A phase-separated nuclear GBPL circuit controls immunity in plants.

Liquid-liquid phase separation (LLPS) has emerged as a central paradigm for understanding how membraneless organelles compartmentalize diverse cellular activities in eukaryotes1-3. Here we identify a superfamily of plant guanylate-binding protein (GBP)-like GTPases (GBPLs) that assemble LLPS-driven condensates within the nucleus to protect against infection and autoimmunity. In Arabidopsis thaliana, two members of this family-GBPL1 and GBPL3-undergo phase-transition behaviour to control transcriptional responses as part of an allosteric switch that is triggered by exposure to biotic stress. GBPL1, a pseudo-GTPase, sequesters catalytically active GBPL3 under basal conditions but is displaced by GBPL3 LLPS when it enters the nucleus following immune cues to drive the formation of unique membraneless organelles termed GBPL defence-activated condensates (GDACs) that we visualized by in situ cryo-electron tomography. Within these mesoscale GDAC structures, native GBPL3 directly bound defence-gene promoters and recruited specific transcriptional coactivators of the Mediator complex and RNA polymerase II machinery to massively reprogram host gene expression for disease resistance. Together, our study identifies a GBPL circuit that reinforces the biological importance of phase-separated condensates, in this case, as indispensable players in plant defence.

Analysis of Intracellular Vesicles in B Lymphocytes: Antigen Traffic in the Spotlight.

All eukaryotic cells are delimited by the plasma membrane, separating the cell from its environment. Two critical cellular pathways, the endocytic and the exocytic vesicle networks, shuttle material in and out the cell, respectively. The substantial development of cell biological imaging techniques, along with improved fluorescent probes and image analysis tools, has been instrumental in increasing our understanding of various functions and regulatory mechanisms of various intracellular vesicle subpopulations and their dynamics. Here, using B lymphocytes (B cells) as a model system, we provide a protocol for 3D analysis of the intracellular vesicle traffic in either fixed or living cells using spinning disk confocal microscopy. We also describe the usage of image deconvolution to improve the resolution, particularly important for vesicular networks in lymphocytes due to the small size of these cells. Lastly, we describe two types of quantitative analysis: vesicle distribution/clustering toward the microtubule organizing center (MTOC), and colocalization analysis with endolysosomal markers.

Immune Cell Connection by Tunneling Nanotubes: The Impact of Intercellular Cross-Talk on the Immune Response and Its Therapeutic Applications.

Direct intercellular communication is an important prerequisite for the development of multicellular organisms, the regeneration of tissue, and the maintenance of various physiological activities. Tunnel nanotubes (TNTs), which have diameters of approximately 50-1500 nm and lengths of up to several cell diameters, can connect cells over long distances and have emerged as one of the most important recently discovered types of efficient communication between cells. Moreover, TNTs can also directly transfer organelles, vehicles, proteins, genetic material, ions, and small molecules from one cell to adjacent and even distant cells. However, the mechanism of intercellular communication between various immune cells within the complex immune system has not been fully elucidated. Studies in the past decades have confirmed the existence of TNTs in many types of cells, especially in various kinds of immune cells. TNTs display different structural and functional characteristics between and within different immunocytes, playing a major role in the transmission of signals across various kinds of immune cells. In this review, we introduce the discovery and structure of TNTs, as well as their different functional properties within different immune cells. We also discuss the roles of TNTs in potentiating the immune response and their potential therapeutic applications.

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.

Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl- and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

Eye on ion channels in immune cells.

Ion channels facilitate the movement of ions across the plasma and organellar membranes. A recent symposium brought together scientists who study ion channels and transporters in immune cells, which highlighted advances in this emerging field and served to chart new avenues for investigating the roles of ion channels in immunity.

Concise Review: Intercellular Communication Via Organelle Transfer in the Biology and Therapeutic Applications of Stem Cells.

The therapeutic potential of stem cell-based therapies may be largely dependent on the ability of stem cells to modulate host cells rather than on their differentiation into host tissues. Within the last decade, there has been considerable interest in the intercellular communication mediated by the transfer of cytoplasmic material and organelles between cells. Numerous studies have shown that mitochondria and lysosomes are transported between cells by various mechanisms, such as tunneling nanotubes, microvesicles, and cellular fusion. This review will focus on the known instances of organelle transfer between stem cells and differentiated cells, what effects it has on recipient cells and how organelle transfer is regulated. Stem Cells 2019;37:14-25.

Improving the Phototherapeutic Efficiencies of Molecular and Nanoscale Materials by Targeting Mitochondria.

Mitochondria-targeted cancer phototherapy (PT), which works by delivering photoresponsive agents specifically to mitochondria, is a powerful strategy to improve the phototherapeutic efficiency of anticancer treatments. Mitochondria play an essential role in cellular apoptosis, and are relevant to the chemoresistance of cancer cells. Furthermore, mitochondria are a major player in many cellular processes and are highly sensitive to hyperthermia and reactive oxygen species. Therefore, mitochondria serve as excellent locations for organelle-targeted phototherapy. In this review, we focus on the recent advances of mitochondria-targeting materials for mitochondria-specific PT. The combination of mitochondria-targeted PT with other anticancer strategies is also summarized. In addition, we discuss both the challenges currently faced by mitochondria-based cancer PT and the promises it holds.

Quantifying Heterogeneity of Individual Organelles in Mixed Populations via Mass Cytometry.

Macroautophagy is a complex degradative intracellular process by which long-lived proteins and damaged organelles are cleared. Common methods for the analysis of autophagy are bulk measurements which mask organelle heterogeneity and complicate the analysis of interorganelle association and trafficking. Thus, methods for individual organelle quantification are needed to address these deficiencies. Current techniques for quantifying individual autophagy organelles are either low through-put or are dimensionally limited. We make use of the multiparametric capability of mass cytometry to investigate phenotypic heterogeneity in autophagy-related organelle types that have been isolated from murine brain, liver, and skeletal muscle. Detection and phenotypic classification of individual organelles were accomplished through the use of a lanthanide-chelating membrane stain and organelle-specific antibodies. Posthoc sample matrix background correction and nonspecific antibody binding corrections provide measures of interorganelle associations and heterogeneity. This is the first demonstration of multiparametric individual organelle analysis via mass cytometry. The method described here illustrates the potential for further investigation of the inherently complex interorganelle associations, trafficking, and heterogeneity present in most eukaryotic biological systems.

New advances in autophagy in plants: Regulation, selectivity and function.

Autophagy is a major and conserved pathway for delivering unwanted proteins or damaged organelles to the vacuole for degradation and recycling. In plants, it functions as a housekeeping process to maintain cellular homeostasis under normal conditions and is induced by stress and senescence; it thus plays important roles in development, stress tolerance and metabolism. Autophagy can both execute bulk degradation and be highly selective in targeting cargos under specific environmental conditions or during certain developmental processes. Here, we review recent research on autophagy in plants, and discuss new insights into its core mechanism, regulation, selectivity and physiological roles. Potential future directions are also highlighted.

Interaction of the innate immune system with positive-strand RNA virus replication organelles.

The potential health risks associated with (re-)emerging positive-strand RNA (+RNA) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. The innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (PAMPs). Viral RNA is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (IFNs) and pro-inflammatory cytokines. However, +RNA viruses developed various methods to avoid detection and downstream signaling, including isolation of viral RNA replication in membranous viral replication organelles (ROs). These structures therefore play a central role in infection, and consequently, loss of RO integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. This review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ROs and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein 1A/1B-light chain 3 (LC3) and ubiquitin, resulting in the recruitment of IFN-inducible GTPases. Further studies should evaluate whether this process forms a general effector mechanism in +RNA virus infection, thereby creating the opportunity for development of novel antiviral therapies.

EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples.

BACKGROUND: Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones. RESULTS: Based on large scale proteomics analyses using the SILProNAQ methodology, tandem mass spectrometry favoured the characterization of thousands of peptides. Data processing using the EnCOUNTer tool provided an efficient and rapid way to extract the most reliable mature protein N-termini. Selected peptides were subjected to N-terminus acetylation yield determination. In an A. thaliana cell lysate, 1232 distinct proteotypic N-termini were characterized of which 648 were located at the predicted protein N-terminus (position 1/2) and 584 were located further downstream (starting at position > 2). A large number of these N-termini were associated with various well-defined maturation processes occurring on organelle-targeted proteins (mitochondria, chloroplast and peroxisome), secreted proteins or membrane-targeted proteins. It was also possible to highlight some protein alternative starts, splicing variants or erroneous protein sequence predictions. CONCLUSIONS: The EnCOUNTer tool provides a unique way to extract accurately the most relevant mature proteins N-terminal peptides from large scale experimental datasets. Such data processing allows the identification of the exact N-terminus position and the associated acetylation yield.

Diacylglycerol kinase ζ limits the polarized recruitment of diacylglycerol-enriched organelles to the immune synapse in T cells.

The antigen-induced formation of an immune synapse (IS) between T cells and antigen-presenting cells results in the rapid generation of the lipid second messenger diacylglycerol (DAG) in T cells. Diacylglycerol kinase ζ (DGKζ) converts DAG into phosphatidic acid (PA). Cytotoxic T lymphocytes (CTLs) from mice deficient in DGKζ have enhanced antiviral and antitumor activities, indicating that the amount of DAG controls the effectiveness of the T cell response. We characterized the second C1 domain of protein kinase Cθ (PKCθ), a DAG-binding protein that is specifically recruited to the IS, as a biological sensor to observe the generation of a DAG gradient during IS formation. In experiments with transgenic mouse CTLs expressing the OT-I T cell receptor (TCR), we showed that both strong and weak interactions between antigen and the TCR led to the rapid generation of DAG, whereas only strong interactions induced the movement of DAG-enriched organelles toward the IS. In DGKζ-deficient CTLs, antigen stimulation led to the enhanced accumulation of DAG-containing organelles at the IS; however, impaired activation of the PA effector PKCζ resulted in lack of reorientation of the microtubule-organizing center toward the IS, a process needed for effective T cell activation. Together, these data suggest that the activation of DGKζ downstream of antigen recognition provides a mechanism that ensures the activation of PA-dependent signaling as a direct result of the strength of TCR-dependent DAG mobilization.

Key roles of autophagy in regulating T-cell function.

In the past 10 years, autophagy has emerged as a crucial regulator of T-cell homeostasis, activation, and differentiation. Through the ability to adjust the cell's proteome in response to different stimuli, different forms of autophagy have been shown to control T-cell homeostasis and survival. Autophagic processes can also determine the magnitude of the T-cell response to TCR engagement, by regulating the cellular levels of specific signaling intermediates and modulating the metabolic output in activated T cells. In this review we will examine the mechanisms that control autophagy activity in T cells, such as ROS signaling and signaling through common gamma-chain cytokine receptors, and the different aspect of T-cell biology, including T-cell survival, effector cell function, and generation of memory, which can be regulated by autophagy.

Apical Organelle Secretion by Toxoplasma Controls Innate and Adaptive Immunity and Mediates Long-Term Protection.

Apicomplexan parasites have unique apical rhoptry and microneme secretory organelles that are crucial for host infection, although their role in protection against Toxoplasma gondii infection is not thoroughly understood. Here, we report a novel function of the endolysosomal T. gondii sortilin-like receptor (TgSORTLR), which mediates trafficking to functional apical organelles and their subsequent secretion of virulence factors that are critical to the induction of sterile immunity against parasite reinfection. We further demonstrate that the T. gondii armadillo repeats-only protein (TgARO) mutant, which is deficient only in apical secretion of rhoptries, is also critical in mounting protective immunity. The lack of TgSORTLR and TgARO proteins completely inhibited T-helper 1-dependent adaptive immunity and compromised the function of natural killer T-cell-mediated innate immunity. Our findings reveal an essential role for apical secretion in promoting sterile protection against T. gondii and provide strong evidence for rhoptry-regulated discharge of antigens as a key effector for inducing protective immunity.

SMOCs: supramolecular organizing centres that control innate immunity.

The diverse receptor families of the innate immune system activate signal transduction pathways that are important for host defence, but common themes to explain the operation of these pathways remain undefined. In this Opinion article, we propose--on the basis of recent structural and cell biological studies--the concept of supramolecular organizing centres (SMOCs) as location-specific higher-order signalling complexes in which increased local concentrations of signalling components promote the intrinsically weak allosteric interactions that are required for enzyme activation. We suggest that SMOCs are assembled on various membrane-bound organelles or other intracellular sites, which may assist signal amplification to reach a response threshold and potentially define the specificity of cellular responses that are induced in response to infectious and non-infectious insults.

Temporal evolution of human autoantibody response to cytoplasmic rods and rings structure during anti-HCV therapy with ribavirin and interferon-α.

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-α) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic "rod/ring" (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-α + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6-12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. In conclusion, this study showed that the temporal kinetics of IFN-α + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans.

Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H2 and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 µm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O2. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles.

Attenuating gene expression (AGE) for vaccine development.

Live attenuated vaccines are adept in stimulating protective immunity. Methods for generating such vaccines have largely adopted strategies used with Salmonella enterica. Yet, when similar strategies were tested in other gram-negative bacteria, the virulence factors or genes responsible to incapacitate Salmonella often failed in providing the desired outcome. Consequently, conventional live vaccines rely on prior knowledge of the pathogen's virulence factors to successfully attenuate them. This can be problematic since such bacterial pathogens normally harbor thousands of genes. To circumvent this problem, we found that overexpression of bacterial appendages, e.g., fimbriae, capsule, and flagella, could successfully attenuate wild-type (wt) Salmonella enterica serovar Typhimurium. Further analysis revealed these attenuated Salmonella strains conferred protection against wt S. Typhimurium challenge as effectively as genetically defined Salmonella vaccines. We refer to this strategy as attenuating gene expression (AGE), a simple efficient approach in attenuating bacterial pathogens, greatly facilitating the construction of live vaccines.

Deciphering the contribution of lipid droplets in leprosy: multifunctional organelles with roles in Mycobacterium leprae pathogenesis

Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves, presenting a singular clinical picture. Across the leprosy spectrum, lepromatous leprosy (LL) exhibits a classical hallmark: the presence of a collection of M. leprae-infected foamy macrophages/Schwann cells characterised by their high lipid content. The significance of this foamy aspect in mycobacterial infections has garnered renewed attention in leprosy due to the recent observation that the foamy aspect represents cells enriched in lipid droplets (LD) (also known as lipid bodies). Here, we discuss the contemporary view of LD as highly regulated organelles with key functions in M. leprae persistence in the LL end of the spectrum. The modern methods of studying this ancient disease have contributed to recent findings that describe M. leprae-triggered LD biogenesis and recruitment as effective mycobacterial intracellular strategies for acquiring lipids, sheltering and/or dampening the immune response and favouring bacterial survival, likely representing a fundamental aspect of M. leprae pathogenesis. The multifaceted functions attributed to the LD in leprosy may contribute to the development of new strategies for adjunctive anti-leprosy therapies.