Activated Src kinase promotes cell cannibalism in Drosophila.

Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.

Septins regulate border cell surface geometry, shape, and motility downstream of Rho in Drosophila.

Septins self-assemble into polymers that bind and deform membranes in vitro and regulate diverse cell behaviors in vivo. How their in vitro properties relate to their in vivo functions is under active investigation. Here, we uncover requirements for septins in detachment and motility of border cell clusters in the Drosophila ovary. Septins and myosin colocalize dynamically at the cluster periphery and share phenotypes but, surprisingly, do not impact each other. Instead, Rho independently regulates myosin activity and septin localization. Active Rho recruits septins to membranes, whereas inactive Rho sequesters septins in the cytoplasm. Mathematical analyses identify how manipulating septin expression levels alters cluster surface texture and shape. This study shows that the level of septin expression differentially regulates surface properties at different scales. This work suggests that downstream of Rho, septins tune surface deformability while myosin controls contractility, the combination of which governs cluster shape and movement.

Who's really in charge: Diverse follower cell behaviors in collective cell migration.

Collective cell migrations drive morphogenesis, wound healing, and cancer dissemination. Cells located at the front are considered leaders while those behind them are defined topologically as followers. Leader cell behaviors, including chemotaxis and their coupling to followers, have been well-studied and reviewed. However, the contributions of follower cells to collective cell migration represent an emerging area of interest. In this perspective, we highlight recent research into the broadening array of follower cell behaviors found in moving collectives. We describe examples of follower cells that possess cryptic leadership potential and followers that lack that potential but contribute in diverse and sometimes surprising ways to collective movement, even steering from behind. We highlight collectives in which all cells both lead and follow, and a few passive passengers. The molecular mechanisms controlling follower cell function and behavior are just emerging and represent an exciting frontier in collective cell migration research.

A Scribble/Cdep/Rac pathway controls follower-cell crawling and cluster cohesion during collective border-cell migration.

Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration in vivo.

A picket fence function for adherens junctions in epithelial cell polarity.

Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. Here we show that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib - similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a 'picket fence' diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.

Tissue topography steers migrating Drosophila border cells.

Moving cells can sense and respond to physical features of the microenvironment; however, in vivo, the significance of tissue topography is mostly unknown. Here, we used Drosophila border cells, an established model for in vivo cell migration, to study how chemical and physical information influences path selection. Although chemical cues were thought to be sufficient, live imaging, genetics, modeling, and simulations show that microtopography is also important. Chemoattractants promote predominantly posterior movement, whereas tissue architecture presents orthogonal information, a path of least resistance concentrated near the center of the egg chamber. E-cadherin supplies a permissive haptotactic cue. Our results provide insight into how cells integrate and prioritize topographical, adhesive, and chemoattractant cues to choose one path among many.

Cell interactions in collective cell migration.

Collective cell migration is the coordinated movement of a physically connected group of cells and is a prominent driver of development and metastasis. Interactions between cells within migrating collectives, and between migrating cells and other cells in the environment, play key roles in stimulating motility, steering and sometimes promoting cell survival. Similarly, diverse heterotypic interactions and collective behaviors likely contribute to tumor metastasis. Here, we describe a sampling of cells that migrate collectively in vivo, including well-established and newer examples. We focus on the under-appreciated property that many - perhaps most - collectively migrating cells move as cooperating groups of distinct cell types.

Coordination of protrusion dynamics within and between collectively migrating border cells by myosin II.

Collective cell migration is emerging as a major driver of embryonic development, organogenesis, tissue homeostasis, and tumor dissemination. In contrast to individually migrating cells, collectively migrating cells maintain cell-cell adhesions and coordinate direction-sensing as they move. While nonmuscle myosin II has been studied extensively in the context of cells migrating individually in vitro, its roles in cells migrating collectively in three-dimensional, native environments are not fully understood. Here we use genetics, Airyscan microscopy, live imaging, optogenetics, and Förster resonance energy transfer to probe the localization, dynamics, and functions of myosin II in migrating border cells of the Drosophila ovary. We find that myosin accumulates transiently at the base of protrusions, where it functions to retract them. E-cadherin and myosin colocalize at border cell-border cell contacts and cooperate to transmit directional information. A phosphomimetic form of myosin is sufficient to convert border cells to a round morphology and blebbing migration mode. Together these studies demonstrate that distinct and dynamic pools of myosin II regulate protrusion dynamics within and between collectively migrating cells and suggest a new model for the role of protrusions in collective direction sensing in vivo.

Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs).

Small micromeres of the sea urchin are believed to be primordial germ cells (PGCs), fated to give rise to sperm or eggs in the adult. Sea urchin PGCs are formed at the fifth cleavage, undergo one additional division during blastulation, and migrate to the coelomic pouches of the pluteus larva. The goal of this chapter is to detail classical and modern techniques used to analyze primordial germ cell specification, gene expression programs, and cell behaviors in fixed and live embryos. The transparency of the sea urchin embryo enables both live imaging techniques and in situ RNA hybridization and immunolabeling for a detailed molecular characterization of these cells. Four approaches are presented to highlight small micromeres with fluorescent molecules for analysis by live and fixed cell microscopy: (1) small molecule dye accumulation during cleavage and blastula stages, (2) primordial germ cell targeted RNA expression using the Nanos untranslated regions, (3) fusing genes of interest with a Nanos2 targeting peptide, and (4) EdU and BrdU labeling. Applications of the live labeling techniques are discussed, including sorting by fluorescence-activated cell sorting for transcriptomic analysis, and, methods to image small micromere behavior in whole and dissociated embryos by live confocal microscopy. Finally, summary table of antibody and RNA probes as well as small molecule dyes to label small micromeres at a variety of developmental stages is provided.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

Development and dynamics of cell polarity at a glance.

Cells exhibit morphological and molecular asymmetries that are broadly categorized as cell polarity. The cell polarity established in early embryos prefigures the macroscopic anatomical asymmetries characteristic of adult animals. For example, eggs and early embryos have polarized distributions of RNAs and proteins that generate global anterior/posterior and dorsal/ventral axes. The molecular programs that polarize embryos are subsequently reused in multiple contexts. Epithelial cells require apical/basal polarity to establish their barrier function. Migrating cells polarize in the direction of movement, creating distinct leading and trailing structures. Asymmetrically dividing stem cells partition different molecules between themselves and their daughter cells. Cell polarity can develop de novo, be maintained through rounds of cell division and be dynamically remodeled. In this Cell Science at a Glance review and poster, we describe molecular asymmetries that underlie cell polarity in several cellular contexts. We highlight multiple developmental systems that first establish cell/developmental polarity, and then maintain it. Our poster showcases repeated use of the Par, Scribble and Crumbs polarity complexes, which drive the development of cell polarity in many cell types and organisms. We then briefly discuss the diverse and dynamic changes in cell polarity that occur during cell migration, asymmetric cell division and in planar polarized tissues.

Functional diversification of sea urchin ABCC1 (MRP1) by alternative splicing.

The multidrug resistance protein (MRP) family encodes a diverse repertoire of ATP-binding cassette (ABC) transporters with multiple roles in development, disease, and homeostasis. Understanding MRP evolution is central to unraveling their roles in these diverse processes. Sea urchins occupy an important phylogenetic position for understanding the evolution of vertebrate proteins and have been an important invertebrate model system for study of ABC transporters. We used phylogenetic analyses to examine the evolution of MRP transporters and functional approaches to identify functional forms of sea urchin MRP1 (also known as SpABCC1). SpABCC1, the only MRP homolog in sea urchins, is co-orthologous to human MRP1, MRP3, and MRP6 (ABCC1, ABCC3, and ABCC6) transporters. However, efflux assays revealed that alternative splicing of exon 22, a region critical for substrate interactions, could diversify functions of sea urchin MRP1. Phylogenetic comparisons also indicate that while MRP1, MRP3, and MRP6 transporters potentially arose from a single transporter in basal deuterostomes, alternative splicing appears to have been the major mode of functional diversification in invertebrates, while duplication may have served a more important role in vertebrates. These results provide a deeper understanding of the evolutionary origins of MRP transporters and the potential mechanisms used to diversify their functions in different groups of animals.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

Transport in technicolor: mapping ATP-binding cassette transporters in sea urchin embryos.

One quarter of eukaryotic genes encode membrane proteins. These include nearly 1,000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multidrug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease.

Migration of sea urchin primordial germ cells.

BACKGROUND: Small micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live-cell imaging of small micromere plasma membranes. RESULTS: Early in gastrulation, small micromeres transition from non-motile epithelial cells, to motile quasi-mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43-49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia. CONCLUSIONS: Previous work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left-right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left-right segregation.

Localization and substrate selectivity of sea urchin multidrug (MDR) efflux transporters.

In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.

Programmed reduction of ABC transporter activity in sea urchin germline progenitors.

ATP-binding cassette (ABC) transporters protect embryos and stem cells from mutagens and pump morphogens that control cell fate and migration. In this study, we measured dynamics of ABC transporter activity during formation of sea urchin embryonic cells necessary for the production of gametes, termed the small micromeres. Unexpectedly, we found small micromeres accumulate 2.32 times more of the ABC transporter substrates calcein-AM, CellTrace RedOrange, BoDipy-verapamil and BoDipy-vinblastine, than any other cell in the embryo, indicating a reduction in multidrug efflux activity. The reduction in small micromere ABC transporter activity is mediated by a pulse of endocytosis occurring 20-60 minutes after the appearance of the micromeres--the precursors of the small micromeres. Treating embryos with phenylarsine oxide, an inhibitor of endocytosis, prevents the reduction of transporter activity. Tetramethylrhodamine dextran and cholera toxin B uptake experiments indicate that micromeres have higher rates of bulk and raft-associated membrane endocytosis during the window of transporter downregulation. We hypothesized that this loss of efflux transport could be required for the detection of developmental signaling molecules such as germ cell chemoattractants. Consistent with this hypothesis, we found that the inhibition of ABCB and ABCC-types of efflux transporters disrupts the ordered distribution of small micromeres to the left and right coelomic pouches. These results point to tradeoffs between signaling and the protective functions of the transporters.