Summer Translational Cardiovascular Science Institute (STSCI)

Research Experience for Undergraduates

Characterizing Dynein A Pivotal

Molecular Motor In Cardiovascular
Development and Health
Thomas Randolph Blanda
Advisor: Dr. William O. Hancock
Department of Biomedical Engineering
The Pennsylvania State University, University Park, PA 16802
Introduction
Background:
1% of US children (~36,000) affected yearly by congenital heart
defects (Garg, 2006).
Examples: Tetralogy of Fallot, Situs Ambiguous

Figure 1: Schematic of Possible Situs Variations (Omran, 2017)

 Introduction
Background continued:
Congenital heart defects are developmental diseases, cilia responsible
for asymmetrical development (Li, Klena, Gabriel, et al. 2015).
Dynactin (green) GFP linker

BICD2 (orange)
Tail region

Heads

Figure 2: Ventral View of Monocilia on the Mouse Node in Early Gastrulation (Wood, 2005)
Stalks

Figure 3: Animated Representation of Cytoplasmic Dyneins (DDB) Structure (Hancock Lab Feng)
Introduction
Background continued:
Dynein (along with other molecular motors) are responsible for the
localization of mitochondria, which are crucial to cardiac myocyte
function (Rosenberg, 2004) (Ong, Hall, and Hausenloy, 2013).

Figure 4: Microtubule-dependent Motor-driven Mitochondrial Transport (Cai and Tammineni, 2016)

Methods
Imaging:
Rhodamine labeled tubulin polymerized to make fluorescent
microtubules when excited with green light.
Placed in flow cell with motors and imaged with upright microscope.
Fluorescence emitted to detector Green laser input

25 nm
Volume ~ 10-15 L

Figure 5: Schematic of Gliding Assay (Hancock Lab Blanda)

 Methods
Image Processing
Videos analyzed using ImageJ, FIESTA, and MATLAB

Mean: 506.97 nm/s

SD: 94.23 nm/s
N=280
(+1 )2 +(+1 )2
=
+1

Velocity nm/s
Figure 6: FIESTA Tracking of Filament Movement for Kinesin 1 (Hancock Lab Blanda) Figure 7: Kinesin 1 Velocity Distribution (Hancock Lab Blanda)
Results
Experiments done in limiting ATP ~ 50 M
First attempt: Dynein done alone, no landing events + no motility
Kinesin1 rigor + dynein, landing events + no motility (Faulty motor?)
Second attempt after new dynein prep:

Mean: 59.53 nm/s

SD: 36.84 nm/s
N=82

Figure 8: Dynein Velocity Distribution (Hancock Lab Blanda)

Conclusions
Dynein is an unstable motor
Can only function in low ATP
Possibly does not store well in -80C
Low ATP velocity = 60 nm/s
Agrees with previous motility assay velocities (Feng, unpublished; Reck-
Peterson, et al. 2006)
Future Directions
Tug-of-war model is important for understanding of antagonistic
motor behavior (kinesin vs. dynein).

Experiments done previously bear reasoning to a certain

codependence of these motors, specifically dyneins dependence on
kinesin for stability.

Figure 9: The Tug-Of-War Model (Hancock, 2014)

Figure 10: Three Hypothetical Mechanisms for Resolving the Paradox of Codependence (Hancock, 2014)
Future Directions Quantum dot
(for imaging)

Mixed motor assays

Repeat kin1 rigor vs. dynein DNA linker
Active kin1 vs. dynein

Kinesin
DNA origami linked motors (Figure 11)

Single molecule experiments

DDB complex

Figure 11: DDB-Kin1 DNA Origami Construct (Hancock Lab

Microtubule Feng)
Acknowledgements
American Heart Association
Grant #: 16UFEL27930008

Advisor: William O. Hancock

Notable Graduate Student(s): Qingzhou Feng, Keith Mickolajczyk

Works Cited
Cai, Qian, and Prasad Tammineni. Microtubule-dependent Motor-driven Mitochondrial Transport. Digital image. ResearchGate. Frontiers in Cellular
Neuroscience, 9 Feb. 2016. Web. 21 July 2017.
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Reck-Peterson, Samara L., Ahmet Yildiz, Andrew P. Carter, Arne Gennerich, Nan Zhang, and Ronald D. Vale. "Single-Molecule Analysis of Dynein
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Rosenberg, Paul. "Mitochondrial Dysfunction and Heart Disease." Mitochondrion 4.5-6 (2004): 621-28. ScienceDirect. Web. 21 July 2017.
Wood, William B. Ventral View of Monocilia on the Mouse Node in Early Gastrulation. Digital image. PubMed. PLOS Biology, 3 Aug. 2005. Web. 21 July
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