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See detailA microtubule-independent role of p150glued in secretory cargo concentration at endoplasmic reticulum exit sites
Verissimo, Fatima; Halavatyi, Aliaksandr UL; Pepperkok, Rainer et al

in Journal of Cell Science (2015), 128(22), 4160-4170

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by ... [more ▼]

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd. [less ▲]

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See detailSimulation and computer-assisted analysis of actin cytoskeleton dynamics in living cells using fluorescence microscopy methods
Halavatyi, Aliaksandr UL

Doctoral thesis (2012)

Controlled in time and space by a variety of actin-binding proteins, assembly and disassembly of the actin cytoskeleton is involved in many biological and biophysical phenomena. In order to understand the ... [more ▼]

Controlled in time and space by a variety of actin-binding proteins, assembly and disassembly of the actin cytoskeleton is involved in many biological and biophysical phenomena. In order to understand the dynamics of such a complex intracellular system quantitative time-lapse imaging approaches are required. This thesis presents the results of the interdisciplinary project aimed at the quantitative evaluation of the effects of actin-binding proteins on actin turnover under physiological conditions. It combines fluorescence microscopy experiments, development of mathematical models and data processing tools to understand how regulatory proteins control actin dynamics. Confocal-microscopy-based Fluorescence Recovery After Photobleaching (FRAP) technique is a major experimental tool to measure the exchange of actin and actin-binding proteins between polymerised and monomeric pools. The developed models and computational algorithms allow to predict actin dynamics depending on regulatory proteins and to connect the experimental data to parameters characterising polymerisation dynamics, length and structures of actin filaments and activities of tested regulatory proteins. To test the models and to evaluate the activities of actin binding proteins without cellular constrains I combined quantitative FRAP analysis with a biomimetic assay which allows to reproduce major features of actin motility. This combination of techniques was utilised to measure the influence of previously characterised actin-binding proteins working together on actin dynamics in a concentration-dependent manner. In particular we investigated how capping and severing of actin filaments influences actin exchange in a bulk meshwork generated from spatially restricted nucleation. The experimental and analysis methods I developed were also used to investigate the dynamics of the actin cytoskeleton at focal adhesions of living cells. Taking as an example the focal adhesion protein zyxin and its binding partner Tes we addressed how the interactions of these proteins with actin regulate cytoskeleton dynamics. Taken together, the developed approaches and collected data help to better understand how regulatory proteins control actin dynamics in living cells. [less ▲]

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See detailA Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition.
Moes, Michèle UL; Le Béchec, Antony UL; Crespo, Isaac UL et al

in PLoS ONE (2012), 7(4), 35440

Background: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as ... [more ▼]

Background: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. Results: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. Conclusion: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression. [less ▲]

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See detailQuantitative kinetic study of the actin-bundling protein L-plastin and of its impact on actin turn-over.
Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth UL; Halavatyi, Aliaksandr UL et al

in PloS one (2010), 5(2), 9210

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers ... [more ▼]

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-delta signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion. [less ▲]

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