L-Plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages

A mathematical model of actin filament turnover for fitting FRAP data

A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the presence of actin nucleating proteins, (3) the FRAP curves may exhibit both a linear and non-linear behaviour depending on the parameters of actin polymerisation, and (4) our model resulted in more accurate parameter estimations of actin dynamics as compared with other FRAP fitting models. Additionally, we provide a computational tool that integrates the model and that can be used for interpretation of FRAP data on actin cytoskeleton. © 2009 European Biophysical Societies' Association

Time-resolved analysis of transcriptional events during SNAI1-triggered epithelial to mesenchymal transition

The transcription regulator SNAI1 triggers a transcriptional program leading to epithelial to mesenchymal transition (EMT), providing epithelial cells with mesenchymal features and invasive properties during embryonic development and tumor progression. To identify early transcriptional changes occurring during SNAI1-induced EMT, we performed a time-resolved genome-scale study using human breast carcinoma cells conditionally expressing SNAI1. The approach we developed for microarray data analysis, allowed identifying three distinct EMT stages and the temporal classification of genes. Importantly, we identified unexpected, biphasic expression profiles of EMT-associated genes, supporting their pivotal role during this process. Finally, we established early EMT gene networks by identifying transcription factors and their potential targets which may orchestrate early events of EMT. Collectively, our work provides a framework for the identification and future systematic analysis of novel genes which contribute to SNAI1-triggered EMT

J Cell Sci

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest by activating nuclear RA receptors (RARs) (alpha, beta and gamma), which are ligand-dependent transcription factors. RARs are also phosphorylated in response to RA. Here, we investigated the in vivo relevance of the phosphorylation of RARs during RA-induced neuronal differentiation of mouse embryonic stem cells (mESCs). Using ESCs where the genes encoding each RAR subtype had been inactivated, and stable rescue lines expressing RARs mutated in phospho-acceptor sites, we show that RA-induced neuronal differentiation involves RARgamma2 and requires RARgamma2 phosphorylation. By gene expression profiling, we found that the phosphorylated form of RARgamma2 regulates a small subset of genes through binding an unusual RA response element consisting of two direct repeats with a seven-base-pair spacer. These new findings suggest an important role for RARgamma phosphorylation during cell differentiation and pave the way for further investigations during embryonic development

A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression