The molecular motor kinesin-1 performs a large number of transport tasks, and the regulatory mechanisms governing those processes are critical. Mis-regulation of kinesin-1 or mis-localization of kinesin-1 cargoes may be implicated in several diseases such as Parkinson?s disease, neurofibromatosis, schizophrenia, and Charcot-Marie-Tooth disease. Kinesin-1?s motile mechanism is well understood, and we now also know that kinesin-1?s C-terminal tail interacts directly with and inhibits the heads when the motor is not needed for cargo transport. However, we do not know how kinesin-1 regulators initiate or stop cargo movement. The tail is certainly involved, as it binds to heads, microtubules, and several distinct kinesin-1 activators that function in different transport complexes. Separate from the tail, the Miro protein has a direct, Ca2+-dependent interaction with kinesin-1?s enzymatic head domains, and Miro is required for Ca2+-dependent suppression of mitochondrial motility. We hypothesize that the tail is an intrinsically disordered domain, having structural flexibility that facilitates multiple binding partner interactions involved in kinesin-1 auto-inhibition and/or activation, while Miro has a distinct mechanism, directly inhibiting the enzymatic mechanism of kinesin-1 heads to suppress mitochondrial movement. To address this hypothesis, we will first gain detailed information in vitro about the structure of the kinesin-1 tail and its interactions with binding partners, by NMR and EPR spectroscopy. We will determine whether Miro is a direct, Ca2+-switchable inhibitor of kinesin-1?s enzymatic activity, assess its effects on kinesin-1 mechanism using EPR, and map its interaction with kinesin-1 heads by cross-linking. After obtaining this structural and mechanistic information on both the tails and Miro, we will determine whether and how they influence mitochondrial movement by controlling kinesin-1 in vivo, by imaging mitochondria in live Drosophila S2 cells.
These Aims together will provide an exciting new bridge between in vitro biophysical and cell biological work on molecular motor transport mechanisms. Furthermore, as Miro and other kinesin-1 regulators have been implicated in several neurological diseases, our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.

Public Health Relevance

Kinesin-1 motors move many types of intracellular cargoes along microtubules. External binding partners of kinesin-1 control cargo movement in response to several cues, by binding to its head and/or tail regions, but we do not know how they work. We will obtain detailed structural information in vitro on the interactions of the kinesin-1 tail with binding partners, and determine how the Miro protein reversibly binds to kinesin-1 heads in the presence of calcium. We will then determine whether, and how, both tails and Miro control mitochondrial movement by inhibiting kinesin-1 in a calcium-dependent manner in vivo, providing an exciting new bridge between biophysical and cell biological work. Miro and other kinesin-1 regulators have been implicated in several neurological diseases, and our work will provide detailed, relevant biochemical information and reagents that will accelerate efforts to develop therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM072656-06A1
Application #
8187600
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Gindhart, Joseph G
Project Start
2005-09-01
Project End
2015-06-30
Budget Start
2011-09-01
Budget End
2012-06-30
Support Year
6
Fiscal Year
2011
Total Cost
$303,814
Indirect Cost
Name
Northwestern University at Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
French, Michael E; Klosowiak, Julian L; Aslanian, Aaron et al. (2017) Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase. J Biol Chem 292:10398-10413
Park, Sungjin; Foote, Peter K; Krist, David T et al. (2017) UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN. J Biol Chem 292:16539-16553
Klosowiak, Julian L; Park, Sungjin; Smith, Kyle P et al. (2016) Structural insights into Parkin substrate lysine targeting from minimal Miro substrates. Sci Rep 6:33019
Krist, David T; Park, Sungjin; Boneh, Galyah H et al. (2016) UbFluor: A Mechanism-Based Probe for HECT E3 Ligases. Chem Sci 7:5587-5595
Waitzman, Joshua S; Rice, Sarah E (2014) Mechanism and regulation of kinesin-5, an essential motor for the mitotic spindle. Biol Cell 106:1-12
Rice, Sarah (2014) Structure of kif14: an engaging molecular motor. J Mol Biol 426:2993-6
Landahl, Eric C; Rice, Sarah E (2013) Model-independent decomposition of two-state data. Phys Rev E Stat Nonlin Soft Matter Phys 88:062713
Klosowiak, Julian L; Focia, Pamela J; Chakravarthy, Srinivas et al. (2013) Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro. EMBO Rep 14:968-74
Seeger, Mark A; Rice, Sarah E (2013) Intrinsic Disorder in the Kinesin Superfamily. Biophys Rev 5:
Seeger, Mark A; Zhang, Yongbo; Rice, Sarah E (2012) Kinesin tail domains are intrinsically disordered. Proteins 80:2437-46

Showing the most recent 10 out of 24 publications