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High-content image-based cell phenotyping provides fundamental insights in a broad variety of life science areas. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, even more importantly with the advent of data sharing initiatives. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy have not been systematically investigated. Here, using high content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells and time points. Significant technical variability occurred between laboratories, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image data and meta-analysis depend on standardized procedures and batch correction applied to studies of perturbation effects.

Colorectal cancer (CRC) is the third most common cancer worldwide. The TP53 gene is mutated in approximately 60% of all CRC cases. Sporadic CRC is characterized by high prevalence of TP53 hotspot missense mutations. In particular, over 20 percent of all TP53-mutated CRC tumors carry either the p53R175H structural mutant or the p53R273H DNA contact mutant. Importantly, clinical data analysis suggests that CRC tumors harboring p53 R273 mutations are more prone to progress to metastatic disease than those with R175 mutations, in association with decreased survival. By combining in vitro CRC cell line models and human CRC data mining, we identified a unique transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse patient outcome. Concordantly, p53R273H selectively promotes rapid CRC cell spreading, migration and invasion in vitro and metastasis in vivo. Mechanistically, the transcriptional output of p53R273H is associated with, and presumably driven by, its preferential binding to regulatory elements of R273 signature genes. Together, this demonstrates that different TP53 missense mutations contribute differently to cancer progression, and that p53R273H possesses distinct gain-of-function activities in CRC that bear on disease course and possibly on patient management strategy. Given that practically all current analytical cancer gene panels include TP53, elucidation of the differential impact of distinct TP53 mutations on disease features is expected to make information on TP53 mutations more actionable and holds potential for better precision-based medicine.

Integrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling ‘adhesome’ components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different – often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their ‘adhesive’ and ‘invasive’ functionalities require distinct sources of biomechanical reinforcement.

Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.

Background & Aims The intestinal barrier protects intestinal cells from microbes and antigens in the lumen—breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. Methods We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. Results The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. Conclusions We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.

Objective: Aggregation and modification of LDLs (low-density lipoproteins) promote their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of agLDL (aggregated LDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse. Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites, such as cholesterol, promotes foam cell formation, a process that drives atherogenesis. Furthermore, there is accumulating evidence for the involvement of TLR4 (Toll-like receptor 4) and its adaptor protein MyD88 (myeloid differentiation primary response 88) in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the lysosomal synapse and foam cell formation. Approach and Results: Using bone marrow–derived macrophages from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment, and foam cell formation. Using siRNA (small interfering RNA), pharmacological inhibition and knockout bone marrow–derived macrophages, we implicate SYK (spleen tyrosine kinase), PI3K (phosphoinositide 3-kinase), and Akt in agLDL catabolism using the lysosomal synapse. Using bone marrow transplantation of LDL receptor knockout mice with TLR4 knockout bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL. Conclusions: We present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the lysosomal synapse, catabolism of agLDL, and lipid accumulation in vitro and in vivo.

ObjectiveAggregation and modification of low-density lipoproteins (LDL) promotes their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of aggregated LDL (agLDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse (LS). Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites such as cholesterol promotes foam cell formation, a process that drives atherogenesis. Further, there is accumulating evidence for the involvement of TLR4 and its adaptor protein MyD88 in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the LS and foam cell formation.Approach and ResultsUsing bone marrow-derived macrophages (BMMs) from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment and foam cell formation. Using siRNA, pharmacological inhibition and knockout BMMs, we implicate SYK, PI3 kinase and Akt in agLDL catabolism using the LS. Using bone marrow transplantation of LDL receptor knockout mice with TLR4KO bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophagesin vivoform an extracellular compartment and exocytose lysosome contents similar to that observedin vitrofor degradation of agLDL.ConclusionsWe present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the LS, catabolism of agLDL and lipid accumulationin vitroandin vivo.

Metabolic syndrome, leaky guts, and infection Metabolic syndrome often accompanies obesity and hyperglycemia and is associated with a breakdown in the integrity of the intestinal barrier and increased risk of systemic infection. Thaiss et al. found that mice with systemic infection of a Salmonella analog, Citrobacter rodentium , also exhibited hyperglycemia. Deletion of the glucose transporter GLUT2 altered sensitivity to chemically induced epithelial permeability and protected mice from pathogen invasion. The authors also found a correlation in humans between glycated hemoglobin (an indicator of hyperglycemia) and serum levels of pathogen recognition receptor ligands. Science , this issue p. 1376

Objective— Although dendritic cells are known to play a role in atherosclerosis, few studies have examined the contribution of the wide variety of dendritic cell subsets. Accordingly, their roles in atherogenesis remain largely unknown. We investigated the ability of different dendritic cell subsets to become foam cells after contact with aggregated low-density lipoprotein (LDL; the predominant form of LDL found in atherosclerotic plaques). Approach and Results— We demonstrate that both murine and human monocyte-derived dendritic cells use exophagy to degrade aggregated LDL, leading to foam cell formation, whereas monocyte-independent dendritic cells are unable to clear LDL aggregates by this mechanism. Exophagy is a catabolic process in which objects that cannot be internalized by phagocytosis (because of their size or association with extracellular structures) are initially digested in an extracellular acidic lytic compartment. Surprisingly, we found that monocyte-derived dendritic cells upregulate exophagy on maturation. This contrasts various forms of endocytic internalization in dendritic cells, which decrease on maturation. Finally, we show that our in vitro results are consistent with dendritic cell lipid accumulation in plaques of an ApoE −/− mouse model of atherosclerosis. Conclusions— Our results show that monocyte-derived dendritic cells use exophagy to degrade aggregated LDL and become foam cells, whereas monocyte-independent dendritic cells are unable to clear LDL deposits. Furthermore, we find that exophagy is upregulated on dendritic cell maturation. Thus, exophagy-mediated foam cell formation in monocyte-derived dendritic cells could play a significant role in atherogenesis.

Despite impressive advances in research, prevention, and treatment, atherosclerotic vascular disease remains the leading cause of death in the developed world. Mechanisms of cholesterol accumulation in the arteries have been studied intensively, but the in vivo contributions of different pathways leading to lipid accumulation and foam cell formation are not understood. In the arteries, low-density lipoprotein (LDL) is aggregated and bound to the extracellular matrix. When such aggregated LDL is presented to macrophages, they form a novel acidic, hydrolytic compartment that is topologically extracellular, to which lysosomal enzymes are secreted. Such compartments are observed in vivo in murine atherosclerotic plaque macrophages interacting with cholesterol rich deposits. Using state-of-the-art quantitative and high resolution microscopy techniques, characterization of compartment morphology reveals how macrophages use local actin polymerization to drive plasma membrane remodeling at the interface with aggregated LDL. This leads to sequestration of aggregated LDL into topologically convoluted structures that allow acidification, catabolism and internalization of LDL. We find that a TLR4/MyD88/Syk/PI3 kinase/Akt dependent signaling pathway in macrophages regulates the formation of such catabolic compartments. Consistent with this, deficiency of TLR4 in vivo can protect macrophages from lipid accumulation in murine atherosclerotic plaques. Herein, we provide compelling evidence for a novel form of catabolism that macrophages use to degrade aggregated LDL in vivo during atherosclerosis and this process leads to foam cell formation, cell death and promotes disease progression.

Monocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub-regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane., Summary: Macrophages use extracellular compartments, termed lysosomal synapses, that are topologically complex, have a convoluted structure and contain sub-regions that are acidified to digest aggregated lipoproteins.

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies., Research in our laboratories is funded by the Spanish Ministerio de Economía y Competitividad and the FEDER programme (BFU2013-48988-C2-1-P to M.D.M.-B, BFU2012-35446 to A.G.-R., BFU2011-30303 and BFU2010-18959 to D.G.M.) and by the Junta de Andalucía (Proyecto de Excelencia P09-CVI-5058). I.G. was supported by a JAE-DOC (CSIC) and D.G.M. by a Ramon y Cajal Fellowship (Ministerio Español de Economía y Competitividad Ref. RYC-2010-07450) and a Marie Curie International Reintegration Grant (EU, Ref. 248346-NMSSBLS).

The armadillo-family protein, p120 catenin (p120), binds to the juxtamembrane domain of classical cadherins and increases cell–cell junction stability. Overexpression of p120 modulates the activity of Rho family GTPases and augments cell migratory ability. Here we show that down-regulation of p120 in epithelial MCF-7 cells by siRNA leads to a striking decrease in lamellipodial persistence and focal adhesion formation. Similar alterations in lamellipodial activity were observed in MCF-7 cells treated with siRNA to cortactin, an activator of Arp2/3-dependent actin polymerization. We found that, in many cell types, p120 is colocalized with cortactin-containing actin structures not only at cell–cell junctions, but also at extrajunctional sites including membrane ruffles and actin-rich halos around endocytotic vesicles. p120 depletion led to dramatic loss of cortactin and its partner, Arp3, from the cell leading edges. Cortactin and p120 are shown to directly interact with each other via the cortactin N-terminal region. We propose that the mechanism underlying p120 functions at the leading edge involves its cooperation with cortactin.

Introduction: One of the initiating events in atherogenesis is the deposition of low-density lipoproteins (LDL) in the arterial wall. This LDL becomes modified, aggregated and retained. Macrophages form a degradative contact (a lysosomal synapse or LS) with the aggregated LDL (agLDL) through local actin polymerization. Degradation of agLDL releases free cholesterol, which is taken up by macrophages and results in foam cell formation. Characterization of proteins regulating actin polymerization that allows LS formation may identify new therapeutic targets to inhibit foam cell formation and halt the progression of atherosclerosis. Hypothesis: Small GTPases of the Rho family such as RhoA are key regulators of actin polymerization. Further, many lipids in the micro-environment of the atherosclerotic plaque, such as ceramide, are known to modulate RhoA activity. Therefore we tested the hypothesis that RhoA is important in actin polymerization and foam cell formation in response to macrophage catabolism of agLDL and that this process can be modulated by ceramide. Methods: We overexpressed RhoA in RAW264.7 macrophages prior to treatment with agLDL to test the role of RhoA in LS formation and foam cell formation. We used bone marrow derived macrophages (BMM) from Sphingosine Kinase 2 knockout mice (SK2 KO) as a model for macrophages loaded with long chain ceramides and wild-type BMM loaded with C2-ceramide to test the role of ceramide. We also used inhibitors of Rho Kinase ROCK and LIM Kinase to assess their role in LS formation. Results and Conclusions: Overexpression of wild-type and constitutive active RhoA in RAW264.7 macrophages significantly impaired actin polymerization and foam cell formation in response to agLDL treatment. Using SK2KO BMM and wild-type BMM loaded with C2- ceramide, we find that macrophages loaded with ceramide display increased levels of activated RhoA. This leads to impaired actin polymerization and foam cell formation which could be rescued by RNAi mediated silencing of RhoA, inhibition of ROCK or inhibition of LIMK. In conclusion, these results suggest that RhoA/ROCK/LIMK signaling negatively regulates agLDL degradation and foam cell formation and that this process can be directly modulated by ceramide through activation of RhoA.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24-48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (microl min-1 mmHg-1) for all anterior segments averaged (mean+/-sem): human, 0.19+/-0.03 (n=12); monkey, 0.36+/-0.02 (n=19). In human anterior segments, transduction with 10(7) plaque forming units of AdGFPCald increased outflow facility by 43+/-21% (por=0.11, n=6) at 66 hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2-3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5x10(7) plaque forming units of AdGFPCald was increased by 35+/-18%, p0.2, n=10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66 hr) and monkey (2-3 days) data, outflow facility was increased by 38+/-13%, p0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66+/-18%, p0.01, n=10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell-matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma.

Cell adhesion molecules of the cadherin family contribute to the regulation of cell shape and fate by mediating strong intercellular adhesion through Ca2+-dependent interaction of their ectodomain and association of their cytoplasmic tail to actin. However, the mechanisms co-ordinating cadherinmediated adhesion with the reorganization of the actin cytoskeleton remain elusive. Here, the formation of de novo contacts was dissected by spreading cells on a highly active N-cadherin homophilic ligand. Cells responded to N-cadherin activation by extending lamellipodium and organizing cadherin-catenin complexes and actin filaments in cadherin adhesions. Lamellipodium protrusion, associated with actin polymerization at the leading edge sustained the extension of cadherin contacts through a phosphoinositide 3-kinase (PI 3-kinase)-Rac1 pathway. Cadherin adhesions were formed by PI 3-kinase-independent, Rac1-dependent co-recruitment of adhesion complexes and actin filaments. The expression and localization of p120 at the plasma membrane, associated with an increase in membrane-associated Rac1 was required for both cell responses, consistent with a major role of p120 in signalling pathways initiated by cadherin activation and contributing to Rac1-dependent contact extension and maturation. These results provide additional information on the mechanisms by which cadherin coordinates adhesion with dynamic changes in the cytoskeleton to control cell shape and intercellular junction organization.

We have characterized a novel process that macrophages use to degrade species such as aggregated lipoproteins (agLDL) that are too large to be internalized via conventional phagocytosis. Macrophage...

Centripetal motion of surface-adherent particles is a classic experimental system for studying surface dynamics on a eukaryotic cell. To investigate bead migration over the entire cell surface, we have developed an experimental assay using multinuclear giant fibroblasts, which provide expanded length scales and an unambiguous frame of reference. Beads coated by adhesion ligands concanavalin A or fibronectin are placed in specific locations on the cell using optical tweezers, and their subsequent motion is tracked over time. The adhesion, as well as velocity and directionality of their movement, expose distinct regions of the cytoplasm and membrane. Beads placed on the peripheral lamella initiate centripetal motion, whereas beads placed on the central part of the cell attach to a stationary cortex and do not move. Careful examination by complementary three-dimensional methods shows that the motion of a bead placed on the cell periphery takes place after engulfment into the cytoplasm, whereas stationary beads, placed near the cell center, are not engulfed. These results demonstrate that centripetal motion of adhering particles may occur inside as well as outside the cell. Inhibition of actomyosin activity is used to explore requirements for engulfment and aspects of the bead movement. Centripetal movement of adherent particles seems to depend on mechanisms distinct from those driving overall cell contractility.

The Spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of Spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). As previously described, we show that α-Spectrin and β-Spectrin are essential to maintain a mono-layered FE, but, contrary to previous work, Spectrins are not required to control proliferation. Furthermore, spectrin cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-spectrin and integrin cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-myosin-II pathway enhances and decreases multi-layering of α-spectrin cells, respectively. Similarly, higher myosin-II activity enhances the integrin multi-layering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. All together, we suggest that a functional Spectrin-Integrin complex is essential to balance adequate forces, in order to maintain a mono-layered epithelium., This work was supported by the Singapore Ministry of Education [Master Scholarship EDUN N23-03-044 to B.F.N.]; the Biotechnology and Biological Sciences Research Council (BBSRC) [RG42522 to I.A.G.; BB/L001748/1 to G.K.S. and I.M.P.]; the Wellcome Trust [087899/Z/08/Z to I.A.G. and I.M.P.]; the Isaac Newton Trust (Cambridge, UK) [11.35(af ) to I.A.-G.]; the FEDER programme [BFU2013-48988-C2-1-P to C.S.-C.M. and M.D.M.-B.]; Junta de Andalucía [Proyecto de Excelencia P09-CVI-5058 to C.S.-C.M. and M.D.M.-B.]; a Superior Council for Scientific Research (CSIC) JAE-DOC (to I.G.); and the Department of Zoology (Cambridge) and the University of Cambridge (I.M.P.).

A critical event in atherogenesis is the interaction of macrophages with subendothelial lipoproteins. Although most studies model this interaction by incubating macrophages with monomeric lipoproteins, macrophages in vivo encounter lipoproteins that are aggregated. The physical features of the lipoproteins require distinctive mechanisms for their uptake. We show that macrophages create an extracellular, acidic, hydrolytic compartment to carry out digestion of aggregated low-density lipoproteins. We demonstrate delivery of lysosomal contents to these specialized compartments and their acidification by vacuolar ATPase, enabling aggregate catabolism by lysosomal acid hydrolases. We observe transient sealing of portions of the compartments, allowing formation of an “extracellular” proton gradient. An increase in free cholesterol is observed in aggregates contained in these compartments. Thus, cholesteryl ester hydrolysis can occur extracellularly in a specialized compartment, a lysosomal synapse, during the interaction of macrophages with aggregated low-density lipoprotein. A detailed understanding of these processes is essential for developing strategies to prevent atherosclerosis.

Objective— Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. Methods and Results— AgLDL resides for prolonged periods in surface-connected compartments. Although agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells because of hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. Conclusions— We describe a novel process that occurs during agLDL–macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathological positive feedback loop for increased catabolism of agLDL and eventual foam cell formation.

Objective— Atherogenesis begins as small subendothelial accumulations of foam cells that develop through unregulated uptake of modified and aggregated low-density lipoprotein (LDL). The reason why foam cells remain in the atherosclerotic plaque rather than migrating out of the area is unclear. We tested the hypothesis that elevated membrane cholesterol levels, which may result from interactions with aggregated LDL, affect macrophage migration. Methods and Results— Cholesterol loading by incubation with cholesterol-chelated methyl-β-cyclodextrin decreased migration of J774A.1 macrophages toward complement 5a (C5a) in transwell migration assays, even though cholesterol-loaded macrophages responded to a bath application of C5a. In a micropipette polarization assay, cholesterol-loaded cells polarized toward a C5a gradient. In a transwell migration assay, cholesterol-loaded cells extended lamellae through the filter pores but were unable to translocate their cell bodies. Cholesterol loading decreased both the cellular levels of GTP-bound active RhoA and the phosphorylation of myosin light chain. Expression of constitutively active RhoA largely prevented the inhibition of cell migration by cholesterol loading. Conclusions— These results suggest that increases in plasma membrane cholesterol content alter RhoA activation, resulting in inhibition of cell migration. These findings provide one possible explanation for the retention of foam cells in atherosclerotic lesions.

Objective—During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions.Methods and Results—We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-β-cyclodextrin (chol-MβCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised.Conclusions—These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.

The molecular basis for contact inhibition of cell locomotion is still largely unknown. Cadherins, the major receptors mediating cell-cell adhesion, associate in the cytoplasm with armadillo family proteins, including beta- and gamma-catenin and p120 catenin (p120ctn). E-cadherin-mediated contact formation was shown to inhibit cellular motility. We examine whether p120ctn may have a role in this regulation. We show here that overexpression of p120ctn in fibroblasts and epithelial cells induces pronounced changes in cell shape, motility and adhesion to the extracellular matrix. p120ctn-transfected cells display increased filopodial/lamellipodial activity, decreased contractility and focal adhesion formation, and augmented migratory ability. These effects of p120ctn are mediated by small GTPases of the Rho family. Direct assessment of the activity of these GTPases in cells expressing a 5-fold higher level of p120ctn as compared to non-transfected control cells revealed significant augmentation of Cdc42 and Rac activity. Moreover, co-transfection of p120ctn with dominant-negative Cdc42 and Rac, or constitutively active Rho suppressed morphological effects of p120ctn. Confocal immunofluorescence visualization of the distribution of endogenous p120ctn in dense cultures showed that formation of cadherin-mediated cell-cell contacts is accompanied by sequestering of p120ctn to the junction regions. In sparse cultures p120ctn is distributed over the cytoplasm. Co-transfection with an excess of E-cadherin leads to sequestration of exogenous p120ctn to cell-cell junctions or to small cadherin-containing vesicles, and abolishes p120ctn effects on cell morphology. Thus, p120ctn may couple the formation and disruption of cadherin-mediated contacts with regulation of cell motility by triggering pathway(s) affecting Rho family GTPases.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Atherosclerosis is the underlying cause of the majority of heart attacks and strokes. Lipoprotein accumulation and degradation by monocyte-derived macrophages is a central event in the pathogenesis of atherosclerosis. Data show that the vast majority of low density lipoprotein (LDL) in atherosclerotic plaques is aggregated and avidly bound to the subendothelial matrix.We employ various microscopy and biochemical techniques in both cell culture and animal models to investigate the mechanism of uptake of aggregated LDL (agLDL) by macrophages. We show that macrophages create an extracellular, acidic compartment where the cells contact the agLDL, and lysosomal contents are delivered to these compartments, thereby forming an extracellular hydrolytic compartment - a lysosomal synapse. Active acidification of these specialized compartments and aggregate catabolism were observed by fluorescence ratiometric time-lapse imaging of an in vitro cell culture model. Biochemical assays, employing radiolabeled agLDL demonstrated an increase in free cholesterol in aggregates contained in the lysosomal synapse. This cholesterol can be delivered to the cell, initiating the process of macrophage cholesterol loading and ultimately causing progression of the atherosclerotic plaque.Although in vitro systems provide insight into macrophage uptake of lipoproteins, ultimately we must strive to understand the function and behavior of macrophages in intact animals. Relatively little is known of the fate of monocytes once they migrate into atherosclerotic lesions. To this end, we employ intravital multiphoton microscopy in mouse models of atherosclerosis to allow observation of macrophages within the artery wall.Our studies elucidate the mechanism of a novel pathway for catabolism of agLDL by macrophages. Better understanding of the mechanisms by which macrophages interact with lipoproteins in the subendothelium may lead to new approaches to inhibit lipid accumulation in macrophages and thus, may be of therapeutic value in preventing atherosclerosis.