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.

Dynamic regulation of LINC complex composition and function across tissues and contexts.

The concept of mechanotransduction to the nucleus through a direct force transmission mechanism has fascinated cell biologists for decades. Central to such a mechanism is the linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the nuclear envelope to couple the cytoplasmic cytoskeleton to the nuclear lamina. In reality, there is not one LINC complex identity, but instead, a family of protein configurations of varied composition that exert both shared and unique functions. Regulated expression of LINC complex components, splice variants, and mechanoresponsive protein turnover mechanisms together shape the complement of LINC complex forms present in a given cell type. Disrupting specific gene(s) encoding LINC complex components therefore gives rise to a range of organismal defects. Moreover, evidence suggests that the mechanical environment remodels LINC complexes, providing a feedback mechanism by which cellular context influences the integration of the nucleus into the cytoskeleton. In particular, evidence for crosstalk between the nuclear and cytoplasmic intermediate filament networks communicated through the LINC complex represents an emerging theme in this active area of ongoing investigation.

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.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Loops and the activity of loop extrusion factors constrain chromatin dynamics.

The chromosomes-DNA polymers and their binding proteins-are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

Integrating mechanical signals into cellular identity.

The large arrays of cell types in a multicellular organism are defined by their stereotypic size and/or morphology, and, for cells in vivo, by their anatomic positions. Historically, this identity-structure-function correlation was conceptualized as arising from distinct gene expression programs that dictate how cells appear and behave. However, a growing number of studies suggest that a cell's mechanical state is also an important determinant of its identity, both in lineage-committed cells and in pluripotent stem cells. Defining the mechanism by which mechanical inputs influence complex cellular programs remains an area of ongoing investigation. Here, we discuss how the cytoskeleton actively participates in instructing the response of the nucleus and genome to integrate mechanical and biochemical inputs, with a primary focus on the role of the actomyosin-LINC (linker of nucleoskeleton and cytoskeleton) complex axis.

Homology-directed repair involves multiple strand invasion cycles in fission yeast.

Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent colocalization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in rqh1Δ cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

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.

Extrusion of chromatin loops by a composite loop extrusion factor.

Chromatin loop extrusion by structural maintenance of chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA. Loop extrusion is enabled by SMC motion along nucleosome-free DNA, created in the wake of the remodeling complex, while nucleosome rebinding behind the SMC acts as a ratchet, holding the SMC close to the remodeling complex. We show that, for a wide range of parameter values, this collection of factors constitutes a composite LEF that extrudes loops with a velocity, comparable to the velocity of remodeling complex translocation on chromatin in the absence of SMC, and much faster than loop extrusion by an isolated SMC that is blocked by nucleosomes.

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.

Covariance distributions in single particle tracking.

Several recent experiments, including our own experiments in the fission yeast, Schizosaccharomyces pombe, have characterized the motions of gene loci within living nuclei by measuring the locus position over time, then proceeding to obtain the statistical properties of this motion. To address the question of whether a population of such single-particle tracks, obtained from many different cells, corresponds to a single mode of diffusion, we derive theoretical equations describing the probability distribution of the displacement covariance, assuming the displacement itself is a zero-mean multivariate Gaussian random variable. We also determine the corresponding theoretical means, variances, and third central moments. Bolstering the theory is good agreement between its predictions and the results obtained for various simulated and measured data sets, including simulated particle trajectories undergoing simple and anomalous diffusion, and the measured trajectories of an optically trapped bead in water, and in a viscoelastic polymer solution. We also show that, for sufficiently long tracks, each covariance distribution in all of these examples is well-described by a skew-normal distribution with mean, variance, and skewness given by the theory. However, for the experimentally measured motion of a gene locus in S. pombe, we find that the first two covariance distributions are wider than predicted, although the third and subsequent covariance distributions are well-described by theory. This observation suggests that the origin of the theory-experiment discrepancy in this case is associated with localization noise, which influences only the first two covariances. Thus, we hypothesized that the discrepancy is caused by locus-to-locus heterogeneity in the localization noise, of independent measurements of the same tagged site. Indeed, simulations implementing heterogeneous localization noise revealed that the excess covariance widths can be largely recreated on the basis of heterogeneous noise. Thus, we conclude that the motion of gene loci in fission yeast is consistent with a single mode of diffusion.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

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.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells.

An abnormal number of chromosomes, or aneuploidy, accounts for most spontaneous abortions, causes developmental defects, and is associated with aging and cancer. The molecular mechanisms by which aneuploidy disrupts cellular function remain largely unknown. Here, we show that aneuploidy disrupts the morphology of the nucleus. Mutations that increase the levels of long-chain bases suppress nuclear abnormalities of aneuploid yeast independent of karyotype identity. Quantitative lipidomics indicates that long-chain bases are integral components of the nuclear membrane in yeast. Cells isolated from patients with Down syndrome also show that abnormal nuclear morphologies and increases in long-chain bases not only suppress these abnormalities but also improve their fitness. We obtained similar results with cells isolated from patients with Patau or Edward syndrome, indicating that increases in long-chain bases improve the fitness of aneuploid cells in yeast and humans. Targeting lipid biosynthesis pathways represents an important strategy to suppress nuclear abnormalities in aneuploidy-associated diseases.

RNA-DNA Hybrids Support Recombination-Based Telomere Maintenance in Fission Yeast.

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 ortholog, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that upregulation of telomere-engaged TERRA, or altered recruitment of shelterin components, can support telomerase-independent telomere maintenance.

Loss of nuclear envelope integrity? No probLEM-BAF has it covered.

Ruptures of the nuclear envelope can drive a catastrophic loss of nucleocytoplasmic compartmentalization. In this issue, Halfmann et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201901116) describe a mechanism for surveilling the integrity of the nuclear barrier and coupling the sensing of nuclear ruptures to the recruitment of the nuclear envelope repair machinery.