The contribution of AP-1gamma2 and PI3KC2alpha for the early-to-late endosome maturation, extracellular vesicles secretion and lysosome related organelles biogenesis

• Grant number: 23/12126-0
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: February 29, 2024
• End date: February 27, 2025
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Luis Lamberti Pinto da Silva
• Grantee: Lucas Alves Tavares
• Supervisor: Graça Raposo
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Institut Curie, France
• Associated to the scholarship: 21/01182-1 - The functional characterization of AP-1 in the endolysosomal system and its relationship with the assembly of HIV-1, BP.PD

Abstract

The adaptor protein (AP) complexes are heterotetrameric complexes that coordinate protein trafficking in the endocytic and secretory pathways. AP-1, a complex of four distinct subunits (gamma, beta1, mi1, and sigma1), is thought to mediate protein trafficking between the trans-Golgi network (TGN) and endosomes and Lysosomes Related Organelles (LROs) though clathrin-coated vesicles (CCV). The human genome encodes two isoforms of the gamma-adaptin (gamma1 and gamma2) subunit that form two variants of AP-1 (AP-1gamma1 and AP-1gamma2). Previous studies demonstrated that gamma2 may form an AP-1 complex variant, but the knowledge is limited regarding the cellular roles played by this alternative AP-1 complex. We previously reported that gamma2 is part of a functional AP-1 complex variant hijacked by HIV-1 Nef for targeting CD4 and MHC-I for lysosomal degradation. Additionally, our previously data suggests that AP-1gamma2 might be involved in the early-to-late endosome maturation and Extracellular Vesicles (EVs) biogenesis and secretion, possible through its binding partner PI3KC2alpha. However, the exactly mechanisms underlying these alterations remain unclear. Here we propose to investigate the participation of AP-1gamma2 and PI3KC2alpha in the dynamics of the endo-lysosomal system. We hypothesize that AP-1gamma1 and AP-1gamma2 are likely to be recruited to different subcellular compartments of the cell and this might explain their different functions. To further shed light on the functions of these different isoforms we will perform complementary cell biological methods. In particular, we will perform indirect immunofluorescence followed by super resolution microscopy and immunoelectron microscopy (EM) of HeLa cells to compare the subcellular distribution of AP-1gamma1, AP-1gamma2 and PI3KC2alpha. To better understand the morphological changes of early and late endosomes in WT cells and gamma2 knockout (KO) cells we will perform in addition conventional electron microscopy after high pressure freezing to optimally preserve morphology. Moreover, we will develop PI3KC2alpha KO cells by CRISPr-Cas9 methodology to investigate whether the ablation of PI3KC2alpha phenocopies the alterations in endosome maturation and EV biogenesis and secretion observed in gamma2 KO cells. Finally, we aim to expand our knowledge acquired so far on gamma2 and PI3KC2alpha to investigate their functions in specialized cells (skin melanocytes) generating LROs (melanosomes). We will study if the ablation of gamma2 and PI3KC2alpha in pigmented melanocytes will compromise the biogenesis of melanosomes, the best characterized LRO and for which a function of AP1 was reported. Therefore, the proposed study has the potential to contribute to the understanding of fundamental processes in the regulation of protein trafficking involved in early-to-late endosome maturation, EVs secretion and LROs biogenesis. (AU)