In cell analysis of Myosin Va function in organelle transport.

In order that the cells of our body function correctly they must establish and maintain a complex arrangement of specialised internal structures (organelles). The function of these organelles is essential for life e.g. energy production. Transport and communication between organelles is vital for both the normal function of organelles, cells and the whole body. In line with the importance of organelle transport blocks and other defects in these processes result in many common human diseases such as Cystic Fibrosis, hypercholesterolemia, neuro-degeneration and some forms of cancer. To allow organelle transport, cells have evolved a complex (protein-based) machinery that can move material between different organelles and areas of the cell. One class of proteins required for this are motor proteins that convert chemical energy, from food, into movement and transport cargo along subcellular protein ?tracks? known as the cytoskeleton. One of these motors, MyosinVa (MyoVa), is the focus of this proposal.
MyoVa is present in many cell types (man, animals, yeasts and plants) and transports a variety of cargo. Defects in its function have been implicated in the life-threatening human neurological and albinism diseases (Griscelli syndrome Type I and Elejalde syndrome). Thus understanding the mechanism of MyoVa function is critical to understanding the function of the human nervous and pigmentation systems. MyoVa has been studied extensively in artificial experimental environments outside the cell (in vitro) that have led to the discovery of many properties that make MyoVa an ideal cargo transporter. However, the cellular environment is much more complex than artificially created environments, so the importance of these motor properties in intracellular transport (in vivo) remains unclear.
In this study melanosome transport in melanocytes, pigment producing skin cells, will be used as a model system to determine which MyoVa properties are essential for cargo transport in living cells. Melanosomes are large, pigment containing organelles that are easily seen using a microscope. They are produced near the cell centre and are transported to the cell periphery in a process that requires MyoVa. This process is essential for skin pigmentation and protection from solar ultraviolet radiation. Loss of MyoVa causes defects in transport resulting in melanosomes clustering near the nucleus. This cellular defect results in skin and hair albinism.
The main experimental system used to address the importance of MyoVa properties in cells will be to introduce versions of MyoVa protein in which specific motor properties have been altered (mutated) into melanocytes lacking their own MyoVa. The altered (or mutant) motor proteins will then be tested for their ability to compensate for the loss of the native protein by restoring normal melanosome transport into the cell periphery. The results of this study will provide information relevant to improvements in human health and development of novel technology.

Importance. Cytoskeleton tracks and motors are vital for intracellular organisation and transport by regulating organelle movement and distribution. In vitro data reveal that dimeric motor MyosinVa (MyoVa) ?walks? along actin filaments, indicating a cargo carrying function in cells. Meanwhile cellular studies indicate that it tethers cargo at the cortical actin. Also MyoVa interacts with other cytoskeleton components, suggesting that it may co-ordinate cytoskeleton structure and function.
Aims. The overall objective of this proposal is to determine the mechanism by which MyoVa functions in organelle transport in cells. Specifically it will determine to what extent MyoVa functions in cells as a motor or a tether (Aims1+2)? And how interactions with other cytoskeleton proteins and intra-molecular regulatory interactions affect its function (Aim3)?
Methods. These issues will be addressed using a recently established melanosome transport assay that reports MyoVa function in the movement of physiologically relevant and easily observable cargo (melanosomes) within primary mammalian cells (melanocytes). In this assay MyoVa, and mutants altered in structural and enzymatic properties e.g. dimerisation, are introduced into MyoVaKO melanocytes using adenovirus expression vectors. The functionality of introduced protein is then tested by assessing its ability to rescue loss of endogenous MyoVa, measured by observing redistribution of pigment from the perinuclear cluster into peripheral dendrites. Beyond this simple assay further functional tests will be performed to better understand the cellular role of the mutated part of the protein. For non-rescuing mutants the basis of loss of function will be further defined by testing cargo targeting and motor activity. For rescuing mutants more subtle alterations of function will be investigated by measuring the efficiency of rescue, indicated by the kinetics of rescue and the expression level required for rescue.
Applicant. The applicant has 10 years experience working with cultured melanocytes, and molecular biology techniques required to fully exploit this assay system. He has successfully developed similar complementation assays to investigate the function of Myosin activating proteins Mlph and Myrip. Additionally he maintains a network of collaborators who will provide essential reagents (e.g. libraries, cell lines, viral vectors and mutant mice).
Outcomes. This program will generate valuable information on the role of MyoVa and other motors by establishing their role as regulators of cytoskeleton activity, tethers or cargo carriers. Many motor proteins, including MyoVa, have been linked to human disease and advances in the basic understanding of their cellular function will aid the development of treatments of these diseases.