Beta-actin is required for proper mouse neural crest ontogeny

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11

Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site

Background: Profilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P-2). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P-2-binding, it was suggested that carboxy terminal basic residues may be involved as well. Results : Using site directed mutagenesis we have refined the PI(4,5)-P-2 binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P-2 micelles. We identified at least two PI(4,5)-P-2-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal a-helix may have dramatic effects on ligand binding. Conclusions : The involvement of the two terminal a-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P-2 and actin and PI(4,5)-P-2 and poly(L-proline) regulate profilin functions

The XTT cell proliferation assay applied to cell layers embedded in three-dimensional matrix

Abstract Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other "3D culture conditions," such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both

An end-to-end software solution for the analysis of high-throughput single-cell migration data

The systematic study of single-cell migration requires the availability of software for assisting data inspection, quality control and analysis. This is especially important for high-throughput experiments, where multiple biological conditions are tested in parallel. Although the field of cell migration can count on different computational tools for cell segmentation and tracking, downstream data visualization, parameter extraction and statistical analysis are still left to the user and are currently not possible within a single tool. This article presents a completely new module for the open-source, cross-platform CellMissy software for cell migration data management. This module is the first tool to focus specifically on single-cell migration data downstream of image processing. It allows fast comparison across all tested conditions, providing automated data visualization, assisted data filtering and quality control, extraction of various commonly used cell migration parameters, and non-parametric statistical analysis. Importantly, the module enables parameters computation both at the trajectory-and at the step-level. Moreover, this single-cell analysis module is complemented by a new data import module that accommodates multiwell plate data obtained from high-throughput experiments, and is easily extensible through a plugin architecture. In conclusion, the end-to-end software solution presented here tackles a key bioinformatics challenge in the cell migration field, assisting researchers in their highthroughput data processing

The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions

The focal adhesion protein testin is a modular scaffold and tumour suppressor that consists of an N-terminal cysteine rich (CR) domain, a PET domain of unknown function and three C-terminal LIM domains. Testin has been proposed to have an open and a closed conformation based on the observation that its N-terminal half and C-terminal half directly interact. Here we extend the testin conformational model by demonstrating that testin can also form an antiparallel homodimer. In support of this extended model we determined that the testin region (amino acids 52-233) harbouring the PET domain interacts with the C-terminal LIM1-2 domains in vitro and in cells, and assign a critical role to tyrosine 288 in this interaction

Taking aim at moving targets in computational cell migration

Cell migration is central to the development and maintenance of multicellular organisms. Fundamental understanding of cell migration can, for example, direct novel therapeutic strategies to control invasive tumor cells. However, the study of cell migration yields an overabundance of experimental data that require demanding processing and analysis for results extraction. Computational methods and tools have therefore become essential in the quantification and modeling of cell migration data. We review computational approaches for the key tasks in the quantification of in vitro cell migration: image pre-processing, motion estimation and feature extraction. Moreover, we summarize the current state-of-the-art for in silico modeling of cell migration. Finally, we provide a list of available software tools for cell migration to assist researchers in choosing the most appropriate solution for their needs

Prevalence of cytoplasmic actin mutations in diffuse large B-cell lymphoma and multiple myeloma : a functional assessment based on actin three-dimensional structures

Mutations in actins have been linked to several developmental diseases. Their occurrence across different cancers has, however, not been investigated. Using the cBioPortal database we show that human actins are infrequently mutated in patient samples of various cancers types. Nevertheless, ranking these studies by mutational frequency suggest that some have a higher percentage of patients with ACTB and ACTG1 mutations. Within studies on hematological cancers, mutations in ACTB and ACTG1 are associated with lymphoid cancers since none have currently been reported in myeloid cancers. Within the different types of lymphoid cancers ACTB mutations are most frequent in diffuse large B-cell lymphoma (DLBCL) and ACTG1 mutations in multiple myeloma. We mapped the ACTB and ACTG1 mutations found in these two cancer types on the 3D-structure of actin showing they are in regions important for actin polymer formation or binding to myosin. The potential effects of the mutations on actin properties imply that mutations in cytoplasmic actins deserve dedicated research in DLBCL and multiple myeloma

Identification and expression analysis of splice variants of mouse enabled homologue during development and in adult tissues

<p>Abstract</p> <p>Background</p> <p>The Enabled/Vasodilator stimulated phosphoprotein (Ena/VASP) gene family comprises three genes in vertebrates: <it>Vasp</it>, Enabled homologue (<it>Enah</it>) and Ena-VASP like (<it>Evl</it>). <it>Enah </it>has the most complex gene structure. It has extra alternatively included exons compared to <it>Vasp </it>and <it>Evl</it>, and possibly one alternatively excluded intron S. The aim of this mapping study was to probe the occurrence of combinations of exon usage in <it>Enah </it>thereby identifying possible vertebrate ENAH splice variants. We investigated this via an <it>in silico </it>analysis and by performing a reverse transcription-polymerase chain reaction (RT-PCR) screen on mouse samples. We further probed the expression pattern of mouse Enah splice variants during development and in a selection of mouse adult tissues and mouse cell lines.</p> <p>Results</p> <p><it>In silico </it>analysis of the vertebrate Ena/VASP gene family reveals that birds do not have <it>Vasp</it>, while fish have two <it>Evl </it>genes. Analysis of expressed sequence tags of vertebrate <it>Enah </it>splice variants confirms that an Enah transcript without alternative exons is ubiquitously expressed, but yields only limited information about the existence of other possible alternatively spliced Enah transcripts. Via a RT-PCR screen, we provide evidence that during mouse development and in adult mice at least eight and maximally sixteen different Enah transcripts are expressed. We also show that tissues and cell lines display specific expression profiles of these different transcripts. Exons previously associated with neuronal expression of Enah splice variants are also present in other tissues, in particular in heart.</p> <p>Conclusions</p> <p>We propose a more uniform nomenclature for alternative exons in <it>Enah</it>. We provide an overview of distinct expression profiles of mouse Enah splice variants during mouse development, in adult mouse tissues and in a subset of mouse cell lines.</p

Combination of EGFR inhibitor lapatinib and MET inhibitor foretinib inhibits migration of triple negative breast cancer cell lines

Triple-negative breast cancer (TNBC) is the most challenging subtype to treat due to the lack of estrogen receptor, progesterone receptor, and HER2 expression, which excludes the usage of directed targeted therapy against them. Promising therapeutic targets are the hepatocyte growth factor receptor (MET) and epidermal growth factor receptor (EGFR), which expression is frequently elevated in TNBC. Inhibitors of these receptors used as monotherapy are often ineffective. Due to that, we studied the efficacy of combined therapy targeting MET and EGFR simultaneously. Two TNBC cell lines were treated with lapatinib (a dual EGFR and HER2 inhibitor), foretinib (a MET inhibitor), or a combination of the two. After the inhibitors treatment, we verified the cell viability (XTT assay), distribution of the cell cycle phases, the activation of signaling pathways (Western blotting), distribution of invadopodia, fluorescent gelatin digestion (immunofluorescence), and the invasion capacity of cells. A combination of foretinib and lapatinib effectively reduced the viability of examined cells, led to G2/M arrest and reduction of pAKT. There was also a decrease in number of invadopodia formed by cells, their ability to digest gelatin and reduction of cells migration/invasion capacity. Therapy targeting of both EGFR and MET receptors was much more effective against tested cells than monotherapy. We selected a combination of drugs that could be successfully used against this breast cancer subtype

The US3 kinase of pseudorabies virus leads to activation of the actin regulator cofilin to induce actin cytoskeleton changes

The US3 kinase is conserved amongst all Alphaherpesvirinae. We and others have shown that this kinase induces dramatic rearrangements of the actin cytoskeleton, including disassembly of actin stress fibers (resulting in cell rounding) and the formation of cellular projections, which are associated with increased viral spread (Favoreel et al., 2005, PNAS). For the alphaherpesvirus pseudorabies virus (PRV), we have found that the US3-induced changes in the actin cytoskeleton are mediated through p21-activated kinases (PAKs), central regulators in RhoGTPase signaling (Van den Broeke et al., 2009, PNAS). Apart from the involvement of PAKs, relatively little is known on the cellular factors that contribute to US3-mediated actin rearrangements. Cofilin, a member of the ADF/cofilin family, is a central player in actin dynamics and is known to be inactivated through phosphorylation on serine residue 3 (S3) (Moriyama et al., 1996, Genes Cells). Our aim is to investigate whether the US3 protein of the alphaherpesvirus pseudorabies virus (PRV) affects cofilin phosphorylation, and, if so, whether this contributes to the US3-mediated effects on the actin cytoskeleton. We report that US3 leads to strong cofilin dephosphorylation, which is inhibited by a PAK inhibitor, and that overexpression of a phosphomimetic cofilin variant interferes with US3-mediated actin rearrangements