The overarching goal of the parent grant has been to gain critical insights into the fundamentals of kinesin motor structure and function and to extrapolate this understanding to the inner workings of the cell. Kinesin superfamily members share a common catalytic domain yet participate in a wide range of cellular functions. Sequence differences modify the catalytic properties and microtubule interactions that are critical for the specific function of a kinesin. In this funding cycle our specific aims are directed to establish the mechanistic, mechanical, and structural features that are shared by Kinesin-2 KIF3AB and KIF3AC, and to define the mechanism by which heterodimerization modulates the distinct motile properties of KIF3AC in comparison to KIF3AB and other processive kinesins. The supplemental funds are requested specifically to purchase a GE Healthcare AKTA pure protein purification system which is essential for our mechanistic studies to define the presteady-state ATPase kinetics of the microtubule-based ATPase cycle. Our present AKTA system is 11 years old, and the model is due to be discontinued in the coming year which will result in a cost increase in the annual service contract followed by a gradual decrease in company support to no support in ~5 years. This supplement will ensure our ability to purify proteins in large amounts required to accomplish the specific aims as outlined in the parent grant.
Parent Project and Contribution of the Supplement Kinesin superfamily members share a common catalytic domain, yet participate in a wide range of essential cellular functions including intracellular transport, mitosis and meiosis, regulation of microtubule (MT) dynamics to remodel the cytoskeleton, and generation of cell polarity. Sequence and domain differences among kinesins modify the mechanochemistry and MT interactions so that different motors may accomplish their specific cellular functions. Most recently we have focused our effort on heterodimeric kinesin-2 KIF3AC and KIF3AB. These are fascinating molecule motors because they result from three different gene products: KIF3A, KIF3B, and KIF3C. While KIF3AB is ubiquitously expressed and best understood for its role in intraflagellar particle transport in cilia and flagella, KIF3AC is expressed in neurons. Moreover, our results have shown that many of the properties of KIF3AC are very different from those of KIF3AB. The experimental approaches we use include presteady-state kinetics (stopped-flow and chemical quench flow methodologies), fluorescence microscopy MT gliding assays, and Total Internal Reflection Fluorescence (TIRF) microscopy for single molecule studies. We collaborate with Ivan Rayment (University of Wisconsin) for the structural studies by X-ray crystallography, Juergen Hahn (Rensselaer) for the computational modeling, and Michael Ostap (University of Pennsylvania) to determine the stepping kinetics of KIF3AC as it works against hindering and assisting loads as it would in neurons. It is a mystery as to why kinesin-2 polypeptides heterodimerize to form two different motors, KIF3AB and KIF3AC, and our hypothesis is that this is a mechanism to affect the motile performance of each. The overall goals of the parent grant are to define how kinesin-2 heterodimerization influences KIF3AC and KIF3AB structure, head-head communication, and mechanochemistry to define the key principles for these kinesin-2s that specify their functional roles in the cell. Contribution of the Supplement The AKTA pure is essential to my research program, and this upgrade will enable us to accomplish the specific aims of the parent grant and beyond.
| Quinn, Sean M; Howsmon, Daniel P; Hahn, Juergen et al. (2018) Kinesin-2 heterodimerization alters entry into a processive run along the microtubule but not stepping within the run. J Biol Chem 293:13389-13400 |
| Woll, Kellie A; Guzik-Lendrum, Stephanie; Bensel, Brandon M et al. (2018) An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules. J Biol Chem 293:11283-11295 |
| Gilbert, Susan P; Guzik-Lendrum, Stephanie; Rayment, Ivan (2018) Kinesin-2 motors: Kinetics and biophysics. J Biol Chem 293:4510-4518 |
| Bensel, Brandon M; Guzik-Lendrum, Stephanie; Masucci, Erin M et al. (2017) Common general anesthetic propofol impairs kinesin processivity. Proc Natl Acad Sci U S A 114:E4281-E4287 |
| Guzik-Lendrum, Stephanie; Rayment, Ivan; Gilbert, Susan P (2017) Homodimeric Kinesin-2 KIF3CC Promotes Microtubule Dynamics. Biophys J 113:1845-1857 |
| Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena et al. (2016) Myosin MyTH4-FERM structures highlight important principles of convergent evolution. Proc Natl Acad Sci U S A 113:E2906-15 |
| Zhang, Pengwei; Rayment, Ivan; Gilbert, Susan P (2016) Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC. J Biol Chem 291:4407-16 |
| Albracht, Clayton D; Guzik-Lendrum, Stephanie; Rayment, Ivan et al. (2016) Heterodimerization of Kinesin-2 KIF3AB Modulates Entry into the Processive Run. J Biol Chem 291:23248-23256 |
| Phillips, Rebecca K; Peter, Logan G; Gilbert, Susan P et al. (2016) Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil. J Biol Chem 291:20372-86 |
| Zhang, Pengwei; Dai, Wei; Hahn, Juergen et al. (2015) Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s. Proc Natl Acad Sci U S A 112:6359-64 |
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