Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser435 by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser897 by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma—whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis—indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo

Alcohol-induced retrograde facilitation renders witnesses of crime less suggestible to misinformation

RATIONALE: Research has shown that alcohol can have both detrimental and facilitating effects on memory: intoxication can lead to poor memory for information encoded after alcohol consumption (anterograde amnesia) and may improve memory for information encoded before consumption (retrograde facilitation). This study examined whether alcohol consumed after witnessing a crime can render individuals less vulnerable to misleading post-event information (misinformation). METHOD: Participants watched a simulated crime video. Thereafter, one third of participants expected and received alcohol (alcohol group), one third did not expect but received alcohol (reverse placebo), and one third did not expect nor receive alcohol (control). After alcohol consumption, participants were exposed to misinformation embedded in a written narrative about the crime. The following day, participants completed a cued-recall questionnaire about the event. RESULTS: Control participants were more likely to report misinformation compared to the alcohol and reverse placebo group. CONCLUSION: The findings suggest that we may oversimplify the effect alcohol has on suggestibility and that sometimes alcohol can have beneficial effects on eyewitness memory by protecting against misleading post-event information

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease

The Rho-Family GTPase Rac1 Regulates Integrin Localization in Drosophila Immunosurveillance Cells

BACKGROUND: When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response. RESULTS: In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization. SIGNIFICANCE: We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes

Endosomal integrin signals for survival.

The mechanisms underlying integrin-dependent signalling are a topic of continued study. Endocytosed integrins are now shown to drive assembly of signalling complexes on the cytoplasmic face of endocytic membranes to promote cancer cell survival and increase metastatic capacity following cell detachment

Interplay between cell adhesion and growth factor receptors: from the plasma membrane to the endosomes

The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling

Cells Assemble Invadopodia-Like Structures and Invade into Matrigel in a Matrix Metalloprotease Dependent Manner in the Circular Invasion Assay

The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment

Beta 1-integrin-c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/R.B.M. was a recipient of a UK Medical Research Council (MRC) studentship, MRC Centenary Award, Barts and The London Charity (472/1711) and Rosetrees Trust (M314), N.K. was a recipient of an MRC studentship (MR/J500409/1), C.J. was a recipient of the Barts and The London Charitable Foundation Scholarship (RAB 05/PJ/07), L.M. was supported by CR-UK, Breast Cancer Now (2008NovPR10) and Rosetrees Trust (M346), A.H. was a recipient of a CR-UK studentship (C236/A11795). P.J.P. was supported by CR-UK. J.I. was supported by grants from the Academy of Finland, ERC Starting grant, Finnish Cancer Organisations and Sigrid Juselius Foundation. S.K. was supported by the MRC (G0501003) and The British Lung Foundation (CAN09-4)

ARRDC3 suppresses breast cancer progression by negatively regulating integrin β4

Large-scale genetic analyses of human tumor samples have been used to identify novel oncogenes, tumor suppressors and prognostic factors, but the functions and molecular interactions of many individual genes have not been determined. In this study we examined the cellular effects and molecular mechanism of the arrestin family member, ARRDC3, a gene preferentially lost in a subset of breast cancers. Oncomine data revealed that the expression of ARRDC3 decreases with tumor grade, metastases and recurrences. ARRDC3 overexpression represses cancer cell proliferation, migration, invasion, growth in soft agar and in vivo tumorigenicity, whereas downregulation of ARRCD3 has the opposite effects. Mechanistic studies showed that ARRDC3 functions in a novel regulatory pathway that controls the cell surface adhesion molecule, β-4 integrin (ITGβ4), a protein associated with aggressive tumor behavior. Our data indicates ARRDC3 directly binds to a phosphorylated form of ITGβ4 leading to its internalization, ubiquitination and ultimate degradation. The results identify the ARRCD3-ITGβ4 pathway as a new therapeutic target in breast cancer and show the importance of connecting genetic arrays with mechanistic studies in the search for new treatments