It Walks The Walk
The St. Dominic School S.M.A.R.T. Team: Jessie Austin, Jenna Brockman, Hannah Brown, Ben Caballero, Ryan Chaffee, Luke Emery, Amanda Hodgson, Finola Hughes,
Jackie Jarosz, Kevin Kohl, Connor Lagore, Chris Malliet, Kerri Jo Mark, Emily Ott, Andrew Rusnak, Mitchell Sauer, Josh Schmirler, Graydon Schroeder, Katie Seim, Aaron
Siehr, Joe Valentyn, Keegan von Estorff, Michael von Estorff, Evan Wetzel.
Teacher: Donna LaFlamme
Mentor: Jason Bader, Ph.D. , Medical College of Wisconsin
How Does Dynein Walk? One Hypothesis [6]
Abstract: Cytoplasmic Dynein
Cytoplasmic dynein is a multi-subunit motor protein
powered by ATP hydrolysis that “walks” along the
microtubules (MTs) of a cell’s cytoskeleton carrying cargo
that is too large to diffuse such as lysosomes, endosomes
and parts of the Golgi complex. With the help of accessory
proteins dynein can transport cell components as large as
the nucleus. During mitosis dynein associates with the
kinetochore of chromosomes and captures spindle MTs so
that chromosomes can be positioned correctly. Because of
this crucial role in cell division, lack of dynein is lethal for
mammalian embryos and death occurs 5-7 days after
fertilization.
Cytoplasmic dynein assembles as a
homodimeric complex consisting of a tail where cargo is
attached and a force producing head known as the motor
domain. The head consists of a motor domain composed of
six AAA ATPase subunits arranged in a ring. In addition,
the head contains two microtubule binding domains
(MTBD’s) which are connected to the motor domain by
coiled coil stalks. The MTBD, stalk, and motor domain
form the “legs” of dynein that walk along microtubules.
The stalks are composed of two anti-parallel alpha helices
that can move relative to each other. Changes in
conformation in the motor domain caused by ATP binding
to the AAA ring are thought to be transmitted along the
stalk to the MTBD causing it to be pulled off the
microtubule while conformational changes in the six
helices of the MTBD upon binding to the microtubule are
thought to be transmitted back along the stalk to the motor
domain readying it for ATP binding.
H1
+
_
Microtubule
H6
MTBD
Stalk
H3
5
Head
4
+ATP
Hydrolysis of ATP
2
Microtubule Binding
Domain (MTBD)
Based on PDB ID: 3ERR [3]
Motor
Domain
+
+
1
_
Power Stroke
Adapted
Adaptedfrom[4]
from [6]
1. Dynein binds to the microtubule strongly when the stalk
is in the α registry (black).
MTBD
Tail
3
Recovery Stroke
ADP +Pi
[5]
(α registry = a conformation of stalk resulting in strong binding).
The Structure of Dynein
•
1
AAA+
Motor Domain
•
2
3
3
Stalk
4
•
MTBD
•
[4]
What is Cytoplasmic Dynein?
2. ATP binds to the head causing the stalk to shift to the
+β registry (orange).
MTBD
(+β registry = the weak-binding conformation of the stalk)
The two identical heavy chains of dynein
each have a head, tail, stalk and a
microtubule binding domain (MTBD).
Each head is a motor domain consisting of
six subunits. Subunits 1 through 4 bind ATP.
ATP binding and hydrolysis in subunit 1 is
required for dynein motility.
The microtubule binding domain (MTBD) is
attached to the motor domain by the stalk.
The stalk allows two way communication
between the MTBD and the motor domain.
Stalk
3. Recovery stroke with bound ATP in the head.
Head
4. ATP hydrolysis in the head causes a shift in the stalk
registry from the +β to the α registry. Dynein binds
again to the microtubule.
5. Release of ADP and Pi from the head triggers the power
stroke. The process repeats.
Dynein Activities During Mitosis.
Dynein helps position and separate chromosomes.
MTBD
Cargo
2
Arg3337
Dynein
Arg3306
H1
H1
H3
Image A [2]
+
Ser3338
]
H6
Image B [1]
Image A: Cytoplasmic dynein, a two headed
molecular motor found in all eukaryotic cells, walks
toward the minus end of microtubules carrying cargo
that are too large to diffuse such as lysosomes, mRNA,
organelles, even viruses. In long cells such as
neurons, dynein can carry its cargo up to one meter.
This would be like a person walking about 40,000 km.
[5]
Image B: The network of microtubules forming the
cytoskeleton of this cell is green. The nucleus is blue.
Dynein carries cargo toward the nucleus where the
minus ends of microtubules are located.
[1] Image of cells with microtubules in green and nucleus in blue. National Institute of General Medical Sciences
[2] R.J. Lye Homepage. University of Virginia Health Sciences Center. (2002)
[3] Primary Citation: PDB ID: 3ERR
A.P. Carter, J.E. Garbarino,E.M. Wilson-Kubalek, W.E. Shipley, C. Cho, R.A. Milligan, R.D. Vale, I.R.Gibbons
Structure and Functional Role of Dynein’s Microtubule-Binding Domain. Science 322 pp 1691-1695 (2008)
[4] Predicted Structure of Dynein. R.D. Vale Cell 112 p 467 (2003)
[6] T.Kon, K. Imamula, A. J Roberts, R. Ohkura, P. J. Knight, I. R. Gibbons,S. A. Burgess & K. Sutoh
Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding.
Nature Structural &Molecular Biology 16 pp325-333 (2009)
H6
H3
Glu3304
Lys3299
Lys3298
Arg3382
α
[8] [8]
β
_
α
Microtubule
1.
2.
3.
Adapted from [3].
4.
Microtubule Binding Domain
Helices 1, 3, and 6 have amino
acid residues that when mutated
to alanine eliminate dynein’s
ability to bind microtubules. [3]
Dynein’s MTBD docked on a
microtubule protofilament.
Helix 1 and Helix 3 interact with
the microtubule between the α
tubulin and β tubulin monomers.
[3]
[5] David Bradley. Cell 3 pp 485-495 (2009[6] T. Kon, K. Imamula, A. J Roberts, R. Ohkura, P. J. Knight, I. R
Gibbons,S. A. Burgess & K. Sutoh
Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding.
Nature Structural &Molecular Biology 16 pp325-333 (2009)
[7] D. J. Sharp, G. C. Rogers and J. M. Scholey
Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila
Embryos. Nature Cell Biology p 923 (2000)
[8] Jason Bader, Medical College of Wisconsin
3
4
Dynein During Metaphase
Anchors microtubules (spindle
fibers) to the cell cortex.
Anchors microtubules by their
minus ends to the spindle poles.
Captures microtubules at the
kinetochores of chromosomes
during prometaphase and helps
position chromosomes.
Silences checkpoint proteins by
pulling them off the kinetochore
allowing mitosis to proceed to
anaphase.
1
[7]
Embryonic Cells at Metaphase
Image a: Microtubules are
greenish yellow. Cytoplasmic
dynein at the each cell’s equator is
bright red. Background red is
associated with each cell’s cortex.
Image b: Dynein on an individual
spindle.
Images c,d,e: Chromosomes are
blue. Microtubules are green. Red
dots on chromosomes are
cytoplasmic dynein associated with
the kinetochores. [7]
What Happens in the Absence of Functioning Dynein?
Mouse embryos lacking cytoplasmic dynein die before implantation
5 to 7 days after fertilization. Embryonic development involves continuous
cell division for which dynein is essential. In humans, the disease
lissencephaly in which the embryonic development of the brain is stunted,
has been associated with the cytoplasmic dynein. Cytoplasmic dynein has
also been associated with neurodegenerative diseases.
SMART Teams are supported by the National Institutes of Health (NIH)- National Center for Research Resources-Science
Education Partnership Award (NCRR-SEPA), and an NIH CTSA Award to the Medical College of Wisconsin.