VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3- positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.

The role of the recycling endosome in regulating lamellipodia formation and macrophage migration

Cell migration is a highly complex process that requires the extension of cell membrane in the direction of travel. This membrane is continuously remodeled to expand the leading edge and alter its membrane properties. For a long time it has been known that there is a continual flow of polarized membrane traffic towards the leading edge during migration and that this trafficking is essential for cell migration. However, there is little information on how the cell coordinates exocytosis at the leading edge. It is also unclear whether these internal membranes are incorporated into the leading edge or are just delivering the necessary proteins for migration to occur. We have shown that recycling endosome membrane is incorporated into the plasma membrane at the leading edge to expand the membrane and at the same time delivers receptors to the leading edge to mediate migration. In order for this to happen the surface Q-SNARE complex Stx4/SNAP23 translocates to the leading edge where it binds to the R-SNARE VAMP3 on the recycling endosome allowing incorporation into the plasma membrane. Loss of any one of the components of this complex reduces efficient lamellipodia formation and restrains cell migration.

Recycling endosome membrane incorporation into the leading edge regulates lamellipodia formation and macrophage migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

Mean-field descriptions of collective migration with strong adhesion

Random walk models based on an exclusion process with contact effects are often used to represent collective migration where individual agents are affected by agent-to-agent adhesion. Traditional mean field representations of these processes take the form of a nonlinear diffusion equation which, for strong adhesion, does not predict the averaged discrete behavior. We propose an alternative suite of mean-field representations, showing that collective migration with strong adhesion can be accurately represented using a moment closure approach.

Mathematics funds of knowledge : sotmaute and sermaute fish in a Torres Strait Islander community

The purpose of this article is to describe a project with one Torres Strait Islander Community. It provides some insights into parents’ funds of knowledge that are mathematical in nature, such as sorting shells and giving fish. The idea of funds of knowledge is based on the premise that people are competent and have knowledge that has been historically and culturally accumulated into a body of knowledge and skills essential for their functioning and well-being. This knowledge is then practised throughout their lives and passed onto the next generation of children. Through adopting a community research approach, funds of knowledge that can be used to validate the community’s identities as knowledgeable people, can also be used as foundations for future learnings for teachers, parents and children in the early years of school. They can be the bridge that joins a community’s funds of knowledge with schools validating that knowledge.

Statistical phylogeographic tests of competing 'Lake Carpentaria hypotheses' in the mouth-brooding freshwater fish, Glossamia aprion (Apogonidae)

Glacial cycles during the Pleistocene reduced sea levels and created new land connections in northern Australia, where many currently isolated rivers also became connected via an extensive paleo-lake system, 'Lake Carpentaria'. However, the most recent period during which populations of freshwater species were connected by gene flow across Lake Carpentaria is debated: various 'Lake Carpentaria hypotheses' have been proposed. Here, we used a statistical phylogeographic approach to assess the timing of past population connectivity across the Carpentaria region in the obligate freshwater fish, Glossamia aprion. Results for this species indicate that the most recent period of genetic exchange across the Carpentaria region coincided with the mid- to late Pleistocene, a result shown previously for other freshwater and diadromous species. Based on these findings and published studies for various freshwater, diadromous and marine species, we propose a set of 'Lake Carpentaria' hypotheses to explain past population connectivity in aquatic species: (1) strictly freshwater species had widespread gene flow in the mid- to late Pleistocene before the last glacial maximum; (2) marine species were subdivided into eastern and western populations by land during Pleistocene glacial phases; and (3) past connectivity in diadromous species reflects the relative strength of their marine affinity.

Insights into historical drainage evolution based on the phylogeography of the chevron snakehead fish (Channa striata) in the Mekong Basin

1. The phylogeography of freshwater taxa is often integrally linked with landscape changes such as drainage re-alignments that may present the only avenue for historical dispersal for these taxa. Classical models of gene flow do not account for landscape changes and so are of little use in predicting phylogeography in geologically young freshwater landscapes. When the history of drainage formation is unknown, phylogeographical predictions can be based on current freshwater landscape structure, proposed historical drainage geomorphology, or from phylogeographical patterns of co-distributed taxa. 2. This study describes the population structure of a sedentary freshwater fish, the chevron snakehead (Channa striata), across two river drainages on the Indochinese Peninsula. The phylogeographical pattern recovered for C. striata was tested against seven hypotheses based on contemporary landscape structure, proposed history and phylogeographical patterns of codistributed taxa. 3. Consistent with the species ecology, analysis of mitochondrial and microsatellite loci revealed very high differentiation among all sampled sites. A strong signature of historical population subdivision was also revealed within the contemporary Mekong River Basin (MRB). Of the seven phylogeographical hypotheses tested, patterns of co-distributed taxa proved to be the most adequate for describing the phylogeography of C. striata. 4. Results shed new light on SE Asian drainage evolution, indicating that the Middle MRB probably evolved via amalgamation of at least three historically independent drainage sections and in particular that the Mekong River section centred around the northern Khorat Plateau in NE Thailand was probably isolated from the greater Mekong for an extensive period of evolutionary time. In contrast, C. striata populations in the Lower MRB do not show a phylogeographical signature of evolution in historically isolated drainage lines, suggesting drainage amalgamation has been less important for river landscape formation in this region.

The effects of dietary fish oil on inflammation, fibrosis and oxidative stress associated with obstructive renal injury in rats.

Scope: We examined whether dietary supplementation with fish oil modulates inflammation, fibrosis and oxidative stress following obstructive renal injury. Methods and results: Three groups of Sprague-Dawley rats (n = 16 per group) were fed for 4 wk on normal rat chow (oleic acid), chow containing fish oil (33 g eicosapentaenoic acid and 26 g docosahexaenoic acid per kg diet), or chow containing safflower oil (60 g linoleic acid per kg diet). All diets contained 7% fat. After 4 wk, the rats were further subdivided into four smaller groups (n = 4 per group). Unilateral ureteral obstruction was induced in three groups (for 4, 7 and 14 days). The fourth group for each diet did not undergo surgery, and was sacrificed as controls at 14 days. When rats were sacrificed, plasma and portions of the kidneys were removed and frozen; other portions of kidney tissue were fixed and prepared for histology. Compared with normal chow and safflower oil, fish oil attenuated collagen deposition, macrophage infiltration, TGF-beta expression, apoptosis, and tissue levels of arachidonic acid, MIP-1 alpha, IL-1 beta, MCP-1 and leukotriene B(4). Compared with normal chow, fish oil increased the expression of HO-1 protein in kidney tissue. Conclusions: Fish oil intake reduced inflammation, fibrosis and oxidative stress following obstructive renal injury.

Stratum basale keratinocyte expression of the cell surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration

Background: Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell to cell adhesion is crucial to the initiation and regulation of these processes. CUB domain containing protein 1 (CDCP1) is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling and is expressed by keratinocytes in native human skin, as well as in primary cultures. Objectives: To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. Methods: We examined both human skin tissue and an in vitro three-dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function blocking anti-CDCP1 antibody and a real-time Transwell™ cell migration assay. Results: Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a 3-dimensional human skin equivalent model CDCP1 was expressed only in the stratum basale, with localization restricted to the cell-cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Further, an anti-CDCP1 function blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. Conclusions: These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.