Characterization of the functional and spatial dynamics of myosin Va in the mitochondrial fission process

Author(s)

Jackeline Souza Araújo

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2021-03-29

Examining board members

Enilza Maria Espreafico; Alícia Juliana Kowaltowski; Mário Tyago Murakami; Luis Lamberti Pinto da Silva

• Advisor: Enilza Maria Espreafico

Abstract

Mitochondrial architecture is involved in several crucial functions to cell viability, such as proliferation, senescence and signaling. In particular, mitochondrial dynamics through the balance between mitochondrial fusion and fission plays a central role in adjusting the cell metabolic needs. Recently, there has been a great focus on the participation of the cytoskeleton and molecular motors in the processes required for mitochondrial homeostasis. In particular, mitoSpire, an actin-nucleating protein that has a mitochondrial location, has been shown to be important in mitochondrial fission. Interestingly, Spire proteins interact with myosin-V molecular motors. Myosin-Va, an actin based molecular motor, is involved in a number of crucial cell functions, and published data from our laboratory indicate that it also has a mitochondrial location. In this context, we hypothesized that myosin-Va could be involved in the processes of mitochondrial dynamics. To answer these questions, we initially confirmed the localization of myosin-Va to mitochondria surface using super-resolution and confocal images. Later, we saw that myosin-Va interacts directly with mitoSpire1, and that its recruitment to the OMM increases with the overexpression of mitoSpire1, making myosin-Va mitochondrial interaction even more evident. To understand if myosin-Va plays a role in the mitochondria, we modulate it in A375 melanoma cells through knockdown with shRNA and also nocaute (KO) using CRISPR. We observed that these cells have elongated mitochondria and fewer fission events after myosin-Va depletion. This phenotype is rescued by overexpressing a construct containing the full length myosin-Va. In addition, through video microscopy we observed that myosin-Va is localized at mitochondrial fission points, and that this location is partially lost in mitoSpire1 KO cells. In search of the mechanism by which myosin-Va interferes with mitochondrial fission, we investigated its interaction with other important proteins in this process. We observed that the localization of myosin-Va at the mitochondrial fission points also coincides with clusters of Drp1, which is the central protein responsible for mitochondrial fission. Our data also show the interesting localization of myosin-Va and Rab11a to the mitochondrial fission points, which is also partially lost in mitoSpire1 KO cells. Finally, we found that KO cells for myosin-Va have lower oxygen consumption rates when compared to control cells, as well as lower levels of ATP production. In search for the consequences of this lower energy efficiency, we evaluated some tumorigenic functions of the cells, such as clonogenicity and migration, we observed that these capacities are reduced after myosin-Va KO. Therefore, our data are indicative of a role for myosin-Va in mitochondrial fission, and indicate that myosin-Va, mitoSpire1 and Rab11a could possibly act as a protein complex that is involved in the regulation of mitochondrial fission. In conclusion, myosin-Va depletion causes an imbalance in mitochondrial fission associated with lower energy efficiency, which leads to a decrease in tumorigenic properties of A375 melanoma cells and may explain some of the severe neuromuscular dysfunctions associated with the Griscelli syndrome-type 1. (AU)

• FAPESP's process: 16/10862-8 - Study of myosin-Va role in the processes of mitochondrial dynamics and mitophagy
• Grantee: Jackeline Souza Araújo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)