Pericytes Elicit Resistance to Vemurafenib and Sorafenib Therapy in Thyroid Carcinoma via the TSP-1/TGFß1 Axis.

PURPOSE: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. EXPERIMENTAL DESIGN: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. RESULTS: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFß1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFß1 expression evoke gene-regulatory networks and pathways (TGFß signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand-VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFß1 axis reduces tumor cell growth and overcomes drug resistance. CONCLUSIONS: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFß1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis.

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils.

Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.

Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes.

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.

TM4SF1: a new vascular therapeutic target in cancer.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.

Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

The tetraspanin CD63 is required for efficient IgE-mediated mast cell degranulation and anaphylaxis.

Mast cell (MC) activation through the high-affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit FcεRI-induced MC degranulation, the intrinsic role of these molecules in FcεRI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow-derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow-derived MC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by ß-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, FcεRI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kit(w/w-v) mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.

Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

The internal architecture of leukocyte lipid body organelles captured by three-dimensional electron microscopy tomography.

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their "cores". After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB "cores" and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans.

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Mesenchymal stem cells transmigrate between and directly through tumor necrosis factor-α-activated endothelial cells via both leukocyte-like and novel mechanisms.

Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM-1-enriched "transmigratory cups". Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms.

Lipid body-phagosome interaction in macrophages during infectious diseases: host defense or pathogen survival strategy?

Phagocytosis of invading microorganisms by specialized cells such as macrophages and neutrophils is a key component of the innate immune response. These cells capture and engulf pathogens and subsequently destroy them in intracellular vacuoles-the phagosomes. Pathogen phagocytosis and progression and maturation of pathogen-containing phagosomes, a crucial event to acquire microbicidal features, occurs in parallel with accentuated formation of lipid-rich organelles, termed lipid bodies (LBs), or lipid droplets. Experimental and clinical infections with different pathogens such as bacteria, parasites, and viruses induce LB accumulation in cells from the immune system. Within these cells, LBs synthesize and store inflammatory mediators and are considered structural markers of inflammation. In addition to LB accumulation, interaction of these organelles with pathogen-containing phagosomes has increasingly been recognized in response to infections and may have implications in the outcome or survival of the microorganism within host cells. In this review, we summarize our current knowledge on the LB-phagosome interaction within cells from the immune system, with emphasis on macrophages, and discuss the functional meaning of this event during infectious diseases.

Revealing the role of phospholipase Cß3 in the regulation of VEGF-induced vascular permeability.

VEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cß3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCß3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

Antigen recognition is facilitated by invadosome-like protrusions formed by memory/effector T cells.

Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.

Vascular hyperpermeability, angiogenesis, and stroma generation.

It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level "basal" permeability of the normal vasculature; (2) the "acute," short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the "chronic" hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the clotting system, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.

TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration.

Transmembrane-4-L-six-family-1 (TM4SF1) is a tetraspanin-like membrane protein that is highly and selectively expressed by cultured endothelial cells (EC) and, in vivo, by EC lining angiogenic tumor blood vessels. TM4SF1 is necessary for the formation of unusually long (up to a 50 µm), thin (~100-300 nm wide), F-actin-poor EC cell projections that we term 'nanopodia'. Immunostaining of nanopodia at both the light and electron microsopic levels localized TM4SF1 in a regularly spaced, banded pattern, forming TM4FS1-enriched domains. Live cell imaging of GFP-transduced HUVEC demonstrated that EC project nanopodia as they migrate and interact with neighboring cells. When TM4SF1 mRNA levels in EC were increased from the normal ~90 mRNA copies/cell to ~400 copies/cell through adenoviral transduction, EC projected more and longer nanopodia from the entire cell circumference but were unable to polarize or migrate effectively. When fibroblasts, which normally express TM4SF1 at ~5 copies/cell, were transduced to express TM4SF1 at EC-like levels, they formed typical TM4SF1-banded nanopodia, and broadened, EC-like lamellipodia. Mass-spectrometry demonstrated that TM4SF1 interacted with myosin-10 and ß-actin, proteins involved in filopodia formation and cell migration. In summary, TM4SF1, like genuine tetraspanins, serves as a molecular organizer that interacts with membrane and cytoskeleton-associated proteins and uniquely initiates the formation of nanopodia and facilitates cell polarization and migration.

Lipid bodies in inflammatory cells: structure, function, and current imaging techniques.

Lipid bodies (LBs), also known as lipid droplets, have increasingly been recognized as functionally active organelles linked to diverse biological functions and human diseases. These organelles are actively formed in vivo within cells from the immune system, such as macrophages, neutrophils, and eosinophils, in response to different inflammatory conditions and are sites for synthesis and storage of inflammatory mediators. In this review, the authors discuss structural and functional aspects of LBs and current imaging techniques to visualize these organelles in cells engaged in inflammatory processes, including infectious diseases. The dynamic morphological aspects of LBs in leukocytes as inducible, newly formable organelles, elicitable in response to stimuli that lead to cellular activation, contribute to the evolving understanding of LBs as organelles that are critical regulators of different inflammatory diseases, key markers of leukocyte activation, and attractive targets for novel anti-inflammatory therapies.

Contributions of electron microscopy to understand secretion of immune mediators by human eosinophils.

Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.