For proper functioning of the cell, the cytoskeleton must be continuously constructed and maintained with high fidelity. This requires the ballet of many different proteins, particularly myosin motors and their actin tracks. One such myosin, Myo18 is known to interact with the proto-typical force producing myosin, Myo2, within stress fibres during migration and muscle sarcomeres. This interaction occurs through a conserved coiled-coil domain allowing for the formation of filaments displaying arrays of active motor heads. Myo18 itself has been shown to have very little motor activity. Yet, it still binds the actin track and is heavily implicated in the proper construction of muscle tissue and cell migration. Despite tantalizing data on Myo18, a structural and mechanistic connection has not been made between the divergent activity of Myo18 and its important cellular roles. This research project aims to bridge this gap and understand the function of Myo18, and in turn how it can modulate the function of Myo2 within mixed filaments. Our consortium will integrate data from the atomic to the cellular scale through structural, biochemical, biophysical and cell biology approaches. Firstly, we aim to elucidate the nature of the interaction between Myo18 and actin. Secondly, we aim to directly assay the interaction between Myo18 and Myo2 from a structural perspective (i.e. how Myo18 can intercalate into large Myo2 architectures) and then how the inclusion of Myo18 can modulate the function of Myo2 filaments in vitro and in vivo. A holistic view upon such a divergent myosin opens new doors onto the changes in enzymatic function of myosins upon mutation and the complex sociology between myosins required for the high-fidelity construction of intricate cytoskeletal architectures.

Force generation by molecular motors powers numerous cellular processes. Activated by partners, motors can use this force for distinct mechanistic roles, such as transport, anchoring and track organization. However, the actual action the motor performs upon generating force has not been demonstrated in situ for most of their cellular roles. The design of the domains that follow the motor (lever arm, dimerization region if the motor is dimeric) greatly influences the coordination of heads and the reach of the motor during stepping. However, little is known about how motors control their action with a given design and the tuning of their motility properties. Even less is known about how the attachment of the motor to particular cargoes can contribute to defining the action it performs in cells. MyoActions will focus on Myosin VI (Myo6), a unique and puzzling motor since its mechanical properties allows it to transport, anchor or organize actin filaments. Myo6 produces force in the reverse direction than all other myosins and can be recruited in cells by distinct tail partners (cargoes). It is thus well-suited for delineating the roles cell partners play in specifying the action of molecular motors. Understanding the control of motor activation in cells is essential to gain information about its cellular function. It is critical to design experiments (1) to probe the exact role the motor plays in its own cellular environment, (2) to study how recruitment is regulated in space and time and (3) how partners dictate the role of the motor, in particular when it experiences reverse force (load). We propose a multi-scale analysis (from structural biology, development of specific inhibitors/modulators of force generation, loaded motility assays and cell biology) to provide critical insights into how motors anchor and organize their tracks and how their partners specify and control these actions. A chemical/genetic approach will be developed with Partner 2 to identify small molecules able to influence the motor properties to generate selective anchors, or Myo6 motors unable to anchor. Taking advantage of the preliminary data recently obtained by Partner 3, we have designed a line of study to gain mechanistic insights into the role. Myo6 plays in the fission of tubular intermediates from melanosomes, a process that is required for their maturation and function. Myosins have been implicated in membrane fission in many different contexts, thus, the proposed studies on Myo6 will provide fundamental new insights into how motors act on membranes. The MyoActions proposal will result in a mechanistic understanding of the Myo6 cellular roles by innovative approaches not previously explored for studying molecular motors. The results will have direct implications for understanding pathologies associated with this motor. Myo6 dysfunction has been associated with deafness due to the degeneration of stereocilia in the inner ear. Similarly, mutation of its adaptors, such as OPTN and GIPC also result in disease pathologies. Finally, Myo6 has also been shown to contribute to cancer progression and may serve as a potential therapeutic target.

In a complex environment, animals and humans learn to navigate between sequences of significant places. Such spatial sequence learning has been shown to involve brain areas including the hippocampus (HC) and the prefrontal cortex (PFC). Recent studies show that spatial navigation in the rat hippocampus involves the replay of place cell firing during awake and sleep states generating small sequences of spatially related place cells firing. These replay episodes occur primarily during sharp-wave-ripple events. Much attention has been paid to replay during sleep in the context of long term memory consolidation. Here we focus on the role of replay during the awake state, as the animal is learning across multiple trials. We hypothesize that the generation of these short sequences of place cells during replay allow for global spatial sequence learning in PFC. We propose to develop an integrated model of the hippocampus-PFC network that is able to form spatial navigation sequences based on replay. Intellectual Merit: The proposed collaborative research will extend an existing spatial cognition model for simpler goal-oriented tasks (Barrera et al, 2011) with 1) a replay-driven model for memory formation in the hippocampus and 2) a model of spatial sequence learning in PFC. In contrast to existing work on sequence learning that relies heavily on sophisticated learning algorithms and synaptic modification rules, we propose to use an alternative computational framework known as ‘reservoir computing’ (Dominey 1995) in which large pools of prewired neural elements process information dynamically through reverberations. This reservoir will consolidate hippocampal replay sequences into larger spatial sequences that may be later recalled by subsets of the original sequences. We will constrain the model using electrophysiological recordings from the rodent hippocampal CA1 area in a multi-goal spatial task that is known to involve the hippocampus and the PFC (Watkins de Jong et al 2011). The proposed work is expected to generate a new understanding of the role of replay in memory acquisition in complex tasks such as sequence learning. That operational understanding will be leveraged and tested on robotic platforms. The originality and contribution of our proposed work includes 1) the use of hippocampal replay to create small chunks of valid trajectories, 2) the use of reservoir computing to learn spatial sequences using inputs generated by our hippocampus model, 3) our constraining and testing of the model using electrophysiological data in rats and 4) the use of the resulting model in the embodied-cognitive framework of a robot. Broader Impact: The proposal involves a multidisciplinary collaboration between a roboticist, a computational neuroscientist and an experimentalist. We plan to exploit this diversity by developing interdisciplinary material for courses and outreach activities. To actively recruit minority students, we will take advantages of REU and other programs at USF and U. of Arizona and in France. Inserm participates in multiple programs to initiate high-school students in basic research, neuroscience and robotics. We plan to explore new venues to reach the general public (especially younger students) through programs such as USF STARS and Brain Awareness Week at UA and Lyon to provide the opportunity for young students and the public to be exposed to research developments in different disciplines. In this framework, we will develop ‘sequence learning’ demonstrations involving the use of commonly found mobile robots or participants. We also propose an annual meeting in each of our 3 institutions that will be open to our respective communities and the public.