RESUMO
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Assuntos
Colesterol , Proteínas Hedgehog , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligantes , Colesterol/metabolismo , Transdução de Sinais , Esteróis/metabolismoRESUMO
In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.
Assuntos
Blastocisto , Embrião de Mamíferos , Endoderma , Animais , Blastocisto/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Membrana Celular/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Endoderma/metabolismo , Mamíferos , Camundongos , Transporte ProteicoRESUMO
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/química , Movimento Celular , Receptor DCC/deficiência , Receptor DCC/genética , Proteínas Ligadas por GPI/química , Cones de Crescimento/fisiologia , Humanos , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed â¼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.
Assuntos
Ligantes , Mapas de Interação de Proteínas/fisiologia , Receptores de Superfície Celular/metabolismo , Receptor DCC/química , Receptor DCC/metabolismo , Humanos , Filogenia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/classificação , Receptores de Interleucina-1/química , Receptores de Interleucina-1/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/química , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome. Using a high-throughput technology to interrogate most single transmembrane receptors for binding to 445 IgSF proteins, we identify over 500 interactions, 82% previously undocumented, and confirm more than 60 receptor-ligand pairs using orthogonal assays. Our study reveals a map of cell-type-specific interactions and the landscape of dysregulated receptor-ligand crosstalk in cancer, including selective loss of function for tumor-associated mutations. Furthermore, investigation of the IgSF interactome in a large cohort of cancer patients identifies interacting protein signatures associated with clinical outcome. The IgSF interactome represents an important resource to fuel biological discoveries and a framework for understanding the functional organization of the surfaceome during homeostasis and disease, ultimately informing therapeutic development.
Assuntos
Imunoglobulinas/metabolismo , Neoplasias/patologia , Mapas de Interação de Proteínas , Antígeno B7-H1/metabolismo , Antígeno Carcinoembrionário/metabolismo , Comunicação Celular , Análise por Conglomerados , Meios de Cultivo Condicionados/química , Células HEK293 , Humanos , Imunoglobulinas/química , Imunoglobulinas/genética , Ligantes , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
Assuntos
Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteômica/métodos , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/metabolismo , Neurogênese/fisiologia , Nervo Olfatório/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/fisiologia , Receptores de Lipoproteínas/metabolismo , Olfato/fisiologiaRESUMO
Iron is essential for the survival of most bacteria but presents a significant challenge given its limited bioavailability. Furthermore, the toxicity of iron combined with the need to maintain physiological iron levels within a narrow concentration range requires sophisticated systems to sense, regulate, and transport iron. Most bacteria have evolved mechanisms to chelate and transport ferric iron (Fe3+) via siderophore receptor systems, and pathogenic bacteria have further lowered this barrier by employing mechanisms to utilize the host's hemoproteins. Once internalized, heme is cleaved by both oxidative and nonoxidative mechanisms to release iron. Heme, itself a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such, pathogenic bacteria have evolved sophisticated cell surface signaling and transport systems to obtain heme from the host. In this review, we summarize the structure and function of the heme-sensing and transport systems of pathogenic bacteria and the potential of these systems as antimicrobial targets.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Heme/antagonistas & inibidores , Ferro/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Expressão Gênica , Heme/metabolismo , Metaloporfirinas/síntese química , Metaloporfirinas/farmacologia , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sideróforos/antagonistas & inibidores , Sideróforos/biossíntese , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismoRESUMO
Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Peptídeos/genética , Peptidil Transferases/genética , Sequência de Aminoácidos/genética , Aminoaciltransferases/química , Proteínas de Bactérias/química , Catálise , Cisteína Endopeptidases/química , Humanos , Peptídeos/química , Peptidil Transferases/química , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Most single animal cells have an internal vector that determines where recycling membrane is added to the cell's surface. Because of the specific molecular composition of this added membrane, a dynamic asymmetry is formed on the surface of the cell. The consequences of this dynamic asymmetry are discussed, together with what they imply for how cells move. The polarity of a single-celled embryo, such as that of the nematode Caenorhabditis elegans, is explored in a similar framework.
Assuntos
Membrana Celular/química , Endocitose , Animais , Biologia/métodos , Caenorhabditis elegans , Movimento Celular , Citoesqueleto/metabolismo , Dictyostelium , Fibroblastos/metabolismo , Células HeLa , Hemaglutininas/química , Humanos , Proteínas de Membrana/química , Proteínas/químicaRESUMO
At its most fundamental level, life is a collection of synchronized cellular processes driven by interactions among biomolecules. Proximity labeling has emerged as a powerful technique to capture these interactions in native settings, revealing previously unexplored elements of biology. This review highlights recent developments in proximity labeling, focusing on methods that push the fundamental technologies beyond the classic bait-prey paradigm, such as RNA-protein interactions, ligand/small-molecule-protein interactions, cell surface protein interactions, and subcellular protein trafficking. The advancement of proximity labeling methods to address different biological problems will accelerate our understanding of the complex biological systems that make up life.
Assuntos
Proteínas de Membrana , Proteômica , Proteômica/métodos , Proteínas de Membrana/metabolismoRESUMO
In this Inaugural Article the author briefly revises its scientific career and how he starts to work with parasitic protozoa. Emphasis is given to his contribution to topics such as a) the structural organization of the surface of protozoa using freeze-fracture and deep-etching; b) the cytoskeleton of protozoa, especially structures such as the subpellicular microtubules of trypanosomatids, the conoid of Toxoplasma gondii, microtubules and inner membrane complex of this protozoan, and the costa of Tritrichomonas foetus; c) the flagellulm of trypanosomatids, that in addition to the axoneme contains a complex network of filaments that constitute the paraflagellar rod; d) special organelles such as the acidocalcisome, hydrogenosome, and glycosome; and e) the highly polarized endocytic pathway found in epimastigote forms of Trypanosoma cruzi.
Assuntos
Eucariotos , Microtúbulos , Masculino , Humanos , Citoesqueleto , Microscopia Eletrônica de Varredura , AxonemaRESUMO
The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs.
Assuntos
Polímeros , RNA , Humanos , RNA/genética , Membrana Celular , Membranas , CitosolRESUMO
Bacteria require a number of systems, including the type VI secretion system (T6SS), for interbacterial competition and pathogenesis. The T6SS is a large nanomachine that can deliver toxins directly across membranes of proximal target cells. Since major reassembly of T6SS is necessary after each secretion event, accurate timing and localization of T6SS assembly can lower the cost of protein translocation. Although critically important, mechanisms underlying spatiotemporal regulation of T6SS assembly remain poorly understood. Here, we used super-resolution live-cell imaging to show that while Acinetobacter and Burkholderia thailandensis can assemble T6SS at any site, a significant subset of T6SS assemblies localizes precisely to the site of contact between neighboring bacteria. We identified a class of diverse, previously uncharacterized, periplasmic proteins required for this dynamic localization of T6SS to cell-cell contact (TslA). This precise localization is also dependent on the outer membrane porin OmpA. Our analysis links transmembrane communication to accurate timing and localization of T6SS assembly as well as uncovers a pathway allowing bacterial cells to respond to cell-cell contact during interbacterial competition.
Assuntos
Sistemas de Secreção Tipo VI , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Proteico , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismoRESUMO
The T/t locus was a major focus of study by mouse geneticists during the 20th century. In the 70s, as the study of cell surface antigens controlling transplantation antigens was taking off, several laboratories hypothesized that alleles of this locus would control cell surface antigens important for embryonic development. One such antigen, the embryonal carcinoma F9 antigen was said to be an example. Other antigens were described on sperm and embryos that were said to be controlled by alleles at the T/t complex. These findings were later found to be false. The history of the findings and their refutation is described.
RESUMO
Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design.
RESUMO
Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.
Assuntos
Retículo Endoplasmático , Polissacarídeos , Transporte Proteico , Humanos , Glicosilação , Retículo Endoplasmático/metabolismo , Polissacarídeos/metabolismo , Glicoproteínas/metabolismo , Membrana Celular/metabolismo , Dobramento de Proteína , Proteômica/métodos , Glicoproteínas de Membrana/metabolismo , Complexo de Golgi/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Deinococcus radiodurans is an atypical diderm bacterium with a remarkable ability to tolerate various environmental stresses, due in part to its complex cell envelope encapsulated within a hyperstable surface layer (S-layer). Despite decades of research on this cell envelope, atomic structural details of the S-layer have remained obscure. In this study, we report the electron cryomicroscopy structure of the D. radiodurans S-layer, showing how it is formed by the Hexagonally Packed Intermediate-layer (HPI) protein arranged in a planar hexagonal lattice. The HPI protein forms an array of immunoglobulin-like folds within the S-layer, with each monomer extending into the adjacent hexamer, resulting in a highly interconnected, stable, sheet-like arrangement. Using electron cryotomography and subtomogram averaging from focused ion beam-milled D. radiodurans cells, we have obtained a structure of the cellular S-layer, showing how this HPI S-layer coats native membranes on the surface of cells. Our S-layer structure from the diderm bacterium D. radiodurans shows similarities to immunoglobulin-like domain-containing S-layers from monoderm bacteria and archaea, highlighting common features in cell surface organization across different domains of life, with connotations on the evolution of immunoglobulin-based molecular recognition systems in eukaryotes.
Assuntos
Proteínas de Bactérias , Deinococcus , Proteínas de Bactérias/metabolismo , Deinococcus/química , Membrana Celular/metabolismo , Parede Celular/metabolismo , Imunoglobulinas/metabolismoRESUMO
Hepatocellular carcinoma (HCC) takes the predominant malignancy of hepatocytes with bleak outcomes owing to high heterogeneity among patients. Personalized treatments based on molecular profiles will better improve patients' prognosis. Lysozyme (LYZ), a secretory protein with antibacterial function generally expressed in monocytes/macrophages, has been observed for the prognostic implications in different types of tumors. However, studies about the explicit applicative scenarios and mechanisms for tumor progression are still quite limited, especially for HCC. Here, based on the proteomic molecular classification data of early-stage HCC, we revealed that the LYZ level was elevated significantly in the most malignant HCC subtype and could serve as an independent prognostic predictor for HCC patients. Molecular profiles of LYZ-high HCCs were typical of those for the most malignant HCC subtype, with impaired metabolism, along with promoted proliferation and metastasis characteristics. Further studies demonstrated that LYZ tended to be aberrantly expressed in poorly differentiated HCC cells, which was regulated by STAT3 activation. LYZ promoted HCC proliferation and migration in both autocrine and paracrine manners independent of the muramidase activity through the activation of downstream protumoral signaling pathways via cell surface GRP78. Subcutaneous and orthotopic xenograft tumor models indicated that targeting LYZ inhibited HCC growth markedly in NOD/SCID mice. These results propose LYZ as a prognostic biomarker and therapeutic target for the subclass of HCC with an aggressive phenotype.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Muramidase/metabolismo , Proteômica , Linhagem Celular Tumoral , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Processos Neoplásicos , Biomarcadores Tumorais/genética , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.
Assuntos
Eritrócitos , Proteínas de Membrana , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Substâncias Macromoleculares/metabolismo , Eritrócitos/metabolismoRESUMO
Spleen dendritic cells (DC) are critical for initiation of adaptive immune responses against blood-borne invaders. Key to DC function is their positioning at sites of pathogen entry, and their abilities to selectively capture foreign antigens and promptly engage T cells. Focusing on conventional DC2 (cDC2), we discuss the contribution of chemoattractant receptors (EBI2 or GPR183, S1PR1, and CCR7) and integrins to cDC2 positioning and function. We give particular attention to a newly identified role in cDC2 for adhesion G-protein coupled receptor E5 (Adgre5 or CD97) and its ligand CD55, detailing how this mechanosensing system contributes to splenic cDC2 positioning and homeostasis. Additional roles of CD97 in the immune system are reviewed. The ability of cDC2 to be activated by circulating missing self-CD47 cells and to integrate multiple red blood cell (RBC)-derived inputs is discussed. Finally, we describe the process of activated cDC2 migration to engage and prime helper T cells. Throughout the review, we consider the insights into cDC function in the spleen that have emerged from imaging studies.