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): Past work has established that many important cargos move bi-directionally along microtubules, and that the distribution and net transport of such cargos can be controlled by regulating the relative contributions of the plus-end versus minus-end motors. It has also established that multiple plus-end and multiple minus-end motors function together. However, both the fundamental mechanism of regulation of such transport is unclear, as is the importance (and regulation) of the number of engaged motors. The work proposed here will lead to a much deeper understanding both of the regulation of transport, and also of the control and importance of motor number in this regulation. Specifically, bi-directional motion of lipid droplets in early Drosophila embryos is investigated. The work can be conceptually divided into two complimentary approaches. In the first, we develop and critically test a theoretical model for how this transport is controlled by tuning single-molecule properties. The theoretical approach is an extension of our past modeling of transport driven by multiple kinesin motors, and rely on Monte Carlo simulations. The theoretical predictions are then tested using genetics to alter protein dosage of the motors, and also specific point mutants. Some of the testing of the model relies on quantifying-in vivo-the motion of individual cargos with high temporal and spatial resolution, and determining if the motion is consistent with the model's prediction. In the second, we focus on the regulation of the number of engaged motors and their force production, as controlled by the klar protein. We use a variety of biophysical characterizations to determine the effects of klar mutations. In particular, we use optical tweezers and particle tracking and analysis to determine the specific physical role of different domains of the klar protein, and then use complementary biochemical and cell biological techniques to determine the molecular interactions underpinning these physical roles. This information will clarify at the molecular level how the biophysically determined functions come about, and because we have already shown that klar regulates force production (and likely number of engaged motors), the study will directly probe how the number of engaged motors is controlled, and the functional implications of this control. PUBLIC HEALTH RELEVANCE: Bi-directional transport is directly related to public health: viruses such as herpes spread through cells in a bi-directional manner; many important cargos like mitochondria and endosomes move bi-directionally. Impaired vesicular transport is implicated in Neuronal degeneration. Finally, a better understanding of transport might allow the design of new drug delivery systems.

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.

CRISP; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Image; In Vitro; Institution; Investigators; Label; Micro-tubule; Microtubules; Molecular Motors; Monitor; Motion; Motor; Movement; NIH; National Institutes of Health; National Institutes of Health (U.S.); Q-Dot; Quantum Dots; Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; body movement; experiment; experimental research; experimental study; imaging; research study; single molecule

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.

PROJECT SUMMARY Many mitotic kinesins play multiple roles in mitosis?each likely requiring distinct mechanical behavior. This multi-functionality implies that cells need to be able to ?fine tune? these motors. We propose that post- translational modifications (PTMs) of the motor domain (MD) of mitotic kinesins serve this role. These PTMs are found in many mitotic kinesins and they cluster in structures that form an allosteric pathway which couples nucleotide and microtubule binding to movement. In this proposal, we will apply a combination of transient kinetics, single molecule mechanics, and cell biology to examine how MD PTMs affect three multifunctional kinesins?Kif11, Kif18A, and Kif22. Results of these studies should allow us to develop an integrated model for how cells modulate their kinesins to meet diverse needs.