Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

Functional basis for calmodulation of the TRPV5 calcium channel.

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.

FAM111A is dispensable for electrolyte homeostasis in mice.

Autosomal dominant mutations in FAM111A are causative for Kenny-Caffey syndrome type 2. Patients with Kenny-Caffey syndrome suffer from severe growth retardation, skeletal dysplasia, hypoparathyroidism, hypocalcaemia, hyperphosphataemia and hypomagnesaemia. While recent studies have reported FAM111A to function in antiviral response and DNA replication, its role in regulating electrolyte homeostasis remains unknown. In this study, we assessed the role of FAM111A in the regulation of serum electrolyte balance using a Fam111a knockout (Fam111a-/-) C57BL/6 N mouse model. Fam111a-/- mice displayed normal weight and serum parathyroid hormone (PTH) concentration and exhibited unaltered magnesium, calcium and phosphate levels in serum and 24-hour urine. Expression of calciotropic (including Cabp28k, Trpv5, Klotho and Cyp24a1), magnesiotropic (including Trpm6, Trpm7, Cnnm2 and Cnnm4) and phosphotropic (Slc20a1, Slc20a2, Slc34a1 and Slc34a3) genes in the kidneys, duodenum and colon were not affected by Fam111a depletion. Only Slc34a2 expression was significantly upregulated in the duodenum, but not in the colon. Analysis of femurs showed unaffected bone morphology and density in Fam111a-/- mice. Kidney and parathyroid histology were also normal in Fam111a-/- mice. In conclusion, our study is the first to characterise the function of FAM111A in vivo and we report that mice lacking FAM111A exhibit normal electrolyte homeostasis on a standard diet.

Tetraspanin CD53 controls T cell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate T cell receptor signaling. Although the critical role of CD45 in T cells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in T cell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and T cell activation. CD53-negative T cells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing T cells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for T cell immunity.

Fibroblast growth factor 23 is independently associated with renal magnesium handling in patients with chronic kidney disease.

Background: Disturbances in magnesium homeostasis are common in patients with chronic kidney disease (CKD) and are associated with increased mortality. The kidney is a key organ in maintaining normal serum magnesium concentrations. To this end, fractional excretion of magnesium (FEMg) increases as renal function declines. Despite recent progress, the hormonal regulation of renal magnesium handling is incompletely understood. Fibroblast Growth Factor 23 (FGF23) is a phosphaturic hormone that has been linked to renal magnesium handling. However, it has not yet been reported whether FGF23 is associated with renal magnesium handling in CKD patients. Methods: The associations between plasma FGF23 levels, plasma and urine magnesium concentrations and FEMg was investigated in a cross-sectional cohort of 198 non-dialysis CKD patients undergoing renal biopsy. Results: FGF23 was significantly correlated with FEMg (Pearson's correlation coefficient = 0.37, p<0.001) and urinary magnesium (-0.14, p=0.04), but not with plasma magnesium. The association between FGF23 and FEMg remained significant after adjusting for potential confounders, including estimated glomerular filtration rate (eGFR), parathyroid hormone and 25-hydroxyvitamin D. Conclusions: We report that plasma FGF23 is independently associated with measures of renal magnesium handling in a cohort of non-dialysis CKD patients. A potential causal relationship should be investigated in future studies.

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

The structure of CLEC-2: mechanisms of dimerization and higher-order clustering.

The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics.

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation.

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function.

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

BACKGROUND: Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. OBJECTIVES: Several formin proteins are expressed in platelets so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. METHODS: Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. RESULTS: Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. CONCLUSIONS: These data therefore demonstrated an important role for formin-mediated actin polymerization in platelet spreading and highlighted the importance of formins in cross-talk between the actin and tubulin cytoskeletons.

Mucor circinelloides induces platelet aggregation through integrin αIIbß3 and FcγRIIA.

Thrombosis is a hallmark of the fatal fungal infection mucormycosis. Yet, the platelet activation pathway in response to mucormycetes is unknown. In this study we determined the platelet aggregation potential of Mucor circinelloides (M. circinelloides) NRRL3631, characterized the signaling pathway facilitating aggregation in response to fungal spores, and identified the influence of the spore developmental stage upon platelet aggregation potential. Using impedance and light-transmission aggregometry, we showed that M. circinelloides induced platelet aggregation in whole blood and in platelet-rich plasma, respectively. The formation of large spore-platelet aggregates was confirmed by light-sheet microscopy, which showed spores dispersed throughout the aggregate. Aggregation potential was dependent on the spore's developmental stage, with the strongest platelet aggregation by spores in mid-germination. Inhibitor studies revealed platelet aggregation was mediated by the low affinity IgG receptor FcγRIIA and integrin αIIbß3; Src and Syk tyrosine kinase signaling; and the secondary mediators TxA2 and ADP. Flow cytometry of antibody stained platelets showed that interaction with spores increased expression of platelet surface integrin αIIbß3 and the platelet activation marker CD62P. Together, this is the first elucidation of the signaling pathways underlying thrombosis formation during a fungal infection, highlighting targets for therapeutic intervention.

Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics.

The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.

Salmonella-induced thrombi in mice develop asynchronously in the spleen and liver and are not effective bacterial traps.

Thrombosis is a frequent, life-threatening complication of systemic infection associated with multiple organ damage. We have previously described a novel mechanism of inflammation-driven thrombosis induced by Salmonella Typhimurium infection of mice. Thrombosis in the liver develops 7 days after infection, persisting after the infection resolves, and is monocytic cell dependent. Unexpectedly, thrombosis was not prominent in the spleen at this time, despite carrying a similar bacterial burden as the liver. In this study, we show that thrombosis does occur in the spleen but with strikingly accelerated kinetics compared with the liver, being evident by 24 hours and resolving rapidly thereafter. The distinct kinetics of thrombosis and bacterial burden provides a test of the hypothesis that thrombi form in healthy vessels to trap or remove bacteria from the circulation, often termed immunothrombosis. Remarkably, despite bacteria being detected throughout infected spleens and livers in the early days of infection, immunohistological analysis of tissue sections show that thrombi contain very low numbers of bacteria. In contrast, bacteria are present throughout platelet aggregates induced by Salmonella in vitro. Therefore, we show that thrombosis develops with organ-specific kinetics and challenge the universality of immunothrombosis as a mechanism to capture bacteria in vivo.

Differential expression of tetraspanin superfamily members in dendritic cell subsets.

Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC) and human DC subsets (CD1c+, CD141+, pDC) at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.

Tetraspanin microdomains control localized protein kinase C signaling in B cells.

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCß. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCß from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that Cd53-deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human CD53-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.

Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development.

The generation of high-affinity antibodies requires germinal center (GC) development and differentiation of long-lived plasma cells in a multilayered process that is tightly controlled by the activity of multiple transcription factors. Here, we reveal a new layer of complexity by demonstrating that dynamic changes in Id3 and E-protein activity govern both GC and plasma cell differentiation. We show that down-regulation of Id3 in B cells is essential for releasing E2A and E2-2, which in a redundant manner are required for antigen-induced B cell differentiation. We demonstrate that this pathway controls the expression of multiple key factors, including Blimp1, Xbp1, and CXCR4, and is therefore critical for establishing the transcriptional network that controls GC B cell and plasma cell differentiation.

The tetraspanin web revisited by super-resolution microscopy.

The spatial organization of membrane proteins in the plasma membrane is critical for signal transduction, cell communication and membrane trafficking. Tetraspanins organize functional higher-order protein complexes called 'tetraspanin-enriched microdomains (TEMs)' via interactions with partner molecules and other tetraspanins. Still, the nanoscale organization of TEMs in native plasma membranes has not been resolved. Here, we elucidated the size, density and distribution of TEMs in the plasma membrane of human B cells and dendritic cells using dual color stimulated emission depletion (STED) microscopy. We demonstrate that tetraspanins form individual nanoclusters smaller than 120 nm and quantified that a single tetraspanin CD53 cluster contains less than ten CD53 molecules. CD53 and CD37 domains were adjacent to and displayed only minor overlap with clusters containing tetraspanins CD81 or CD82. Moreover, CD53 and CD81 were found in closer proximity to their partners MHC class II and CD19 than to other tetraspanins. Although these results indicate that tetraspanin domains are adjacently positioned in the plasma membrane, they challenge the current view of the tetraspanin web of multiple tetraspanin species organized into a single domain. This study increases the molecular understanding of TEMs at the nanoscale level which is essential for comprehending tetraspanin function in cell biology.

Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs.

Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERα-positive breast cancer cells.

Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent prognostic marker in ESR1 (ERα gene)-positive breast cancer patients. In this study, we further investigated the molecular mechanism on how DC-SCRIPT inhibits breast cancer cell growth. DC-SCRIPT mRNA levels from 190 primary ESR1-positive breast tumors were related to global gene expression, followed by gene ontology and pathway analysis. The effect of DC-SCRIPT on breast cancer cell growth and cell cycle arrest was investigated using novel DC-SCRIPT-inducible MCF7 breast cancer cell lines. Genome-wide expression profiling of DC-SCRIPT-expressing MCF7 cells was performed to investigate the effect of DC-SCRIPT on cell cycle-related gene expression. Findings were validated by real-time PCR in a cohort of 1,132 ESR1-positive breast cancer patients. In the primary ESR1-positive breast tumors, DC-SCRIPT expression negatively correlated with several cell cycle gene ontologies and pathways. DC-SCRIPT expression strongly reduced breast cancer cell growth in vitro, breast tumor growth in vivo, and induced cell cycle arrest. In addition, in the presence of DC-SCRIPT, multiple cell cycles related genes were differentially expressed including the tumor suppressor gene CDKN2B. Moreover, in 1,132 primary ESR1-positive breast tumors, DC-SCRIPT expression also correlated with CDKN2B expression. Collectively, these data show that DC-SCRIPT acts as a novel regulator of CDKN2B and induces cell cycle arrest in ESR1-positive breast cancer cells.

Microdomains in the membrane landscape shape antigen-presenting cell function.

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.