Structure/function analysis of Rab- and Myosin-interacting proteins in organelle motility

Many important biological processes that underlie the normal function of our cells and tissues rely upon the movement of intracellular structures or organelles. This movement is regulated by motor proteins that transport organelles along intracellular tracks known as cytoskeleton. The activity and targeting of one class of motor protein known as Myosins to specific intracellular organelles is regulated by organelle specific proteins known as Rabs. In the case of pigment granules in the skin and eye one Rab protein called Rab27a recruit Myosins known as MyoVa or MyoVIIa to the granule indirectly via interaction with a third class of protein known as Mlph or MyRIP, respectively. Normal intracellular transport of pigment in the pigment producing cells of the skin (melanocytes) is critical in forming skin colour and protection from ultraviolet radiation from the sun, while in the eye it is important to maintain the function of the light sensing photoreceptor cells that lie adjacent to the retinal pigment epithelium (RPE). The aim of the proposed work is to determine the mechanism by which Mlph/MyRIP proteins interact with and activate MyoVa/VIIa. In particular Mlph interacts with and activates only MyoVa while MyRIP interacts with and activates both MyoVa and MyoVIIa. Comparison of the amino acid sequence of Mlph and MyRIP indicate that both proteins contain common parts and that MyRIP contains extra parts that are not present in Mlph. We will test the idea that common parts or protein domains are involved in interaction with/activation of MyoVa while MyRIP-specific domains are involved in interaction with/activation of MyoVIIa. To do this we will produce and purify the common and MyRIP specific domains and test the strength of their interaction with MyoVa and MyoVIIa proteins in a test-tube. This should provide us with information about the key components of Mlph and MyRIP required for interaction with MyoVa and MyoVIIa. We will then conduct cell biological experiments to confirm the results of our interaction studies and also to measure the effects of these components on the activation of MyoVa and MyoVIIa in living cells. As indicated above movement of pigment granules in pigment producing cells, known as melanocytes, in the skin is dependent upon the activity of MyoVa and in particular its recruitment to the pigment granule by Mlph. Meanwhile in the eye movement of pigment granules is regulated by MyoVIIa following its recruitment to the granule surface by MyRIP. In parallel studies we will introduce common domains and MyRIP specific domains to skin melanocytes and RPE cells that lack Mlph and MyRIP, respectively, and test the ability of introduced proteins to restore the pigment transport defects resulting from loss of these proteins. This will allow us to understand the basis of the interaction of Mlph/MyRIP with Myosin family proteins, the mechanism by which Mlph/MyRIP allow activation of the motors and lay the groundwork for future studies of the three-dimensional structure of Mlph/MyRIP in complex with Myosin motors.

Mlph/MyRIP are a subfamily of Rab27-interacting proteins that control the activation of MyosinVa/VIIa motors that regulate organelle movement. We aim to study the molecular basis of Mlph/MyRIP interaction with MyoVa/VIIa both in vitro and in a cellular setting. We will use an established in vitro binding assay to measure the affinity of functional domains of Mlph, the corresponding regions of MyRIP and MyRIP-specific regions for recombinant cargo-binding tails of MyoVa and MyoVIIa. This will allow a description of domains common to Mlph and MyRIP involved in interaction with MyoVa, and MyRIP-specific sequences involved in interaction with MyoVIIa. Based on in vitro data, we will use cellular assays whose readouts reflect either MyoVa or MyoVIIa activity to test the role of the identified domains in interaction with, and indirectly activation of, native MyoVa/VIIa. To test interaction with and activation of MyoVIIa, we will overexpress Mlph/MyRIP proteins in mouse primary RPE cells that lack MyRIP. These cells exhibit defects in melanosome dynamics that result from loss of MyRIP-dependent recruitment of MyoVIIa to the melanosome. We will use immunofluorescence and time-lapse microscopy to measure the effects of introduced Mlph/MyRIP proteins on melanosome dynamics. To test interaction with and activation of MyoVa, we will overexpress mutated Mlph/MyRIP proteins in skin melanocytes from leaden mice. These cells exhibit perinuclear clustering of melanosomes due to lack of Mlph and are unable to recruit MyoVa to the melanosome. Overexpression of MyRIP rescues this melanosome transport defect by recruitment of MyoVa to melanosomes. Thus we will use immunofluorescence and time-lapse microscopy to determine the ability of Mlph/MyRIP proteins to interact with and activate MyoVa. This powerful approach will allow us to dissect the specificity of MyRIP interaction with Myosin motors and identify Mlph/MyRIP determinants that regulate Myosin activation and organelle movement.