Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy.

In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5' end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to gene-rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation.

Assessing resolution in super-resolution imaging.

Resolution is a central concept in all imaging fields, and particularly in optical microscopy, but it can be easily misinterpreted. The mathematical definition of optical resolution was codified by Abbe, and practically defined by the Rayleigh Criterion in the late 19th century. The limit of conventional resolution was also achieved in this period, and it was thought that fundamental constraints of physics prevented further increases in resolution. With the recent development of a range of super-resolution techniques, it is necessary to revisit the concept of optical resolution. Fundamental differences in super-resolution modalities mean that resolution is not a directly transferrable metric between techniques. This article considers the issues in resolution raised by these new technologies, and presents approaches for comparing resolution between different super-resolution methods.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis.

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes.

YAP condensates are highly organized hubs.

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

The nuclear envelope protein emerin binds directly to histone deacetylase 3 (HDAC3) and activates HDAC3 activity.

Organization of the genome is critical for maintaining cell-specific gene expression, ensuring proper cell function. It is well established that the nuclear lamina preferentially associates with repressed chromatin. However, the molecular mechanisms underlying repressive chromatin formation and maintenance at the nuclear lamina remain poorly understood. Here we show that emerin binds directly to HDAC3, the catalytic subunit of the nuclear co-repressor (NCoR) complex, and recruits HDAC3 to the nuclear periphery. Emerin binding stimulated the catalytic activity of HDAC3, and emerin-null cells exhibit increased H4K5 acetylation, which is the preferred target of the NCoR complex. Emerin-null cells exhibit an epigenetic signature similar to that seen in HDAC3-null cells. Emerin-null cells also had significantly less HDAC3 at the nuclear lamina. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear periphery by increasing the catalytic activity of HDAC3.

Transcriptional condensates and phase separation: condensing information across scales and mechanisms.

Transcription is the fundamental process of gene expression, which in eukaryotes occurs within the complex physicochemical environment of the nucleus. Decades of research have provided extreme detail in the molecular and functional mechanisms of transcription, but the spatial and genomic organization of transcription remains mysterious. Recent discoveries show that transcriptional components can undergo phase separation and create distinct compartments inside the nucleus, providing new models through which to view the transcription process in eukaryotes. In this review, we focus on transcriptional condensates and their phase separation-like behaviors. We suggest differentiation between physical descriptions of phase separation and the complex and dynamic biomolecular assemblies required for productive gene expression, and we discuss how transcriptional condensates are central to organizing the three-dimensional genome across spatial and temporal scales. Finally, we map approaches for therapeutic manipulation of transcriptional condensates and ask what technical advances are needed to understand transcriptional condensates more completely.

Quantitative 3D structured illumination microscopy of nuclear structures.

3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.

Strategic and practical guidelines for successful structured illumination microscopy.

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.

SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.

BACKGROUND: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). RESULTS: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an 'autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. CONCLUSIONS: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.