuities up to 550 nm, suggesting mechanisms to prevent diffusion through these holes. We implicate SPB proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodeling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to protect the nuclear compartment.

cell biology↗

bioRxiv2021

Kap-β2/Transportin mediates β-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. {beta}-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 {beta}-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 {beta}-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 orthologue of Kap-{beta}2/Transportin-1 (TNPO1), was required for {beta}-catenin nuclear import. We further demonstrate direct binding between TNPO1 and {beta}-catenin that is mediated by a conserved amino acid sequence that resembles a PY NLS. Finally, using Xenopus secondary axis and TCF/LEF reporter assays, we demonstrate that our results in yeast can be directly translated to vertebrates. By elucidating the NLS in {beta}-catenin and its cognate NTR, our study provides 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 towards therapeutics.

developmental biology↗

bioRxiv2021

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

Mechanisms that turnover 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 shows 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 an integral INM protein. Interestingly, correlative light and electron tomography 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.

cell biology↗

bioRxiv2021

A DNA-origami NanoTrap for studying the diffusion barriers formed by Phe-Gly-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 NPCs central transport channel impede the diffusion of 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 observe that diffusion barriers formed with cohesive FG-interactions dominate in mixed-FG-nup scenarios. Our DNA-origami platform thus sheds light on how NPCs sieve inert macromolecules and will provide a valuable tool for studying nuclear transport.

synthetic biology↗

bioRxiv2020

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

The capsid of human immunodeficiency virus 1 (HIV-1) plays a pivotal role in viral nuclear import, but the mechanism by which the viral core passages the nuclear pore complex (NPC) is poorly understood. Here, we use DNA-origami mimics of the NPC, termed NuPODs (NucleoPorins Organized by DNA), to reveal the mechanistic underpinnings of HIV-1 capsid nuclear entry. We found that trimeric interface formed via three capsid protein hexamers is targeted by a triple-arginine (RRR) motif but not the canonical phenylalanine-glycine (FG) motif of NUP153. As NUP153 is located on the nuclear face of the NPC, this result implies that the assembled capsid must cross the NPC in vivo. This hypothesis is corroborated by our observations of tubular capsid assemblies penetrating through NUP153 NuPODs. NUP153 prefers to bind highly curved capsid assemblies including those found at the tips of viral cores, thereby facilitating capsid insertion into the NPC. Furthermore, a balance of capsid stabilization by NUP153 and deformation by CPSF6, along with other cellular factors, may allow for the intact capsid to pass NPCs of various sizes. The NuPOD system serves as a unique tool for unraveling the previously elusive mechanisms of nuclear import of HIV-1 and other viruses.

microbiology↗

bioRxiv2020

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 be affinity purified with major scaffold components of the nuclear pore complex (NPC), specifically the inner ring complex, in evolutionarily distant yeasts. Interactions between Heh2 and nucleoporins is mediated by its C-terminal winged-helix (WH) domain and are distinct from interactions required for INM targeting. Disrupting interactions between Heh2 and the NPC leads to NPC clustering. Interestingly, Heh2s association with NPCs can also be broken by knocking out Nup133, a component of the outer ring that does not physically interact with Heh2. Thus, Heh2s association 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.

cell biology↗

bioRxiv2020

Direct PA-binding by Chm7 is required for nuclear envelope surveillance at 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 activity in the nuclear envelope-specific ESCRT, Chm7, in budding yeast. PA-binding is mediated through a conserved hydrophobic stretch of amino acids, which confers specific binding to the inner nuclear membrane (INM). This INM-binding is independent but nonetheless required for interaction with the LAP2-emerin-MAN1 (LEM) domain protein, Heh1 (LEM2). Consistent with the functional importance of PA-binding, mutation of this region inhibits recruitment of Chm7 to the INM and abolishes Chm7 function in the context of nuclear envelope herniations or "blebs" that form during defective nuclear pore complex (NPC) biogenesis. In fact, we show that PA accumulates at nuclear envelope herniations. We suggest that local control of PA metabolism is important for ensuring productive nuclear envelope remodeling and that its dysregulation may contribute to pathologies associated with defective NPC assembly.

cell biology↗

bioRxiv2019

A physicochemical roadmap of yeast replicative aging

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding, are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show the macromolecular crowding changes less in longer-lived cells in contrast to shorter-lived cells. While the average pH and crowding levels change only modestly with aging, we observe drastic changes in organellar volume, leading to crowding on the {micro}m scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively-aged yeast cells.

biophysics↗