Cell division in tissues enables macrophage infiltration.

Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Mechanics of developmental migration.

The ability to migrate is a fundamental property of animal cells which is essential for development, homeostasis and disease progression. Migrating cells sense and respond to biochemical and mechanical cues by rapidly modifying their intrinsic repertoire of signalling molecules and by altering their force generating and transducing machinery. We have a wealth of information about the chemical cues and signalling responses that cells use during migration. Our understanding of the role of forces in cell migration is rapidly evolving but is still best understood in the context of cells migrating in 2D and 3D environments in vitro. Advances in live imaging of developing embryos combined with the use of experimental and theoretical tools to quantify and analyse forces in vivo, has begun to shed light on the role of mechanics in driving embryonic cell migration. In this review, we focus on the recent studies uncovering the physical basis of embryonic cell migration in vivo. We look at the physical basis of the classical steps of cell migration such as protrusion formation and cell body translocation and review the recent research on how these processes work in the complex 3D microenvironment of a developing organism.

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion.

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva's vertebrate ortholog, MFSD1, rescues the minerva mutant's migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis.

Drosophila TNF Modulates Tissue Tension in the Embryo to Facilitate Macrophage Invasive Migration.

Migrating cells penetrate tissue barriers during development, inflammatory responses, and tumor metastasis. We study if migration in vivo in such three-dimensionally confined environments requires changes in the mechanical properties of the surrounding cells using embryonic Drosophila melanogaster hemocytes, also called macrophages, as a model. We find that macrophage invasion into the germband through transient separation of the apposing ectoderm and mesoderm requires cell deformations and reductions in apical tension in the ectoderm. Interestingly, the genetic pathway governing these mechanical shifts acts downstream of the only known tumor necrosis factor superfamily member in Drosophila, Eiger, and its receptor, Grindelwald. Eiger-Grindelwald signaling reduces levels of active Myosin in the germband ectodermal cortex through the localization of a Crumbs complex component, Patj (Pals-1-associated tight junction protein). We therefore elucidate a distinct molecular pathway that controls tissue tension and demonstrate the importance of such regulation for invasive migration in vivo.

Tools Allowing Independent Visualization and Genetic Manipulation of Drosophila melanogaster Macrophages and Surrounding Tissues.

Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses, and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid a fuller understanding of these processes, but are lacking. Here, we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, the cytoplasm, or the actin cytoskeleton from embryonic stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae, and adults. They permit efficient GAL4-independent Fluorescence-Activated Cell Sorting (FACS) analysis/sorting of plasmatocytes throughout life. To facilitate genetic studies of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that, in combination with extant GAL4 drivers, allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and GAL80 lines that block GAL4 drivers from affecting plasmatocytes, all of which function from the early embryo to the adult.

Drosophila immune cell migration and adhesion during embryonic development and larval immune responses.

The majority of immune cells in Drosophila melanogaster are plasmatocytes; they carry out similar functions to vertebrate macrophages, influencing development as well as protecting against infection and cancer. Plasmatocytes, sometimes referred to with the broader term of hemocytes, migrate widely during embryonic development and cycle in the larvae between sessile and circulating positions. Here we discuss the similarities of plasmatocyte developmental migration and its functions to that of vertebrate macrophages, considering the recent controversy regarding the functions of Drosophila PDGF/VEGF related ligands. We also examine recent findings on the significance of adhesion for plasmatocyte migration in the embryo, as well as proliferation, trans-differentiation, and tumor responses in the larva. We spotlight parallels throughout to vertebrate immune responses.

Type II phosphatidylinositol 4-kinase ß is an integral signaling component of early T cell activation mechanisms.

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase ß in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase ß with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase ß in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase ß with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase ß is a key component in early T cell activation signaling cascades.

Coordinating Rho and Rac: the regulation of Rho GTPase signaling and cadherin junctions.

Cadherin-based cell-cell adhesions are dynamic structures that mediate tissue organization and morphogenesis. They link cells together, mediate cell-cell recognition, and influence cell shape, motility, proliferation, and differentiation. At the cellular level, operation of classical cadherin adhesion systems is coordinated with cytoskeletal dynamics, contractility, and membrane trafficking to support productive interactions. Cadherin-based cell signaling is critical for the coordination of these many cellular processes. Here, we discuss the role of Rho family GTPases in cadherin signaling. We focus on understanding the pathways that utilize Rac and Rho in junctional biology, aiming to identify the mechanisms of upstream regulation and define how the effects of these activated GTPases might regulate the actin cytoskeleton to modulate the cellular processes involved in cadherin-based cell-cell interactions.

A WAVE2-Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens.

The epithelial zonula adherens (ZA) is a specialized adhesive junction where actin dynamics and myosin-driven contractility coincide. The junctional cytoskeleton is enriched in myosin II, which generates contractile force to support junctional tension. It is also enriched in dynamic actin filaments, which are replenished by ongoing actin assembly. In this study we sought to pursue the relationship between actin assembly and junctional contractility. We demonstrate that WAVE2-Arp2/3 is a major nucleator of actin assembly at the ZA and likely acts in response to junctional Rac signaling. Furthermore, WAVE2-Arp2/3 is necessary for junctional integrity and contractile tension at the ZA. Maneuvers that disrupt the function of either WAVE2 or Arp2/3 reduced junctional tension and compromised the ability of cells to buffer side-to-side forces acting on the ZA. WAVE2-Arp2/3 disruption depleted junctions of both myosin IIA and IIB, suggesting that dynamic actin assembly may support junctional tension by facilitating the local recruitment of myosin.

A bigger picture: classical cadherins and the dynamic actin cytoskeleton.

Classical cadherin adhesion receptors influence tissue integrity in health and disease. Their biological function is intimately linked to the actin cytoskeleton. To date, research has largely focused on identifying the molecular mechanisms that physically couple cadherin to cortical actin filaments. However, the junctional cytoskeleton is dynamic. Recent developments in understanding how filament dynamics and organization in the junctional cytoskeleton are controlled provide new insights into how the actin cytoskeleton regulates cadherin junctions in health and disease.

Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens.

The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. This can potentially be determined by the balance between molecules that promote nucleotide exchange or GTP hydrolysis. However, how these activities may be coordinated is poorly understood. We now report a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. We identify an extramitotic activity of the centralspindlin complex, better understood as a cytokinetic regulator, which localizes to the interphase zonula adherens by interacting with the cadherin-associated protein, α-catenin. Centralspindlin recruits the RhoGEF, ECT2, to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localization of p190 B RhoGAP, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is used in interphase cells to control the Rho GTPase cycle at the zonula adherens.

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway.

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.

Doing cell biology in embryos: regulated membrane traffic and its implications for cadherin biology.

Regulated trafficking of cadherin adhesion molecules is often invoked as a mechanism to generate dynamic adhesive cell-cell contacts for tissue modeling and morphogenesis. The past 2-3 years have seen several important papers that tackle the cell biology of cadherin trafficking in organismal systems to provide new insights into both mechanism and morphogenetic impact.

Pentoxifylline inhibits integrin-mediated adherence of 12(S)-HETE and TNFalpha-activated B16F10 cells to fibronectin and endothelial cells.

BACKGROUND: Pentoxifylline (PTX), a phosphodiesterase inhibitor, inhibits homing of metastatic B16F10 melanoma cells to the lung. Studies on the mechanism of action of PTX showed inhibition of adhesion of cultured melanoma cells to various extracellular matrix substrates and inhibition of cell surface integrin expression. The aim of this study was to determine the effect of PTX on surface expression of integrin and integrin-mediated adhesion induced by biological mediators, tumour necrosis factor (TNF) alpha and 12(S)-hydroxyeicosatetraenoic acid (HETE), in B16F10 cells. MATERIALS AND METHODS: B16F10 cells were treated with 12(S)-HETE (1 microM, 1 h), TNFalpha (5 ng/ml, 2 h) and phorbol 12-myristate 13-acetate (400 nM, 20 min), and the effect of PTX on these treatments was studied by flow cytometry, adhesion assay and confocal microscopy. RESULTS: 12(S)-HETE and TNFalpha brought about an increase in the surface expression of beta(1) integrins and F10 cell adhesion to fibronectin and endothelial cells; this increased adhesion was mediated at least in part by alterations in the localization of beta(1) integrins. Pretreatment with PTX was able to completely abrogate this induction in integrin expression. CONCLUSION: PTX can inhibit surface expression of integrin and integrin-mediated adhesion induced by several biological mediators, and this might be a possible mechanism for its antimetastatic action, in vivo.

Antimetastatic Action of Pentoxifylline, a Methyl Xanthine Derivative, Through its Effect on PKC Mediated Integrin Transport in B16F10 Melanoma Cells.

BACKGROUND: Integrins are adhesion molecules known to regulate cellular processes like adhesion, migration and proliferation. At the same time role of integrin in progress of cancer metastasis is well established, increased integrin expression is reported to be linked to high metastasis potential of cells. Pentoxifylline a methyl xanthine derivative is a potent antimetastatic agent. Studies on the mechanism of inhibition of lung homing of B16F10 melanoma cells by PTX shows that it can inhibit cell- Extracellular Matrix adhesion, cell surface integrin expression as well as Protein kinase C activity. Previous study from our laboratory have shown PTX treatment can selectively inhibit the cell surface expression of α5 integrin in B16F10 cells without affecting its total cellular protein levels. Numerous studies have documented that differences in surface expression and distribution of integrins affects metastasis. The purpose of present study is to observe the effect of PTX on cellular distribution/ redistribution of integrins and to study the underlying molecular mechanism of PTX action. METHODS: Integrin internalization and transport was observed using immunofluorescence confocal microscopy. PKC activity was determined using MBP4-14 as a substrate. Immunoprecipitation and western blotting was used to show association between PKC and α5 integrin, cell adhesion assay was performed using fibronectin/fibrinogen as substrate. RESULTS: Immunofluorescence studies showed that PTX treatment caused a redistribution of α5 integrins from the plasma membrane to a perinuclear compartment where it colocalized with Transferrin receptor and Rab-11 GTPase. Rate of integrin internalization and recycling showed that PTX inhibited the recycling of α5 integrins from perinuclear recycling endosomes. PTX is reported to affect kinases; here we showed that PTX inhibited total PKC activity. Association between α5ß1 integrin and PKC is studied using Immunoprecipitation which show that PTX affects α5ß1 integrin associated PKC activity without affecting the levels of PKC. Studying the effect of delay in integrin recycling on cell functionality showed that it affects spreading of cells on fibronectin/fibrinogen. CONCLUSIONS: Data in the present study shows that PTX interferes with PKC activity bringing about a change in integrin distribution, and there by affecting the functionality of the cell. And this may possibly serve as one of the mechanisms for antimetastatic action of PTX.

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components.

Our previous studies demonstrated that Pentoxifylline (PTX), a phosphodiesterase inhibitor, could inhibit the lung homing of B16-F10 melanoma cells in C57BL/6 mice. In this study we have looked at the effect of PTX on cell surface integrin expression and integrin mediated adhesion of B16-F10 melanoma cells. B16-F10 cells treated with PTX when injected through the tail vein of mice showed a 75% reduction in pulmonary nodules as compared to control untreated cells. PTX brought about a significant reduction in the integrin mediated adhesion of F10 cells to Fibronectin and Vitronectin (58.75% +/- 3.4 S.E and 60% +/- 1.7 S.E respectively if control was considered as 100%). This inhibition in adhesion was evident up to four hours only and treatment for 24 hours brought about an increase in adhesion (135.5% +/- 0.5 S.E). Flow cytometric analysis showed higher surface expressions of alphav, alpha5 and alphaIIb integrin subunits in B16-F10 as compared to the low metastatic cell line B16-F1 suggesting a role for these integrins in determining the metastatic potential. PTX brought about a significant decrease in the cell surface expression of alpha5, alphaIIb and beta1 integrin subunits but not that of the alphav subunit on B16-F10 cells. PTX also brought about a reduction in the total cellular protein levels of beta1 and alphav integrin subunits. Various isoforms of Protein Kinase C (PKC) has been shown to regulate integrin expression, localization and activity. Hence we looked at the effect of PTX on total cellular PKC activity. PTX brought about a significant reduction in total cellular PKC activity (82.66 +/- 0.593). Collectively our results indicate that the antimetastatic action of PTX is mediated, at least in part through its effects on adhesion and the surface expression of specific integrin receptors.

Resveratrol inhibits type II phosphatidylinositol 4-kinase: a key component in pathways of phosphoinositide turn over.

Resveratrol has anti-inflammatory, cardio protective and cancer chemopreventive properties. The molecular targets for resveratrol in early signaling cascades are not well understood. Resveratrol inhibits type II PtdIns 4-kinase but not PtdIns 3-kinase activity in vitro. Resveratrol directly binds to the enzyme with a Kd of 7.2 microM. Kinetic studies show that resveratrol competes with PtdIns binding. Inhibition of PtdIns 4-kinase activity by resveratrol/phenylarsine oxide reduces Jurkat cell adhesion to matrigel/fibronectin coated surfaces, suggesting a role for type II PtdIns 4-kinase in lymphocyte infiltration to the sites of inflammation.