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This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Motile cells such as immune and cancer cells experience large deformation events that result from the physical constraints they encounter while migrating within tissues or circulating between organs. It has become increasingly clear that these cells can survive and adapt to these changes in cell shape using dedicated shape sensing pathways. However, how shape sensing impacts their function and fate remains largely unknown. Here we identify a shape sensing mechanism that couples cell motility to expression of CCR7, the chemokine receptor that guides immune cells to lymph nodes. We found that this mechanism is controlled by the lipid metabolism enzyme cPLA2, requires an intact nuclear envelop and exhibits an exquisitely sensitive activation threshold tuned by ARP2/3 and its inhibitor Arpin. We further show that shape sensing through the ARP2/3-cPLA2axis controls Ikkβ-NFκB-dependent transcriptional reprogramming of dendritic cells, which instructs them to migrate to lymph nodes in an immunoregulatory state compatible with their homeostatic tolerogenic function. These results highlight that the cell shape changes experienced by motile cells evolving within the complex environment of tissues can dictate their behavior and fate.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here we report that septins shape T cells for amoeboid motility. Specifically, septins form F-actin and alpha-actinin-rich cortical rings at the sites of cortex-indenting collisions of T cells with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient ‘hourglass’-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results demonstrate that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots for amoeboid motility of T cells in tissue-like microenvironments.GLOSSARYSteric interactions - interactions by the means of their spatial collision dependent on objects’ shapes.Steric guidance - cell navigation within crowded 3D environments, determined by the available passages around and between steric hindrances.Peristaltic treadmilling - locomotion mode by the means of a repeated sequence of polarized cell cortex extension, stabilization, and retraction, accompanied by translocation of nucleus and cytoplasmviacircumferential cortex contractility.Significance StatementT cells can be highly motile, searching for cognate antigens or better yet targets in chimeric antigen receptor therapy settings. However, mechanisms of motility remain elusive for T cells migrating in structurally and biochemically diverse tissues. Here we address one pivotal question of basic and clinical immunology - How T cells achieve the ‘all-terrain’ motility? Here we decipher and report septin-based T cell motility in a 3D tissue-like environment. Specifically, we show that septins facilitate cell morphological responsiveness to the steric obstacles,i.e., collagen fiber-wise partitioning of actomyosin cortex contractility and cell-obstacle steric interactions. These responses coordinate peristaltic propulsion of the lymphocyte’s cytosolic content along its individually contracting cell segments, forming the obstacle-avoiding motility,i.e.,circumnavigation, shared across various tested lymphocytes.

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Scientific conferences are important avenues for researchers to share and discuss research findings, to exchange ideas and insights, and to network for collaboration and career development. Organizing inclusive and useful scientific meetings is a significant responsibility shared by researchers, scientific societies, and other organizations worldwide. Prior to 2020, most conferences were held in-person and provided limited attendance opportunities for many researchers from diverse socioeconomic backgrounds, particularly early career researchers (ECRs), researchers from low- and middle-income countries, and junior principal investigators (PIs)1. Some conferences in select disciplines have implemented valuable changes for the community, becoming more receptive to attendees with families. Due to the COVID-19 pandemic, a major shift has occurred towards virtual conferencing formats, triggering discussions in the scientific community on the current and future formats and organization of conferences. Here we provide recommendations for improving scientific meetings towards more efficient, sustainable, and equitable gatherings. Instituto de Patología Vegetal Fil: Sarabipour, Sarvenaz. Johns Hopkins University. Institute for Computational Medicine. Department of Biomedical Engineering; Estados Unidos Fil: Khan, Aziz. University of Oslo. Nordic EMBL Partnership. Centre for Molecular Medicine Norway (NCMM); Noruega Fil: Khan, Aziz. Stanford University. School of Medicine. Stanford Cancer Institute; Estados Unidos Fil: Yu Fen, Samantha Seah. European Molecular Biology Laboratory. Genome Biology Unit; Alemania Fil: Mwakilili, Aneth D. Swedish University of Agricultural Sciences. Plant Protection Department; Suecia Fil: Mwakilili, Aneth D. University of Dar es Salaam. Department of Molecular Biology and Biotechnology; Tanzania Fil: Mumoki, Fiona N. University of Pretoria. Department of Zoology and Entomology; Sudáfrica Fil: Sáez, Pablo J. Paris Sciences & Lettres Research University. Institut Curie; Francia Fil: Sáez, Pablo J. University Medical Center Hamburg-Eppendorf. Department of Biochemistry and Molecular Cell Biology; Alemania Fil: Schwessinger, Benjamin. Australian National University. Research School of Biology; Australia Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Mestrovic, Tomislav. Dr Zora Profozic Polyclinic. Clinical Microbiology and Parasitology Unit; Croacia Fil: Mestrovic, Tomislav. University Centre Varazdin. University North; Croacia

Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2α) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler–based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

Scientific conferences provide valuable opportunities for researchers across career stages and disciplines to present their latest work and to network with their peers. The advent of the internet has opened new possibilities for interaction, collaboration and networking, yet the uptake of tools enabling remote participation at scientific meetings has been slow. Academic conferences have become more international with high proliferation in the number of meetings and attendees, but the format and quality of their organization lags behind what is possible. As a result, the experience of attending conferences in many disciplines today is not appreciably different in many respects. A few conferences in some disciplines have recently implemented valuable changes for the community and have become more receptive to attendees with families. Many meetings could still be improved significantly in terms of diversity, inclusivity, promoting early career researcher (ECR) networking and career development, venue accessibility, and more importantly, reducing the meetings’ carbon footprint. These issues are openly visible and to some extent discussed among researchers on social media. It is important to accelerate and mandate these efforts so that researchers in all disciplines, in particular ECRs, consistently benefit from scientific gatherings, for years to come. We examined the current state of over 260 national and international academic meetings in various disciplines for features of inclusivity and sustainability and propose solutions to make conferences more modern, effective, equitable and intellectually productive for the research community and environmentally sustainable for our planet. “They always say time changes things, but you actually have to change them yourself.” — Andy Warhol

Cell migration depends on a combination of the cell's intrinsic capacity to move and the proper interpretation of external cues. This multistep process enables leukocytes to travel long distances in organs in just a few hours. This fast migration is partly due to the leukocytes' high level of plasticity, which helps them to adapt to a changing environment. Here, we review recent progress in understanding the mechanisms used by leukocytes to move rapidly and efficiently in intricate anatomical landscapes. We shall focus on specific cytoskeletal rearrangements used by neutrophils and dendritic cells to migrate within confined environments. Lastly, we will describe the properties that facilitate the rapid migration of leukocyte in complex tissue geometries.

Objective Pregestational maternal obesity (PGMO) associates with foetoplacental vascular endothelial dysfunction and higher risk for insulin resistance in the neonate. We characterised the PGMO consequences on the insulin response of the human foetoplacental vasculature. Methods Umbilical veins were from pregnancies where the mother was with PGMO (body mass index 30–42.3 kg/m2, n = 33) or normal pregestational weight (PGMN) (body mass index 19.5–24.4 kg/m2, n = 21) with total gestational weight gain within the physiological range. Umbilical vein ring segments were mounted in a myograph for isometric force measurements. Primary cultures of human umbilical vein endothelial cells were used in passage 3. Vessel rings and cells were exposed to 1 nmol/L insulin (20 min) in the absence or presence of 100 μmol/L NG-nitro- l -arginine methyl ester (inhibitor of nitric oxide synthase, NOS). Results Vessel rings from PGMO showed reduced nitric oxide synthase-activity dependent dilation to insulin or calcitonin-gene related peptide compared with PGMN. PGMO associated with higher inhibitor phosphorylation of the insulin receptor substrate 1 (IRS-1) and lower activator phosphorylation of protein kinase B/Akt (Akt). Cells from PGMO also showed lower nitric oxide level and reduced activator serine1177 but increased inhibitor threonine495 phosphorylation of endothelial nitric oxide synthase (eNOS) and saturable transport of l -arginine. HUVECs from PGMO were not responsive to insulin. Conclusion The lack of response to insulin by the foetoplacental endothelium may result from reduced IRS-1/Akt/eNOS signalling in PGMO. These findings may result in higher risk of insulin resistance in neonates to PGMO pregnancies.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of in vitro reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration in vivo. These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.

We propose a statistical inference approach designed to detect the presence of collective behavior in cell motility experiments., Migration of cells can be characterized by two prototypical types of motion: individual and collective migration. We propose a statistical inference approach designed to detect the presence of cell-cell interactions that give rise to collective behaviors in cell motility experiments. This inference method has been first successfully tested on synthetic motional data and then applied to two experiments. In the first experiment, cells migrate in a wound-healing model: When applied to this experiment, the inference method predicts the existence of cell-cell interactions, correctly mirroring the strong intercellular contacts that are present in the experiment. In the second experiment, dendritic cells migrate in a chemokine gradient. Our inference analysis does not provide evidence for interactions, indicating that cells migrate by sensing independently the chemokine source. According to this prediction, we speculate that mature dendritic cells disregard intercellular signals that could otherwise delay their arrival to lymph vessels.

Significance CD4 T cells are major regulators of immune responses against both self and pathogens. Understanding pathways that govern CD4 T cell differentiation and regulation are thus key for the discovery of new immunoregulatory drug targets. Here, we have identified an epigenetic pathway that regulates the expression of a set of proteins that determine T cell responsiveness. By silencing enhancers distal to a set of genes known to be involved in regulatory T cell function, the epigenetic modifiers TRIM28 and HP1β/γ regulate T cell receptor signaling. This leads to defective metabolic reprograming and inefficient effector differentiation of naive T cells. This mechanism provides an exciting opportunity to regulate T cell responsivity in both autoimmunity and T cell-based immunodeficiencies., Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 β and γ isoforms (HP1β/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K–AKT–mTOR axis and switch to glycolysis. While differentiation of naive TRIM28−/− T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28−/− regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1β/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.

The first junior European Calcium Society online meeting, held October 20–21, 2020, aimed to promote junior researchers in the Ca 2+ community. The meeting included four scientific sessions, covering Ca 2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca 2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli., Cell polarity is marked by re-orientation of the centrosome, but the mechanisms governing centrosome polarization are poorly understood. Here Obino et al. show that in lymphocytes centrosome-associated Arp2/3 nucleates actin that tethers the centrosome to the nucleus; activation depletes Arp2/3 from the centrosome and frees it from the nucleus.

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules [1]. Although [2], how forces organize and mechanics has been shown to play an important role in this process impact on synapse function is unknown. We found that mechanical forces are spatio-temporally patterned at the immune synapse: global contractile forces are observed at the synapse periphery and local point-like forces are detected at its centre. The global contractile forces result from a pulsatile centripetal actomyosin flow that leads to formation of F-actin protrusions from which the central point like forces emerge. Noticeably, these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis. Accordingly, deletion of the myosin IIA gene leads to impaired B cell responses. The interplay between global and local forces governed by the actomyosin cytoskeleton therefore controls the endocytic function of the immune synapse and might constitute a more general mechanism in the physical regulation of cell-cell interactions.

Calcium ion (Ca2+) is an essential second messenger involved in multiple cellular and subcellular processes. Ca2+ can be released and sensed globally or locally within cells, providing complex signals of variable amplitudes and time-scales. The key function of Ca2+ in the regulation of acto-myosin contractility has provided a simple explanation for its role in the regulation of immune cell migration. However, many questions remain, including the identity of the Ca2+ stores, channels and upstream signals involved in this process. Here, we focus on dendritic cells (DCs), because their immune sentinel function heavily relies on their capacity to migrate within tissues and later on between tissues and lymphoid organs. Deciphering the mechanisms by which cytoplasmic Ca2+ regulate DC migration should shed light on their role in initiating and tuning immune responses.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

Obesity associates with the endoplasmic reticulum (ER) stress-induced endothelial dysfunction. Pregnant women with pre-pregnancy maternal obesity (PGMO) may transfer this potential risk to their offspring; however, whether ER stress occurs and associates with foetoplacental endothelial dysfunction in PGMO is unknown. We studied the L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs) from women with PGMO or with a normal pre-pregnancy weight. We analysed the expression and activation of the ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1 alpha), and activating transcription factor 6 (ATF6). PGMO associated with lower endothelial NO synthase activity due to increased Thr(495)-inhibitor and decreased Ser(1177)-stimulator phosphorylation. However, higher expression and activity of the human cationic amino acid transporter 1 was found. PGMO caused activation of PERK and its downstream targets eukaryotic initiation factor 2 (eIF2 alpha), C/EBP homologous protein 10 (CHOP), and tribbles-like protein 3 (TRB3). Increased IRE1 a protein abundance (but not its phosphorylation or X-box binding protein 1-mRNA splicing) and increased c-Jun N-terminal kinase 1 phosphorylation was seen in PGMO. A preferential nuclear location of the activating transcription factor 6 (ATF6) was found in HUVECs from PGMO. All the changes seen in PGMO were blocked by TUDCA but unaltered by tunicamycin. Thus, PGMO may determine a state of ER stress via upregulation of the PERK-eIP2 alpha-CHOP-TRB3 axis signalling in HUVECs. This phenomenon results in foetoplacental vascular endothelial dysfunction at birth.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ∼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ∼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

Upon its release from injured cells, such as infected, transformed, inflamed, or necrotic cells, extracellular adenosine-5'-triphosphate (ATP) acts as a danger signal that recruits phagocytes, such as neutrophils, macrophages, and dendritic cells (DCs), to the site of injury. The sensing of extracellular ATP occurs through purinergic (P2) receptors. We investigated the cellular mechanisms linking purinergic signaling to DC motility. We found that ATP stimulated fast DC motility through an autocrine signaling loop, which was initiated by the activation of P2X

Despite decades of research, how mammalian cell size is controlled remains unclear because of the difficulty of directly measuring growth at the single-cell level. Here we report direct measurements of single-cell volumes over entire cell cycles on various mammalian cell lines and primary human cells. We find that, in a majority of cell types, the volume added across the cell cycle shows little or no correlation to cell birth size, a homeostatic behavior called “adder”. This behavior involves modulation of G1 or S-G2 duration and modulation of growth rate. The precise combination of these mechanisms depends on the cell type and the growth condition. We have developed a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells. This reveals that a near-adder behavior is the most common type of size control and highlights the importance of growth rate modulation to size control in mammalian cells., The size of cells fluctuates but there are limited experimental methods to measure live mammalian cell sizes. Here, the authors track single cell volume (FXm) over the cell cycle and generate a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria., Highlights • MLN64 overexpression induces an increase in mitochondrial cholesterol content. • MLN64 protein expression is increased in NPC cells. • Down-regulation of MLN64 restores mitochondrial membrane potential and superoxide levels in NPC cells. • MLN64 overexpression produces mitochondrial dysfunction., Graphical abstract fx1

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

Extraction of immobilized antigens by B-cells relies on the fusion and secretion of lysosomes at the immune synapse. This process depends on the local recruitment of the SNARE protein Vamp-7 to the immune synapse, which is critical for B-cells to acquire their antigen presentation function., Recognition of surface-tethered antigens (Ags) by B-cells leads to the formation of an immune synapse that promotes Ag uptake for presentation onto MHC-II molecules. Extraction of immobilized Ags at the immune synapse of B-cells relies on the local secretion of lysosomes, which are recruited to the Ag contact site by polarization of their microtubule network. Although conserved polarity proteins have been implicated in coordinating cytoskeleton remodeling with lysosome trafficking, the cellular machinery associated with lysosomal vesicles that regulates their docking and secretion at the synaptic interface has not been defined. Here we show that the v-SNARE protein Vamp-7 is associated with Lamp-1+ lysosomal vesicles, which are recruited and docked at the center of the immune synapse of B-cells. A decrease in Vamp-7 expression does not alter lysosome transport to the synaptic interface but impairs their local secretion, a defect that compromises the ability of B-cells to extract, process, and present immobilized Ag. Thus our results reveal that B-cells rely on the SNARE protein Vamp-7 to promote the local exocytosis of lysosomes at the immune synapse, which is required for efficient Ag extraction and presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

Satellite glial cells (SGCs) are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system (CNS), astrocytes present connexin43 (Cx43) hemichannels and pannexin1 (Panx1) channels, and the opening of these channels allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complexes (NPJcs) using confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and in sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to a Ca(2+)/Mg(2+)-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT). Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because in those from Panx1 knockout mice showed a reduced increase of neuronal activity induced by Ca(2+)/Mg(2+)-free extracellular conditions. The data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons. ispartof: Frontiers in Cellular Neuroscience vol:8 issue:JUN ispartof: location:Switzerland status: published

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ∼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ∼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

The global epidemics of obesity during pregnancy and excessive gestational weight gain (GWG) are major public health problems worldwide. Obesity and excessive GWG are related to several maternal and fetal complications, including diabetes (pregestational and gestational diabetes) and intrauterine programming of insulin resistance (IR). Maternal obesity (MO) and neonatal IR are associated with long-term development of obesity, diabetes mellitus, and increased global cardiovascular risk in the offspring. Multiple mechanisms of insulin signaling pathway impairment have been described in obese individuals, involving complex interactions of chronically elevated inflammatory mediators, adipokines, and the critical role of the endoplasmic reticulum (ER) stress-dependent unfolded protein response (UPR). However, the underlying cellular processes linking MO and IR in the offspring have not been fully elucidated. Here, we summarize the state-of-the-art evidence supporting the possibility that adverse metabolic postnatal outcomes such as IR in the offspring of pregnancies with MO and/or excessive GWG may be related to intrauterine activation of ER stress response.

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X 7 receptors (P2X 7Rs). However, a link between P2X 7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1-/- mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1-/- mice, in which levels of P2X 7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X 7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.

Adverse microenvironmental stimuli can trigger the endoplasmic reticulum (ER) stress pathway, which initiates the unfolded protein response (UPR), to restore protein-folding homeostasis. Several studies show induction of ER stress during obesity. Chronic UPR has been linked to different mechanisms of disease in obese and diabetic individuals, including insulin resistance (IR) and impaired angiogenesis. Endothelial cell (EC) migration is an initial step for angiogenesis, which is associated with remodeling of existing blood vessels. EC migration occurs according to the leader-follower model, involving coordinated processes of chemotaxis, haptotaxis, and mechanotaxis. Thus, a fine-tuning of EC migration is necessary to provide the right timing to form the required vessels during angiogenesis. ER stress modulates EC migration at different levels, usually impairing migration and angiogenesis, although different effects may be observed depending on the tissue and/or microenvironment. In the context of pregnancy, maternal obesity (MO) induces IR in the offspring. Interestingly, several proteins associated with obesity-induced IR are also involved in EC migration, providing a potential link with the ER stress-dependent alterations observed in obese individuals. Different signaling cascades that converge on cytoskeleton regulation directly impact EC migration, including the Akt and/or RhoA pathways. In addition, ER is the main intracellular reservoir for Ca(2+), which plays a pivotal role during EC migration. Therefore, ER stress-related alterations in Ca(2+) signaling or Ca(2+) levels might also produce distorted EC migration. However, the above findings have been studied in the context of adult obesity, and no information has been reported regarding the effect of MO on fetal EC migration. Here we summarize the state of knowledge about the possible mechanisms by which ER stress and IR might impact EC migration and angiogenesis in fetal endothelium exposed to MO during pregnancy.

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1−/− astrocytes also showed more intracellular Ca2+ signal oscillations mediated by functional connexin 43 hemichannels and P2Y1 receptors. Therefore, Npc1−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.