In collaboration with Philipp Kukura of Oxford University, we have used a light microscopy based interferometric scattering technique to examine the processive movement of myosin 5 HMM on actin. Using this technique we were able to image single, unlabeled molecules of myosin 5 HMM move along actin with a precision of a few nanometers. The molecule took 36 nm steps and moved at the same speed as previously reported for fluorescently-labeled myosin 5. By attaching a 20 nm gold particle to the amino-terminus we are able to measure the movement at sampling rates up to 1000 Hz and follow the movement of the unattached labeled myosin head. Interesting, even with a 20 nm gold particle attached the myosin moves at the same velocity as the unlabeled molecule. The unlabeled head does not freely diffuse as previously proposed, but rather spends most of its time in a fixed, off actin, position from which it occasionaly explores the forward actin binding sites. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. More detailed observations of the step sizes reveals that the technique can precisely detect how many actin monomers are spanned during each step which allows us to examine the effect of head-head spacing on the stepping kinetics. In collaboration with the lab of Tom Friedman we have examined the molecular properties of a myosin-15 bearing a deafness associated mutation. We have expressed and purified myosin-5B and have measured the ATPase and in vitro motility of this protein at NIH and have collaborated with Marco Capitanio's lab in Italy measuring the mechanical properties of this myosin in an optical trap. We show that imposed strain, either assisting or resistive lowers the run length of the molecule. We have begun to analyze the structure and stepping pattern of myosin-6, a processive myosin which moves in the opposite direction on actin compared to other myosins. EM studies show that the angle between the two heads is more variable than in most myosins and that when bound to actin, the motors can be spaced at 13 actins (preferred) or 11 or 15 actin monomers apart. Optical trapping and EM studies demonstrate that the molecule can sometimes take an inchworm like step where the two heads occupy closely spaced binding sites.

Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
2017
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
Zip Code
Gardini, Lucia; Heissler, Sarah M; Arbore, Claudia et al. (2018) Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level. Nat Commun 9:2844
Zheng, Wei; Wu, Yicong; Winter, Peter et al. (2017) Adaptive optics improves multiphoton super-resolution imaging. Nat Methods 14:869-872
Heissler, Sarah M; Chinthalapudi, Krishna; Sellers, James R (2017) Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease. J Biol Chem 292:18372-18385
Heissler, Sarah M; Sellers, James R (2016) Various Themes of Myosin Regulation. J Mol Biol 428:1927-46
Martin, Andreas; Berger, James M; Sellers, James R et al. (2016) Mechanisms and Functional Diversity of Macromolecular Remodeling by ATP-Dependent Motors. J Mol Biol 428:1819-21
Heissler, Sarah M; Sellers, James R (2016) Kinetic Adaptations of Myosins for Their Diverse Cellular Functions. Traffic 17:839-59
Kengyel, András; Bécsi, Bálint; Kónya, Zoltán et al. (2015) Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity. Eur Biophys J 44:207-18
Bond, Lisa M; Sellers, James R; McKerracher, Lisa (2015) Rho kinase as a target for cerebral vascular disorders. Future Med Chem 7:1039-53
Andrecka, Joanna; Ortega Arroyo, Jaime; Takagi, Yasuharu et al. (2015) Structural dynamics of myosin 5 during processive motion revealed by interferometric scattering microscopy. Elife 4:
Ortega Arroyo, J; Andrecka, J; Spillane, K M et al. (2014) Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett 14:2065-70

Showing the most recent 10 out of 19 publications