A cell migration device that maintains a defined surface with no cellular damage during wound edge generation

Studying the rate of cell migration provides insight into fundamental cell biology as well as a tool to assess the functionality of synthetic surfaces and soluble environments used in tissue engineering. The traditional tools used to study cell migration include the fence and wound healing assays. In this paper we describe the development of a microchannel based device for the study of cell migration on defined surfaces. We demonstrate that this device provides a superior tool, relative to the previously mentioned assays, for assessing the propagation rate of cell wave fronts. The significant advantage provided by this technology is the ability to maintain a virgin surface prior to the commencement of the cell migration assay. Here, the device is used to assess rates of mouse fibroblasts (NIH 3T3) and human osteosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. The device design enables rapid and simplistic assessment of multiple repeats on a single chip, where surfaces have not been previously exposed to cells or cellular secretions.

Neuronal nicotinic acetylcholine receptors as pharmacotherapeutic targets for the treatment of alcohol use disorders

Alcohol use disorders (AUDs) are complex and developing effective treatments will require the combination of novel medications and cognitive behavioral therapy approaches. Epidemiological studies have shown there is a high correlation between alcohol consumption and tobacco use, and the prevalence of smoking in alcoholics is as high as 80% compared to about 30% for the general population. Both preclinical and clinical data provide evidence that nicotine administration increases alcohol intake and nonspecific nicotinic receptor antagonists reduce alcohol-mediated behaviors. As nicotine interacts specifically with the neuronal nicotinic acetylcholine receptor (nAChR) system, this suggests that nAChRs play an important role in the behavioral effects of alcohol. In this review, we discuss the importance of nAChRs for the treatment of AUDs and argue that the use of FDA approved nAChR ligands, such as varenicline and mecamylamine, approved as smoking cessation aids may prove to be valuable treatments for AUDs. We also address the importance of combining effective medications with behavioral therapy for the treatment of alcohol dependent individuals.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Partial agonists of the [alpha]3[beta]4[ast] neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2(*) nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

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.

Neuronal nicotinic acetylcholine receptors : neuroplastic changes underlying alcohol and nicotine addictions

Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug's reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine's effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies.