Steven P. Gross - US grants

The goal of this proposal is to explore further the interaction between microtubules (MTs) and microtubule-associated proteins (MAPs), particularly the ability of certain MAPs to alter the mechanical properties of MTs. It is possible that one of the key functions of MAP binding is to produce concomitant changes in MT stiffness, affecting neuronal development (MAP2 and Tau) or mitotic processes (MAP4), and there is preliminary evidence that MAP binding does result in an increase of MT stiffness. Details of how MAP binding changes MT mechanical properties, and how MAP phosphorylation alters such effects, are potentially useful in understanding certain neuronal diseases like Alzheimer's disease (where infected patients exhibit a hyperphosphorylated form of Tau), and may also help understand certain details of mitosis, during which MAP4's state of phosphorylation is changed by MPF. To accurately measure MT stiffness, a state-of-the-art dual-beam optical trap will be built for these experiments. The trap will be used to bend individual MTs, by exerting force on beads bound to each end of the MT. Included in the trap design is the ability to accurately measure applied force. Knowing the applied force and resultant curve in the MT will enable the determination of the MT stiffness. These measurements will be carried out in the absence and presence of MAPs, so that their effect on stiffness can be measured. Phosphorylated and genetically engineered MAPs will also be used in these studies, to examine their relative effects.

DESCRIPTION (provided by applicant): Cytoplasmic dynein is poorly understood in vitro, which makes it hard to understand its function and regulation in vivo. For instance, because its single-molecule properties are not known, it is unclear what roles various accessory proteins might need to play in order to achieve correct in vivo function. Further, although some in vivo evidence suggests that multiple dynein motors work together (and with dynactin), the functional significance of employing multiple motors is unknown. We address these questions by studying dynein function in a controlled environment, first at the single-molecule level and then in increasingly complex situations. We will employ an in vitro bead assay that uses an optical trap to quantify how single---or multiple--dynein molecules move along microtubules, both in the presence or absence of proteins that might alter dynein function such as dynactin and MAPs. Initial results published in a recent paper showed that Dynein functions in a fundamentally different manner than kinesin, and appears to have a gear mechanism enabling it to adjust force production to meet external applied load. Further, our unpublished preliminary results suggest that unlike kinesin, at the single molecule level dynein is not a very good efficient cargo transporter. The research will fully investigate dynein's single-molecule function (aim 1), clarify how this function can be altered (aims 2 and 3), and further study how the proposed gear might function (aim 4). We proposed the following specific aims: Aim 1: Elucidate the single-molecule functions of dynein, combining measurements including force-velocity curve, processivity vs load, dependence of function on ATP, etc. with modeling. Aim 2: Determine how multiple dyneins function together Aim 3: Determination of the role of some accessory proteins in regulating altering dynein function. Investigate the functional ramifications of the presence of the dynactin complex and or MAPs. Is a single dynein-dynactin pair an effective transport system ? Do MAPs impair motor-driven transport? Aim 4: Test our hypothesis explaining the gear's function Understanding dynein is directly related to public health: correct dynein function and regulation is essential for development, alteration of dynein function likely occurs in many cancers, and dynein and dynactin mutations cause neuronal degeneration.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is of evaluation type where in we are trying to test the efficiency of fluorophore in the form of quantum dots. Further investigations will include the two color imaging of the lipid droplets and mitochondria each taged with different fluorophores.