Loss of strumpellin in the melanocytic lineage impairs the WASH Complex but does not affect coat colour

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin

Development of an inducible mouse model of iRFP713 to track recombinase activity and tumour development in vivo

While the use of bioluminescent proteins for molecular imaging is a powerful technology to further our understanding of complex processes, fluorescent labeling with visible light fluorescent proteins such as GFP and RFP suffers from poor tissue penetration and high background autofluorescence. To overcome these limitations, we generated an inducible knock-in mouse model of iRFP713. This model was used to assess Cre activity in a Rosa Cre-ER background and quantify Cre activity upon different tamoxifen treatments in several organs. We also show that iRFP can be readily detected in 3D organoid cultures, FACS analysis and in vivo tumour models. Taken together we demonstrate that iRFP713 is a progressive step in in vivo imaging and analysis that widens the optical imaging window to the near-infrared spectrum, thereby allowing deeper tissue penetration, quicker image acquisition without the need to inject substrates and a better signal to background ratio in genetically engineered mouse models (GEMMs)

(2,0) theory on circle fibrations

We consider (2,0) theory on a manifold M_6 that is a fibration of a spatial S^1 over some five-dimensional base manifold M_5. Initially, we study the free (2,0) tensor multiplet which can be described in terms of classical equations of motion in six dimensions. Given a metric on M_6 the low energy effective theory obtained through dimensional reduction on the circle is a Maxwell theory on M_5. The parameters describing the local geometry of the fibration are interpreted respectively as the metric on M_5, a non-dynamical U(1) gauge field and the coupling strength of the resulting low energy Maxwell theory. We derive the general form of the action of the Maxwell theory by integrating the reduced equations of motion, and consider the symmetries of this theory originating from the superconformal symmetry in six dimensions. Subsequently, we consider a non-abelian generalization of the Maxwell theory on M_5. Completing the theory with Yukawa and phi^4 terms, and suitably modifying the supersymmetry transformations, we obtain a supersymmetric Yang-Mills theory which includes terms related to the geometry of the fibration.Comment: 24 pages, v2 References added, typos correcte

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, levels of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts have been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis

T-duality Covariance of SuperD-branes

T-duality realized on SuperD-brane effective actions probing in constant $G_{mn}$ and $b_{mn}$ backgrounds is studied from a pure world volume point of view. It is proved that requiring {\em T-duality covariance} of such actions ``fixes'' the T-duality transformations of the world volume dynamical fields, and consequently, of the NS-NS and R-R coupling superfields. The analysis is extended to uncover the mapping of the symmetry structure associated with these SuperD-brane actions. In particular, we determine the T-duality transformation properties of kappa symmetry and supersymmetry, which allow us to prove that bosonic supersymmetric world volume solitons of the original theory generate, through T-duality, the expected ones in the T-dual theory. The latter proof is generalized to arbitrary bosonic backgrounds. We conclude with some comments on extensions of our approach to arbitrary kappa symmetric backgrounds, non-BPS D-branes and non-abelian SuperD-branes.Comment: 36 pages, no figures, LaTex file; vs2 minor changes, conclucions unchanged, two references added. Final version to appear in Nucl. Phys.

Design and Development of a Cell Marking System in Transgenic Mice

The aim of this project was to develop a marker system to analyse cell lineage in mouse skin. This should provide useful information on the usual size and organisation of clones in the epidermis and give some clues as to how cell replacement is normally controlled in the epidermis and to the possible location of the stem and progenitor cells. As there is no marker gene currently available in the murine epidermis a marker gene had to be designed and introduced. This was accomplished by generating transgenic mice with a novel marker gene inserted into their DNA. An Escherichia coli lac Z gene was used as a cell marker and activated in single cells following treating the mice with a chemical mutagen. The marker could be activated by a point mutation in the sequence of an oligonucleotide inserted at the 5' end of the lac Z gene, replacing the ATG initiation codon of the gene. Two different marking strategies were devised. One specific for the chemical mutagen N-nitro-N'-methyl-N-nitosoguanidine (MNNG) and the other for the chemical mutagen acetoxyaminofluorene (AAF). Oligonucleotides were designed, synthesised and cloned in front of the lac Z gene to generate oligollac Z fusion marker genes. Test constructs without initiation codons and control constructs with initiation codons were designed and cloned at the same time for both the MNNG and AAF strategies. The fusion genes were then tested in vitro by transfection in to murine epidermal cell lines. No expression was detected in the inactive marker gene designed to be activated by MNNG, whereas high expression was obtained from two control constructs which both contained and in-frame ATG initiation codon in the sequence of the oligonucleotide cloned into the lac Z gene. The MNNG-responsive marker gene was shown to be activated in stably transfected murine epidermal cell lines in response to treatment with MNNG at a frequency of approximately 4.3 x 10-3 per clonogenic cell. Subsequently the work with the AAF strategy was discontinued as there was insufficient time to pursue both strategies and positive results had been obtained with the MNNG construct. Twenty-two transgenic founders were generated with three marker gene constructs (Act-LacZA, K5-LacZD and K5-LacZE by pronuclear microinjection of fertilised embryos. Lines of transgenic mice were bred from the founders and the expression of the marker genes were analysed. Expression from a control lac Z fusion gene driven by a BKIII (keratin 5) promoter was detected by staining skin tissue with the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal). The expression of the beta-galactosidase protein was observed in the epidermal basal layer and the hair follicles of transgenic mice. The expression was highest in newborn mice and gradually diminished as the mice reached adulthood. It was subsequently shown that targeting the expression of an activated H-ras oncogene to the epidermis with the same promoter results in a high frequency of malignant tumours. The implications of these results to the biology of the skin are discussed. Expression of the MNNG-activated test marker gene under the control of a beta-actin promoter was also detected. Activation of the marker gene was detected in serial sections of murine brain tissue from transgenic mice treated topically with MNNG at birth. No activation was detected in other tissues probably due to the low level of expression of the marker gene. The explanation of these results and the implications for developing a marker system to work efficiently in the epidermis are discussed

Generalized Geometry and Partial Supersymmetry Breaking

This review article consists of two parts. In the first part we use the formalism of (exceptional) generalized geometry to derive the scalar field space of SU(2)xSU(2)-structure compactifications. We show that in contrast to SU(3)xSU(3) structures, there is no dynamical SU(2)xSU(2) structure interpolating between an SU(2) structure and an identity structure. Furthermore, we derive the scalar manifold of the low-energy effective action for consistent Kaluza-Klein truncations as expected from N=4 supergravity. In the second part we then determine the general conditions for the existence of stable Minkowski and AdS N=1 vacua in spontaneously broken gauged N=2 supergravities and construct the general solution under the assumption that two appropriate commuting isometries exist in the hypermultiplet sector. Furthermore, we derive the low-energy effective action below the scale of partial supersymmetry breaking and show that it satisfies the constraints of N=1 supergravity. We then apply the discussion to special quaternionic-Kahler geometries which appear in the low-energy limit of SU(3)xSU(3)-structure compactifications and construct Killing vectors with the right properties. Finally we discuss the string theory realizations for these solutions.Comment: 117 pages, Ph.D. thesis (Advisor: Jan Louis); v2: references adde

Lectures on Non-BPS Dirichlet branes

A comprehensive introduction to the boundary state approach to Dirichlet branes is given. Various examples of BPS and non-BPS Dirichlet branes are discussed. In particular, the non-BPS states in the duality of Type IIA on K3 and the heterotic string on T4 are analysed in detail.Comment: 46 pages, 5 figures, LaTeX; lectures given at the TMR network school on `Quantum aspects of gauge theories, supersymmetry and quantum gravity', Torino, 26 January - 2 February 2000, and at the `Spring workshop on Superstrings and related matters', Trieste, 27 March - 4 April 2000; references adde

Towards a tensionless string field theory for the N=(2,0) CFT in d=6

We describe progress in using the field theory of tensionless strings to arrive at a Lagrangian for the six-dimensional $\mathcal N=(2,0)$ conformal theory. We construct the free part of the theory and propose an ansatz for the cubic vertex in light-cone superspace. By requiring closure of the $(2,0)$ supersymmetry algebra, we fix the cubic vertex up to two parameters.Comment: 46 pages, 2 figures. V2: references added; minor changes and improvement

Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation

Abstract: Mammalian embryos change shape dramatically upon implantation. The cellular and molecular mechanism underlying this transition are largely unknown. Here, we show that this transition is directed by cross talk between the embryonic epiblast and the first extra-embryonic tissue, the trophectoderm. Specifically, we show via visualisation of a Cdx2-GFP reporter line and pharmacologically mediated loss and gain of function experiments that the epiblast provides FGF signal that results in differential fate acquisition in the multipotent trophectoderm leading to the formation of a tissue boundary within this tissue. The trophectoderm boundary becomes essential for expansion of the tissue into a multi-layered epithelium. Folding of this multi-layered trophectoderm induces spreading of the second extra-embryonic tissue, the primitive endoderm. Together, these events remodel the pre-implantation embryo into its post-implantation cylindrical shape. Our findings uncover how communication between embryonic and extra-embryonic tissues provides positional cues to drive shape changes in mammalian development during implantation