Susan H. Brown - US grants

A large number of actin regulatory proteins have been studied, but in general, they are from different organisms, making it difficult to know what fraction of these proteins might be necessary for the functioning of actin in a single type of cell. The long term objective of this proposal is to completely characterize all of the actin-regulatory proteins from a single cell type, Dictyostelium discoideum, including both soluble and membrane-associated proteins. The first soluble protein to be characterized is a 30,000 dalton protein which crosslinks actin in a calcium-independent fashion. This protein will be compared with the previously described actin-crosslinking proteins by immunological cross-reactivity and peptide mapping, to look for relatedness (which might explain why there are several proteins of overlapping function in this organism). Interactions among the various crosslinking proteins will also be looked for, and evidence that they are under separate regulation will be sought. The next step is to proceed with a systematic search for more soluble actin-regulatory proteins, using effects on the low shear viscosity of actin as an assay. Subfractionation of each fraction will continue until a single peak of activity is obtained. The first approach to be used with membranes is to make antibodies vs. whole membranes, and look for the ability of this antibody to block the effect of membranes on the low shear viscosity of actin. If blocking activity is obtained, the antibody will be fractionated by adsorbing it to portions of a lane of Dictyostelium membrane proteins run on a gel. If blocking activity is not obtained, several alternate approaches to the question of membrane actin-binding proteins are proposed.

A large number of actin regulatory proteins have been studied, but in general, they are from different organisms, making it difficult to know what fraction of these proteins might be necessary for the functioning of actin in a single type of cell. The long term objective of this proposal is to completely characterize all of the actin-regulatory proteins from a single cell type, Dictyostelium discoideum, including both soluble and membrane-associated proteins. The first soluble protein to be characterized is a 30,000 dalton protein which crosslinks actin in a calcium-independent fashion. This protein will be compared with the previously described actin-crosslinking proteins by immunological cross-reactivity and peptide mapping, to look for relatedness (which might explain why there are several proteins of overlapping function in this organism). Interactions among the various crosslinking proteins will also be looked for, and evidence that they are under separate regulation will be sought. The next step is to proceed with a systematic search for more soluble actin-regulatory proteins, using effects on the low shear viscosity of actin as an assay. Subfractionation of each fraction will continue until a single peak of activity is obtained. The first approach to be used with membranes is to make antibodies vs. whole membranes, and look for the ability of this antibody to block the effect of membranes on the low shear viscosity of actin. If blocking activity is obtained, the antibody will be fractionated by adsorbing it to portions of a lane of Dictyostelium membrane proteins run on a gel. If blocking activity is not obtained, several alternate approaches to the question of membrane actin-binding proteins are proposed.

An increasing number of both myosins and kinesin-like proteins are being discovered in a given organism, including yeast. The proteins in each of these superfamilies have very similar motor domains, attached to unique domains that may link the motors to different cargos. Yeast is especially well suited for sorting out why there are so many of these motor proteins, and what their separate functions and inter-relationships are. This proposal will study a subset of yeast motor proteins, including two myosins (MYO2p and MYO4p) that show some similarity even outside the motor domain, and a kinesin-like protein (SMY1p) that surprisingly corrects a defect in MYO2p. Several approaches will be used to elucidate their functions: Their motor functions will be tested using in vitro motility assays, and other predicted biochemical properties (calmodulin binding, phosphorylation, and dimerization in the case of the myosins) will be tested for. Antibodies will be made in order to immunolocalize these proteins, and a variety of molecular/genetic techniques will be used (e.g., deletion or overexpression of the genes; screens for suppressors, second-site non-complementors, synthetic lethality; making hybrid proteins or altering them by site-directed mutagenesis).

The studies in this application focus on the use of rhythmic sensory stimuli to improve motor performance in chronic motor dysfunction conditions. Two population groups characterized by progressive loss in the ability to coordinate complex movements will be examined: (1) idiopathic Parkinson's Disease patients of 3-5 years duration with decreased responsiveness to L-Dopa therapy and (2) healthy elderly subjects. Specific aims will (1) compare the effectiveness of auditory versus visual stimuli in facilitating movement sequencing, (2) identify motor tasks most responsive to sensory cueing and (3) establish a framework for the future development of effective rehabilitative techniques using rhythmic sensory cueing to improve motor performance in a variety of motor dysfunction conditions. These studies will also provide valuable insights into the mechanisms by which temporal information is utilized by the central nervous system in movement planning and execution and how such sensorimotor transformations are disrupted with aging and in Parkinson's Disease. Auditory, visual and combined auditory-visual stimuli will be used as external timing cues during the generation of rhythmic, sequential arm movements. Motor tasks will include unilateral and bilateral movements in which task complexity will be modified by manipulation of spatio-temporal parameters. By examining a broad repertoire of movements, it will be possible to delineate those motor tasks most sensitive to sensory cueing. Decreased variability in movement duration and end-point position as well as task-specific improvement in speed and inter limb coordination will serve as functional outcome measures of sensory facilitation in the production of sequential motor tasks. Enhanced control of movement dynamics will be assessed by examining changes in the relationship between joint angular rotation, movement trajectories and timing of opposing muscle groups. A three dimensional motion analysis system and surface electromyography will be used to obtain kinematic and muscle activity data. All data will be collected on-line and interactive computer software will be used for off-line quantification. These studies represent a unique cross-disciplinary approach to movement rehabilitation by combining expertise in motor physiology, physical medicine and neurology and will be the first in-depth, quantitative evaluation of sensory enhanced motor performance in elderly subjects and Parkinson's Disease.

DESCRIPTION (provided by applicant): Aging is associated with a decline in motor ability which has been typically described in terms of muscle weakness leading to postural instability and locomotor difficulties. In contrast, our understanding of how age-related changes in somatosensory feedback affects motor function is remarkably poor. This is particularly true for the upper limbs despite the importance of arm and hand use in many activities of daily activity and the maintenance of functional independence. In particular, proprioceptive feedback arising from muscle stretch, cutaneous, and force receptors is critical not only in providing an awareness of body position, but in the generation, learning, and on-going control of goal-directed limb movements. Given the paucity of data in this area, the aims of this study are: 1) to provide much needed baseline information regarding upper limb proprioceptive acuity in older individuals and to determine whether changes in motor function are related to changes in proprioceptive function, 2) to determine whether physical activity impacts proprioceptive acuity in older individuals, and 3) to determine if proprioceptive acuity differs between the dominant and non-dominant hands. Young (20-30 yrs) and two groups of elderly subjects 70 yrs and older (sedentary vs active) will be examined. Wrist position and hand grasp force matching paradigms will be used in which movements/forces are produced based solely on limb position or force reference information. Matching tasks will vary in complexity by requiring subjects to match the movement or force with the same or opposite limb. Instrumented levers and force transducers will be used to measure wrist movement and grasp force, respectively. In the limb matching conditions, a servomotor will be used to passively displace the joint at a constant slow velocity. This study will form the foundation for a longer-term research program examining sensorimotor function in the elderly, including disease conditions which impact mobility, and with a focus on maintenance and/or improvement in upper limb function through, for example, training-based interventions.