Kinesin and myosin are so-called `motor proteins' that can use two `feet' to walk along microtubule and actin filaments (respectively) that make up the cytoskeleton. These motors support many vital functions with the cell, including pulling DNA structures apart during cell division and resupplying nerve junctions (the synapse) with neurotransmitters (such as seratonin). The precise mechanisms by which these elaborate molecular machines, which are composed of tens of thousands of exquisitely arranged atoms, are able to `walk' are complex and incompletely understood. To see how they work, it is necessary to visualize these complex structures in three dimensions at sufficient levels of detail to resolve individual atoms? and to follow molecular rearrangements that happen while the motors step forward. This goal, however, has long remained out of reach due to the extreme technical challenges involved. We have addressed this problem by developing new methods to analyze images of frozen motor-filament assemblies collected by latest-generation electron microscopes. This approach, known as cryo-electron microscopy, allows us to directly visualize the three dimensional shape of individual molecular motor proteins attached to their partner filaments. During the previous funding period, we solved 3D structures of truncated single `feet' (one motor domain) of kinesin and myosin motors attached to their partner filaments, showing in atomic detail how these structures changed when molecules of ATP fuel were bound and consumed. We also captured a 3D structure of an intact pair of kinesin molecules (dimer) caught in mid- step on a microtubule. This allowed us to visualize, for the first time, a way in which the two `feet' of kinesin can pull on each other in a way to stay coordinated while walking. In our ongoing research we are improving our methods to capture more intermediates in the stepping process of kinesin, in order to gain a complete more understanding of how it walks. We are improving our analysis methods to better resolve precise chemical details within these structures. Finally, we are extending our approach to understand how a pair of myosin molecules can walk along the actin filament. Results of our studies are expected to aid the development of a new generation of pharmaceutical agents for treating cancer and a wide variety of other diseases.

Public Health Relevance

The kinesin and myosin motor proteins are involved in nearly every step in mitosis and cytokinesis, a trait that has led to their targeting by pharmaceutical compounds for the treatment of cancer and a variety of other life-threatening diseases. By studying the detailed chemical structures of kinesin and myosin as these molecules `walk' along their molecular trackways, our studies will give insights into how existing inhibitors of these motors work, and facilitate efforts to design new inhibitors. Results of our studies will also aid in better understanding illnesses related to defects in myosin and kinesin function, such as cardiomyopathy, spastic paraplegia and others.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Ainsztein, Alexandra M
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Yale University
Schools of Medicine
New Haven
United States
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Mentes, Ahmet; Huehn, Andrew; Liu, Xueqi et al. (2018) High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing. Proc Natl Acad Sci U S A 115:1292-1297
Iwamoto, Daniel V; Huehn, Andrew; Simon, Bertrand et al. (2018) Structural basis of the filamin A actin-binding domain interaction with F-actin. Nat Struct Mol Biol 25:918-927
Huehn, Andrew; Cao, Wenxiang; Elam, W Austin et al. (2018) The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments. J Biol Chem 293:5377-5383
Elam, W Austin; Cao, Wenxiang; Kang, Hyeran et al. (2017) Phosphomimetic S3D cofilin binds but only weakly severs actin filaments. J Biol Chem 292:19565-19579
Liu, Daifei; Liu, Xueqi; Shang, Zhiguo et al. (2017) Structural basis of cooperativity in kinesin revealed by 3D reconstruction of a two-head-bound state on microtubules. Elife 6:
Sindelar, Charles V; Liu, Daifei (2017) Tracking Down Kinesin's Achilles Heel with Balls of Gold. Biophys J 112:2454-2456
Bell, Kayla M; Cha, Hyo Keun; Sindelar, Charles V et al. (2017) The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner. J Biol Chem 292:14680-14694
Wang, Jing; Li, Feng; Bello, Oscar D et al. (2017) Circular oligomerization is an intrinsic property of synaptotagmin. Elife 6:
Zanetti, Maria N; Bello, Oscar D; Wang, Jing et al. (2016) Ring-like oligomers of Synaptotagmins and related C2 domain proteins. Elife 5:
Sindelar, Charles; Huehn, Andrew (2016) Vinculin: An Unfolding Tale. J Mol Biol 428:1-4

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