Quantifying spatial dynamics of Mycobacterium tuberculosis infection of human macrophages using microfabricated patterns.

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). A challenge for quantitative characterization of host-pathogen processes in differentially polarized primary human monocyte-derived macrophages (MDMs) is their heterogeneous morphology. Here, we describe the use of microfabricated patterns that constrain the size and shape of cells, mimicking the physiological spatial confinement cells experience in tissues, to quantitatively characterize interactions during and after phagocytosis at the single-cell level at high resolution. Comparing pro-inflammatory (M1) and anti-inflammatory (M2) MDMs, we find interferon-γ stimulation increases the phagocytic contraction, while contraction and bacterial uptake decrease following silencing of phagocytosis regulator NHLRC2 or bacterial surface lipid removal. We identify host organelle position alterations within infected MDMs and differences in Mtb subcellular localization in line with M1 and M2 cellular polarity. Our approach can be adapted to study other host-pathogen interactions and coupled with downstream automated analytical approaches.

Co-transport of the nuclear-encoded Cox7c mRNA with mitochondria along axons occurs through a coding-region-dependent mechanism.

Nuclear-encoded mitochondrial protein mRNAs have been found to be localized and locally translated within neuronal processes. However, the mechanism of transport for those mRNAs to distal locations is not fully understood. Here, we describe axonal co-transport of Cox7c with mitochondria. Fractionation analysis and single-molecule fluorescence in situ hybridization (smFISH) assay revealed that endogenous mRNA encoding Cox7c was preferentially associated with mitochondria in a mouse neuronal cell line and within mouse primary motor neuron axons, whereas other mRNAs that do not encode mitochondrial protein were much less associated. Live-cell imaging of MS2-tagged Cox7c mRNA further confirmed the preferential colocalization and co-transport of Cox7c mRNA with mitochondria in motor neuron axons. Intriguingly, the coding region, rather than the 3' untranslated region (UTR), was the key domain for the co-transport. Our results reveal that Cox7c mRNA can be transported with mitochondria along significant distances and that its coding region is a major recognition feature. This is consistent with the idea that mitochondria can play a vital role in spatial regulation of the axonal transcriptome at distant neuronal sites.

DeepSpot: A deep neural network for RNA spot enhancement in single-molecule fluorescence in-situ hybridization microscopy images.

Detection of RNA spots in single-molecule fluorescence in-situ hybridization microscopy images remains a difficult task, especially when applied to large volumes of data. The variable intensity of RNA spots combined with the high noise level of the images often requires manual adjustment of the spot detection thresholds for each image. In this work, we introduce DeepSpot, a Deep Learning-based tool specifically designed for RNA spot enhancement that enables spot detection without the need to resort to image per image parameter tuning. We show how our method can enable downstream accurate spot detection. DeepSpot's architecture is inspired by small object detection approaches. It incorporates dilated convolutions into a module specifically designed for context aggregation for small object and uses Residual Convolutions to propagate this information along the network. This enables DeepSpot to enhance all RNA spots to the same intensity, and thus circumvents the need for parameter tuning. We evaluated how easily spots can be detected in images enhanced with our method by testing DeepSpot on 20 simulated and 3 experimental datasets, and showed that accuracy of more than 97% is achieved. Moreover, comparison with alternative deep learning approaches for mRNA spot detection (deepBlink) indicated that DeepSpot provides more precise mRNA detection. In addition, we generated single-molecule fluorescence in-situ hybridization images of mouse fibroblasts in a wound healing assay to evaluate whether DeepSpot enhancement can enable seamless mRNA spot detection and thus streamline studies of localized mRNA expression in cells.

Interrogating RNA and protein spatial subcellular distribution in smFISH data with DypFISH.

Advances in single-cell RNA sequencing have allowed for the identification of cellular subtypes on the basis of quantification of the number of transcripts in each cell. However, cells might also differ in the spatial distribution of molecules, including RNAs. Here, we present DypFISH, an approach to quantitatively investigate the subcellular localization of RNA and protein. We introduce a range of analytical techniques to interrogate single-molecule RNA fluorescence in situ hybridization (smFISH) data in combination with protein immunolabeling. DypFISH is suited to study patterns of clustering of molecules, the association of mRNA-protein subcellular localization with microtubule organizing center orientation, and interdependence of mRNA-protein spatial distributions. We showcase how our analytical tools can achieve biological insights by utilizing cell micropatterning to constrain cellular architecture, which leads to reduction in subcellular mRNA distribution variation, allowing for the characterization of their localization patterns. Furthermore, we show that our method can be applied to physiological systems such as skeletal muscle fibers.

Immunoglobulin M in Health and Diseases: How Far Have We Come and What Next?

B lymphocytes are important in secreting antibodies that protect against invading pathogens such as viruses, bacteria, parasites, and also in mediating pathogenesis of allergic diseases and autoimmunity. B lymphocytes develop in the bone marrow and contain heavy and light chains, which upon ligation form an immunoglobulin M (IgM) B cell receptor (BCR) expressed on the surface of naïve immature B cells. Naïve B cells expressing either IgM or IgD isotypes are thought to play interchangeable functions in antibody responses to T cell-dependent and T cell-independent antigens. IgM short-lived plasma cells (SLPCs) and antigen-specific IgM memory B cells (MBCs-M) are critical in the first few days of infection, as well as long-term memory induced by vaccination, respectively. At mucosal surfaces, IgM is thought to play a critical part in promoting mucosal tolerance and shaping microbiota together with IgA. In this review, we explore how IgM structure and BCR signaling shapes B cell development, self and non-self-antigen-specific antibody responses, responses to infectious (such as viruses, parasites, and fungal) and non-communicable diseases (such as autoimmunity and allergic asthma). We also explore how metabolism could influence other B cell functions such as mucosal tolerance and class switching. Finally, we discuss some of the outstanding critical research questions in both experimental and clinical settings targeting IgM.

Pinpointing Cell Identity in Time and Space.

Mammalian cells display a broad spectrum of phenotypes, morphologies, and functional niches within biological systems. Our understanding of mechanisms at the individual cellular level, and how cells function in concert to form tissues, organs and systems, has been greatly facilitated by centuries of extensive work to classify and characterize cell types. Classic histological approaches are now complemented with advanced single-cell sequencing and spatial transcriptomics for cell identity studies. Emerging data suggests that additional levels of information should be considered, including the subcellular spatial distribution of molecules such as RNA and protein, when classifying cells. In this Perspective piece we describe the importance of integrating cell transcriptional state with tissue and subcellular spatial and temporal information for thorough characterization of cell type and state. We refer to recent studies making use of single cell RNA-seq and/or image-based cell characterization, which highlight a need for such in-depth characterization of cell populations. We also describe the advances required in experimental, imaging and analytical methods to address these questions. This Perspective concludes by framing this argument in the context of projects such as the Human Cell Atlas, and related fields of cancer research and developmental biology.

Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners.

RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), controlling key cellular processes, including gene expression and translation of proteins. Current methodologies aimed at identifying and characterizing protein binding partners of specific RNAs of interest typically rely on tagging of the RNA with affinity aptamers, using in vitro transcribed RNA or immobilized oligonucleotides to capture RNA-protein complexes under native conditions. These assays are coupled with mass spectrometry or Western Blot analysis to identify or/and confirm interacting proteins. Here, we describe an alternative approach to identify protein binding partners of mRNAs and large long noncoding RNAs. This approach relies on biochemical pulldown of specific target RNAs and interacting protein partners from cellular lysates coupled with mass spectrometry to identify novel interacting proteins. By using 24-48 ~20 mer biotinylated DNA probes that hybridize to the target RNA, the method ensures high specificity and minimal off target binding. This approach is reproducible and fast and serves as a base for discovery studies to identify proteins that bind to RNAs of interest.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti.

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

Targeting of viral capsids to nuclear pores in a cell-free reconstitution system.

Many viruses deliver their genomes into the nucleoplasm for viral transcription and replication. Here, we describe a novel cell-free system to elucidate specific interactions between viruses and nuclear pore complexes (NPCs). Nuclei reconstituted in vitro from egg extracts of Xenopus laevis, an established biochemical system to decipher nuclear functions, were incubated with GFP-tagged capsids of herpes simplex virus, an alphaherpesvirus replicating in the nucleus. Capsid binding to NPCs was analyzed using fluorescence and field emission scanning electron microscopy. Tegument-free capsids or viral capsids exposing inner tegument proteins on their surface bound to nuclei, while capsids inactivated by a high-salt treatment or covered by inner and outer tegument showed less binding. There was little binding of the four different capsid types to nuclei lacking functional NPCs. This novel approach provides a powerful system to elucidate the molecular mechanisms that enable viral structures to engage with NPCs. Furthermore, this assay could be expanded to identify molecular cues triggering viral genome uncoating and nuclear import of viral genomes.

Blm10 facilitates nuclear import of proteasome core particles.

Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT-like modules and is structurally related to the nuclear import receptor importin/karyopherin ß. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor-cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast.

Proteasomes crossing the nuclear border.

Proteasomes localize to different compartments in eukaryotic cells, but the details of this dynamic distribution are poorly understood. Here, we discuss evidence linking proteasome activity to specific intranuclear locations and the potential mechanisms that may target these large proteolytic machines to the nucleus. Recent work has demonstrated the targeting of intact proteasome particles to newly formed nuclear compartments in a cell-free reconstitution assay. We discuss the difference between open and closed mitosis systems and the targeting of proteasomes to the nuclear periphery, as well as the nucleoplasm. Finally, we present a potential model for regulated assembly of the 26S proteasome holoenzyme inside the nucleus.

Proteasome activator 200: the heat is on...

Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1-6), attaches to the same surface of the CP ring, and likewise, induces pore opening.

Nuclear import of an intact preassembled proteasome particle.

The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated "+" factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development.