Effective guidance of collective migration based on differences in cell states.

Directed cell migration is important for normal animal development and physiology. The process can also be subverted by tumor cells to invade other tissues and to metastasize. Some cells, such as leukocytes, migrate individually; other cells migrate together in groups or sheets, called collective cell migration. Guidance of individually migrating cells depends critically on subcellularly localized perception and transduction of signals. For collective cell migration, guidance could result from cells within a group achieving different signaling levels, with directionality then encoded in the collective rather than in individual cells. Here we subject this collective guidance hypothesis to direct tests, using migration of border cells during Drosophila oogenesis as our model system. These cells normally use two receptor tyrosine kinases (RTKs), PDGF/VEGF-related receptor (PVR) and EGFR, to read guidance cues secreted by the oocyte. Elevated but delocalized RTK signaling in one cell of the cluster was achieved by overexpression of PVR in the absence of ligand or by overexpression of fusion receptors unable to detect Drosophila ligands; alternatively, Rac was photoactivated centrally within a single cell. In each case, one cell within the group was in a high signal state, whereas others were in low signal states. The high signal cell directed cluster movement effectively. We conclude that differences in cell signaling states are sufficient to direct collective migration and are likely a substantial contributor to normal guidance. Cell signaling states could manifest as differences in gene expression or metabolite levels and thus differ substantially from factors normally considered when analyzing eukaryotic cell guidance.

Two distinct modes of guidance signalling during collective migration of border cells.

Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. Here we use Drosophila border cells to examine how collective guidance is performed. We report that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cgamma are used redundantly, and we find that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group.

Actinomyces meyeri causing disseminated actinomycosis in the presence of concurrent bronchogenic carcinoma.

We describe the presentation of a 72-year-old woman with concurrent diagnoses of lung adenocarcinoma in conjunction with disseminated Actinomyces meyeri infection; a rare pathogen which can mimic lung cancer both symptomatically and radiologically. The patient was found to have a pelvic mass initially presumed to be cervical metastases-later confirmed to be of xanthogranulomatous inflammatory origin following transvaginal ultrasound-guided biopsy. The pathogenic cause, identified following pleural aspirate, being a fully sensitive A. meyeri infection; treated with prolonged course amoxicillin.

Pygopus and Legless target Armadillo/beta-catenin to the nucleus to enable its transcriptional co-activator function.

Wnt signalling controls the transcription of genes that function during normal and malignant development. Stimulation by canonical Wnt ligands activates beta-catenin (or Drosophila melanogaster Armadillo) by blocking its phosphorylation, resulting in its stabilization and translocation to the nucleus. Here, Armadillo/beta-catenin binds to TCF/LEF transcription factors and recruits chromatin-modifying and -remodelling complexes to transcribe Wnt target genes. The transcriptional activity of Armadillo/beta-catenin depends on two conserved nuclear proteins recently discovered in Drosophila, Pygopus (Pygo) and Legless/BCL-9 (Lgs). Lgs functions as an adaptor between Pygo and Armadillo/beta-catenin, but how Armadillo/beta-catenin is controlled by Pygo and Lgs is not known. Here, we show that the nuclear localization of Lgs entirely depends on Pygo, which itself is constitutively localized to the nucleus; thus, Pygo functions as a nuclear anchor. Pygo is also required for high nuclear Armadillo levels during Wingless signalling, and together with Lgs increases the transcriptional activity of beta-catenin in APC mutant cancer cells. Notably, linking Armadillo to a nuclear localization sequence rescues pygo and lgs mutant fly embryos. This indicates that Pygo and Lgs function in targeting Armadillo/beta-catenin to the nucleus, thus ensuring its availability to TCF during Wnt signalling.

Birth and life of tissue macrophages and their migration in embryogenesis and inflammation in medaka.

Macrophages detecting and migrating toward sites of injury and infection represent one of the first steps in an immune response. Here we directly image macrophage birth and migration in vivo in transgenic medaka fish. Macrophages are born as frequently dividing, immotile cells with spherical morphology that differentiate into flat, highly motile cells. They retain mitotic activity while spreading over the entire body. Cells follow restricted paths not only in directed migration, but also during patrolling. Along those paths the macrophages rapidly patrol the tissue and respond to wounding and bacterial infection from long distances. Upon injury they increase their speed and migratory persistence. Specifically targeting PI3-kinase isoforms efficiently blocks the wounding response and results in a distinct inhibition of cell motility and chemotaxis. Our study provides in situ insights into the properties of immature and migratory macrophages and presents a unique model to further test modulating compounds in vivo.

A role of Dishevelled in relocating Axin to the plasma membrane during wingless signaling.

Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is beta-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, beta-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble beta-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3 beta [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of beta-catenin. In Wnt signaling, this complex is inhibited; as a consequence, beta-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. Here, we show that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling.

Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.

Understanding the mechanisms of collective cell migration is crucial for cancer metastasis, wound healing and many developmental processes. Imaging a migrating cluster in vivo is feasible, but the quantification of individual cell behaviours remains challenging. We have developed an image analysis toolkit, CCMToolKit, to quantify the Drosophila border cell system. In addition to chaotic motion, previous studies reported that the migrating cells are able to migrate in a highly coordinated pattern. We quantify the rotating and running migration modes in 3D while also observing a range of intermediate behaviours. Running mode is driven by cluster external protrusions. Rotating mode is associated with cluster internal cell extensions that could not be easily characterized. Although the cluster moves slower while rotating, individual cells retain their mobility and are in fact slightly more active than in running mode. We also show that individual cells may exchange positions during migration.

Intracellular shuttling of a Drosophila APC tumour suppressor homolog.

BACKGROUND: The Adenomatous polyposis coli (APC) tumour suppressor is found in multiple discrete subcellular locations, which may reflect sites of distinct functions. In Drosophila epithelial cells, the predominant APC relative (E-APC) is concentrated at the apicolateral adherens junctions. Genetic analysis indicates that this junctional association is critical for the function of E-APC in Wnt signalling and in cellular adhesion. Here, we ask whether the junctional association of E-APC is stable, or whether E-APC shuttles between the plasma membrane and the cytoplasm. RESULTS: We generated a Drosophila strain that expresses E-APC (dAPC2) tagged with green fluorescent protein (GFP-E-APC) and we analysed its junctional association with fluorescence recovery after photobleaching (FRAP) experiments in live embryos. This revealed that the junctional association of GFP-E-APC in epithelial cells is highly dynamic, and is far less stable than that of the structural components of the adherens junctions, E-cadherin, alpha-catenin and Armadillo. The shuttling of GFP-E-APC to and from the plasma membrane is unaltered in mutants of Drosophila glycogen synthase kinase 3 (GSK3), which mimic constitutive Wingless signalling. However, the stability of E-APC is greatly reduced in these mutants, explaining their apparent delocalisation from the plasma membrane as previously observed. Finally, we show that GFP-E-APC forms dynamic patches at the apical plasma membrane of late embryonic epidermal cells that form denticles, and that it shuttles up and down the axons of the optic lobe. CONCLUSIONS: We conclude that E-APC is a highly mobile protein that shuttles constitutively between distinct subcellular locations.

Early detection of antiangiogenic treatment responses in a mouse xenograft tumor model using quantitative perfusion MRI.

Angiogenesis plays a major role in tumor growth and metastasis, with tumor perfusion regarded as a marker for angiogenesis. To evaluate antiangiogenic treatment response in vivo, we investigated arterial spin labeling (ASL) magnetic resonance imaging (MRI) to measure tumor perfusion quantitatively. Chronic and 24-h acute treatment responses to bevacizumab were assessed by ASL and dynamic-contrast-enhanced (DCE) MRI in the A498 xenograft mouse model. After the MRI, tumor vasculature was assessed by CD34 staining. After 39 days of chronic treatment, tumor perfusion decreased to 44.8 ± 16.1 mL/100 g/min (P < 0.05), compared to 92.6 ± 42.9 mL/100 g/min in the control group. In the acute treatment study, tumor perfusion in the treated group decreased from 107.2 ± 32.7 to 73.7 ± 27.8 mL/100 g/min (P < 0.01; two-way analysis of variance), as well as compared with control group post dosing. A significant reduction in vessel density and vessel size was observed after the chronic treatment, while only vessel size was reduced 24 h after acute treatment. The tumor perfusion correlated with vessel size (r = 0.66; P < 0.005) after chronic, but not after acute treatment. The results from DCE-MRI also detected a significant change between treated and control groups in both chronic and acute treatment studies, but not between 0 and 24 h in the acute treatment group. These results indicate that tumor perfusion measured by MRI can detect early vascular responses to antiangiogenic treatment. With its noninvasive and quantitative nature, ASL MRI would be valuable for longitudinal assessment of tumor perfusion and in translation from animal models to human.

Microtubules and Lis-1/NudE/dynein regulate invasive cell-on-cell migration in Drosophila.

The environment through which cells migrate in vivo differs considerably from the in vitro environment where cell migration is often studied. In vivo many cells migrate in crowded and complex 3-dimensional tissues and may use other cells as the substratum on which they move. This includes neurons, glia and their progenitors in the brain. Here we use a Drosophila model of invasive, collective migration in a cellular environment to investigate the roles of microtubules and microtubule regulators in this type of cell movement. Border cells are of epithelial origin and have no visible microtubule organizing center (MTOC). Interestingly, microtubule plus-end growth was biased away from the leading edge. General perturbation of the microtubule cytoskeleton and analysis by live imaging showed that microtubules in both the migrating cells and the substrate cells affect movement. Also, whole-tissue and cell autonomous deletion of the microtubule regulator Stathmin had distinct effects. A screen of 67 genes encoding microtubule interacting proteins uncovered cell autonomous requirements for Lis-1, NudE and Dynein in border cell migration. Net cluster migration was decreased, with initiation of migration and formation of dominant front cell protrusion being most dramatically affected. Organization of cells within the cluster and localization of cell-cell adhesion molecules were also abnormal. Given the established role of Lis-1 in migrating neurons, this could indicate a general role of Lis-1/NudE, Dynein and microtubules, in cell-on-cell migration. Spatial regulation of cell-cell adhesion may be a common theme, consistent with observing both cell autonomous and non-autonomous requirements in both systems.

Cell behaviors regulated by guidance cues in collective migration of border cells.

Border cells perform a collective, invasive, and directed migration during Drosophila melanogaster oogenesis. Two receptor tyrosine kinases (RTKs), the platelet-derived growth factor/vascular endothelial growth factor-related receptor (PVR) and the epidermal growth factor receptor (EGFR), are important for reading guidance cues, but how these cues steer migration is not well understood. During collective migration, front, back, and side extensions dynamically project from individual cells within the group. We find that guidance input from both RTKs affects the presence and size of these extensions, primarily by favoring the persistence of front extensions. Guidance cues also control the productivity of extensions, specifically rendering back extensions nonproductive. Early and late phases of border cell migration differ in efficiency of forward cluster movement, although motility of individual cells appears constant. This is caused by differences in behavioral effects of the RTKs: PVR dominantly induces large persistent front extensions and efficient streamlined group movement, whereas EGFR does not. Thus, guidance receptors steer movement of this cell group by differentially affecting multiple migration-related features.

Recombinase-mediated cassette exchange provides a versatile platform for gene targeting: knockout of miR-31b.

A series of vectors has been designed to enhance the versatility of targeted homologous recombination. Recombinase-mediated cassette exchange permits sequential targeting at any locus and improves flexibility in making user-defined mutations. Application of RMCE to delete an intronic microRNA gene is described.

Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription.

The adenomatous polyposis coli (APC) protein is inactivated in most colorectal tumours. APC loss is an early event in tumorigenesis, and causes an increase of nuclear beta-catenin and its transcriptional activity. This is thought to be the driving force for tumour progression. APC shuttles in and out of the nucleus, but the functional significance of this has been controversial. Here, we show that APC truncations are nuclear in colorectal cancer cells and adenocarcinomas, and this correlates with loss of centrally located nuclear export signals. These signals confer efficient nuclear export as measured directly by fluorescence loss in photobleaching (FLIP), and they are critical for the function of APC in reducing the transcriptional activity of beta-catenin in complementation assays of APC mutant colorectal cancer cells. Importantly, targeting a functional APC construct to the nucleus causes a striking nuclear accumulation of beta-catenin without changing its transcriptional activity. Our evidence indicates that the rate of nuclear export of APC, rather than its nuclear import or steady-state levels, determines the transcriptional activity of beta-catenin.