The discovery of the interaction between myosin Va and RPGRIP1L: characterization of the complex and localization at the centrosome

Author(s)

Leandro Henrique de Paula Assis

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2016-08-18

Examining board members

Mário Tyago Murakami; Carmen Veríssima Ferreira; Daniel Martins de Souza; Rafael Victório Carvalho Guido; Marcos Vicente de Albuquerque Salles Navarro

• Advisor: Mário Tyago Murakami; Jörg Kobarg

Abstract

The human myosin Va (MyoVa) is a dimeric motor protein that uses the energy released from the ATP hydrolysis to move along the actin cytoskeleton. Its main function involves the transport of cargoes whitin the cell, such as vesicles, organelles, messenger RNA and regulatory proteins. To do that, the cargoes are attached to the cargo-binding domain (CBD) situated at the myosin C-terminus. Usually, the binding does not occur directly, but it is mediated by partner proteins, sometimes also called adapter proteins, which are attached on the cargoes surface. For this reason, mutations in the genes encoding for MyoVa or its partner proteins are associated with some pathologies, including the Griscelli syndrome type 1, characterized by albinism and neurological disorders. Although MyoVa has been extensively studied, few MyoVa-binding proteins have been described, hampering the understanding of several MyoVa-dependent cellular processes. In order to fill this gap, we searched for new MyoVa binding-partners, screening a human fetal brain cDNA library by the yeast two-hybrid method. After the validation step, we selected the protein RPGRIP1L as our target. RPGRIP1L is a cilia-centrosomal multidomain protein that plays a key role in the formation of the transition zone at the base of the primary cilium, an organelle that senses a wide variety of extracellular signals. Using GST-pull down and microscale themophoresis assays, we showed that MyoVa-CBD binds to the RPGRIP1L-C2 domains with low affinity (KD between 3 and 9 µM), which is typical of transient protein-protein interactions. Moreover, our yeast two-hybrid data using point mutations on the MyoVa-CBD surface indicated that the residues W1713, Y1721, Q1755 and F1792 are involved in RPGRIP1L recognition. Those residues are fully conserved in MyoVb and our results showed that this myosin also interacts with RPGRIP1L. Proximity ligation assays and confocal microscopy evidenced the physical interaction between MyoVa and RPGRIP1L in the centrosomes during mitosis and at the base of the primary cilium during interphase. Surprisingly, we also noticed MyoVa along the primary cilium, a new and unexpected finding, since this organelle is essentially formed by microtubules. Together, our results unveil RPGRIP1L as a novel binding partner of MyoVa, paving the way for future studies aiming to clarify the role of this molecular motor in the centrosome and primary cilium (AU)

• FAPESP's process: 11/20229-7 - Human myosin-5A and the intracellular transport of cargoes: studies of the myosin-5A and adapter proteins complexes
• Grantee: Leandro Henrique de Paula Assis
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)