Participación de la proteína mediadora de respuesta a Colapsina 2 (CRMP2) en la migración de células de adenocarcinoma de pulmón [Tesis]

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Título:  Participación de la proteína mediadora de respuesta a Colapsina 2 (CRMP2) en la migración de células de adenocarcinoma de pulmón.

Autor: Xabier Morales Urteaga

Director/es: Ana Rouzaut

Resumen: Oriented migration is the basis of major cellular functions such as developmental morphogenesis, tissue repair and tumor metastasis. During cell migration, microtubules are temporarily captured and stabilized near the actin enriched leading by plus end tracking proteins (+TIPs) such as EB1 and CLASP2. As a result, proper cell orientation and vesicle transport to the extending cellular edge is ensured. MTs are constantly switching between phases of polymerization and depolymerization, known as dynamic instability. Collapsin Response Mediator Protein 2 (CRMP2), is a cytosolic phosphoprotein that transports tubulin dimers to the growing +TIP of a MT and contribute the elongation of the tubulin filaments. CRMP2 affinity for tubulin diminishes through its phosphorylation by several protein kinases such as Cdk5 and GSK-3b which phosphorylates CRMP2 on Ser522 and Thr509, Thr514 and Ser518 respectively. Other less frequent CRMP2 phosphorylation events involve the residue Thr555, which are targets of ROCK kinase. In this work, we specifically address the involvement of CRMP2 during cell migration and adhesion using as a model the lung adenocarcinoma cell line A549. We demonstrated that CRMP2 co-localizes strongly with the microtubules in the lamella of migrating cells in a phosphorylation-dependent manner. Furthermore, we observed that aberrant CRMP2 phosphorylation affect tubulin polymerization rate and stability and how these processes modify the capture of microtubules in the membrane. We demonstrated for the first time that CRMP2 is distributed in the plus end of the microtubules where interact with the protein EB1, which regulates microtubule dinamycs. Likewise, we have described that CRMP2 contributed in the anchoring of the microbules with the actin cytoskeleton through its interaction with the adaptator protein IQGAP1. The aberrant CRMP2 phosphorylation affected to these processes and reduced the adherence to different extracellular proteins matrix, especially over collagen. CRMP2 has been reported that regulate traffic of recycling vesicles by its interaction with motor proteins in neurons. We have demonstrated that CRMP2 interact with b1 integrin in lung adenocarcinoma cells during its endocitosis and how the overexpression of phosphorylation mimetic mutants of CRMP2 reduced the percentage of recycled b1 integrin. These deficiencies in b1 integrin recycling are correlated with less ability of migration in these mutants towards chemotactic gradients. In fact, these mutants showed a lower velocity and directness in their trajectories compared to control cells. Importantly, we observed that overexpression of phosphorylation mimetic mutants of CRMP2 in Ser522 switched from a filopodia driven cell migration to amoeboid movement in 3D migration assays. In short, here and for the first time, we show CRMP2 dynamic binding to tubulin during cell migration and demonstrate that altered phosphorylation of CRMP2 affects MT polymerization in the front lamella and cell movement

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