La kazrina c controla el tràfic endocític i es un doble regulador de la polimerització d'actina i el transport per microtúbuls
- Autores: Inés Hernández Pérez
- Directores de la Tesis: María Isabel Geli Fernández-Peñaflor (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Jens Lüders (presid.), José Ramon Bayascas Ramírez (secret.), Oriol Gallego Moli (voc.)
- Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
Eukaryotic cells internalise and redistribute the molecules from their surface through the endocytic pathway. This process is key to nutrient uptake and catabolism, and controls the surface exposure of signalling receptors and cell adhesion complexes, among others. Previous work in the laboratory identified kazrin C as a protein that blocked Clathrin-Mediated Endocytosis (CME) when overexpressed.
The work presented in this thesis further supported the role of kazrin in CME, as kazrin KO cells generated with the CRISPR-cas9 system were defective in the uptake of the endocytic marker Transferrin (Tfn). Kazrin C co-localised with markers of adherence junctions, such as N-cadherin, at the plasma membrane and on intracellular structures. Indeed, subcellular fractionation analysis showed the localisation of kazrin in Early Endosomes (EEs). Consistent with a role of kazrin in EEs, kazrin depletion caused an accumulation of N-cadherin-loaded EEs, which showed a more peripheral distribution as compared to WT cells. Kazrin KO cells loaded with Tfn were unable to transport the cargo towards the Endocytic Recycling Compartment (ERC) and had a concomitant defect in Tfn recycling. All phenotypes on KO cells were recovered by the re-expression of GFP-kazrin C but not GFP. These evidences indicated a role of kazrin C in endosomal recycling and the transport of EEs towards the ERC. In agreement with this hypothesis, kazrin depletion caused defects in cellular processes that strongly depend on recycling through the ERC, such as cell migration and cytokinesis.
This study also analysed the molecular mechanisms of kazrin C function in endocytic traffic. Kazrin C was found to interact with the microtubule motors kinesin-1 and dynein, and directly bind to the dynein Light Intermediate Chain LIC1. In fact, kazrin C contains a coiled-coil domain similar to those found in dynein adaptors. Also similar to those, GFP-kazrin C localised to the pericentriolar region, where it seemed to trap EEs. Therefore, we proposed that kazrin C promoted microtubule-dependent transport of EEs, possibly as an EE dynein adaptor. Accordingly, this and previous studies in the laboratory showed direct interactions and partial co-localisations of kazrin C with EE components, such as the AP-1 clathrin adaptor complex and EHD1/3 GTPases. In addition, kazrin C interacted with PI3P and the class III PI3K, and its depletion caused an increase in the endosomal levels of the PI3P probe GFP-FYVE. Finally, we linked kazrin C with another player in endocytic traffic: the Arp2/3-associated machinery for actin polymerisation. Direct interactions were observed with cortactin and N-WASP, as well as co-localisation of GFP-kazrin C with cortactin at the plasma membrane and intracellular structures. Moreover, kazrin depletion caused a reduction in cortical and an increase in endosomal branched actin.
Altogether, we proved that kazrin C functions in endosomal recycling and propose that this function is mediated by the regulation of microtubule-dependent transport, actin polymerisation and PI3P metabolism.
