Download Abstract Importance Structure of Primary Cilia A B Functional Kif3B

I Like to Move It, Move It:
The Role of Kif3B of Kinesin II in Primary Cilia
Cedarburg SMART Team: Kyle Kohlwey, Meredith Kuhn, Nicole Lang, Kathryn Tiffany, Laura Tiffany,Jacqueline Albrecht, Sarah Clapp, Kayla Fenton,
Austin Gallogly, and Rebecca Jankowski
Teacher: Karen Tiffany
Mentor: Minde Willardsen, Ph.D., Medical College of Wisconsin
Abstract
Structure of Kif3B
Function of Kinesin II in Primary Cilia
Primary cilia are structures found on the surface of most cells and are important for cell
signaling. For cilia to develop properly, materials must be transported in and out of these
structures by motor proteins that travel along microtubules in the cilia. The Kinesin II
motor protein transports cargo toward the tip of the cilia. Our interest lies in a motor
subunit of Kinesin II, Kif3B. Kif3B binds to both ATP and microtubules; hydrolysis of ATP
causes Kif3B to change its shape and move up the microtubules. Cilia development
depends on the movement of materials into the cilia, and research indicates that if Kif3B
is not functioning, cilia formation will not occur properly. Diseases called ciliopathies
result if primary cilia production is altered. These diseases include Bardet-Biedl
syndrome (BBS), Joubert syndrome, and MORM syndrome. Using 3D printing
technology, the Cedarburg SMART (Students Modeling a Research Topic) Team has
designed a model of Kif3B to investigate the interaction of amino acid residues 96
through 104 of Kif3B with ATP and to visualize the neck region in Kif3B important for
dimerization with Kif3A and for microtubule motility.
Function of Primary Cilia
Primary (nonmotile) cilia are structures found on almost every vertebrate cell that
function in cell signaling rather than the cell movement associated with motile cilia. Until
the 1990s, primary cilia were considered to be vestigial structures, but more recent
research implicates these structures in development, proliferation, homeostasis and
maintenance of the differentiated state. Malfunctioning primary cilia can result in
diseases known as ciliopathies.
Structure of Primary Cilia
B
A
microtubule doublet
Primary cilia
3B6U.pbd
Tip of primary cilium
Inactive Dynein
Inactive Kinesin II
Microtubule
Dynein
Kinesin II
Cargo
Based on http://www.nature.com/nrm/journal/v12/n4/fig_tab/nrm3085_F2.html
Fig. 3: Kinesin II (blue) carries its cargo along the microtubule from the base toward
the tip of the primary cilium, while dynein (red), another motor protein, moves
materials in the opposite direction.
Primary cilia are structures used for communication between cells. Primary cilia
do not have organelles of their own, thus materials need to be transported into
and out of the cilia by motor proteins in order for primary cilia to grow and develop.
These motor proteins move along the microtubules within the cilia in a process
called intraflagellar transport (IFT). Kinesin II and dynein are two motor proteins
involved in IFT.
ATP binding
3B6U.pbd
Fig. 5: A backbone model of Kif3B, one of the motor domains of Kinesin II, shows
both alpha helical (cyan) and beta sheet (lime green) secondary structure. The
region involved in binding ATP is shown in purple and the neck region important
for movement along the microtubule is shown in deep pink. ADP is displayed in
yellow ball and stick format.
Amino acid residues 96-104 are part of the ATP binding pocket in Kif3B. Binding
of ATP/ADP is important for the conformational and orientation changes within
the protein that allow the motor protein to “walk” along the microtubule. The neck
linker region (amino acid residues 309-363) are important for dimerization with
Kif3A and for movement along the microtubule.
Functional Kif3B is necessary for the proper
functioning of primary cilia. Primary cilia are
required for normal development.
Basal
Body
Role of ATP Binding and Hydrolysis in the Motion of
Kinesin II
A
http://www.kidneyresearchcenter.org/images/cysts_cilia_02-w197.jpg
B.A. Link, unnpublished data
ATP
Fig. 1A: Electron micrograph showing
primary cilia protruding from the cell
membrane of neuroepithelial cells in
the brain of zebrafish embryos 30
hours post fertilization (hpf).
Fig. 1B: Cross section of primary
cilium displaying the ringed
arrangement of nine microtubule
pairs that make up the central
structural core (the axoneme).
B
B
ADP
Inorganic P
Motor
domains
M.I. Willardsen, unpublished data
Location of Primary Cilia
A
Fig. 6: Knockdown of Kif3B in zebrafish
is accomplished by injecting embryos
with Kif3B morpholino, a molecule that
suppresses the expression of Kif3B
protein. (A) Wild type embryo. (B)
Decreased Kif3B expression results in
the curved body axis phenotype typical of
cilia mutants, suggesting Kif3B is
important for primary cilia formation.
Neck linker
C
Microtubule
Importance
Fig. 4: Binding and hydrolysis of ATP (orange) and release of ADP (yellow) result in
conformational changes in the motor protein domains (blue) and neck linker regions
(pink) of Kinesin II. The motor domains of Kinesin II (Kif3A and Kif3B) bind to the
microtubule if ADP is not bound. Binding of ATP to the motor domain causes the
neck linker region to change orientation thus rotating the complex. Hydrolysis of ATP
results in the motor domain disconnecting from the microtubule.
M.I. Willardsen, unpublished data
Fig. 2: Green fluorescent protein (GFP) labeling the microtubule axoneme of primary
cilia in live zebrafish embryos 28 hpf. A) Primary cilia in the brain. B,C) Primary cilia in
the otic (ear) vesicle. Figure 2C is a magnification of the area within the white box in
2B. In each view, the primary cilia are clearly seen protruding from the membrane of
cells throughout the embryo.
Kinesin II is one of the motor protein complexes involved in IFT. Kinesin II is
composed of three different subunits, Kif3B, Kif3A, and KAP3. Kif3B is one of the
motor proteins in the Kinesin II complex. ATP hydrolysis activates Kif3B, enabling
the complex to “walk” along a microtubule and carry its cargo toward the end of the
primary cilium.
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).
Kif3B is vital in the proper functioning of the Kinesin II. Without Kinesin II, primary
cilia would not be able to function in cell signaling. Some ciliopathes that are caused
by malfunctioning primary cilia include Bardet-Biedl syndrome (BBS), Joubert
syndrome, and MORM (mental retardation, obesity, retinal dystrophy and micropenis)
syndrome. These diseases usually leave the individual with mental retardation,
obesity problems, vision impairment, and other abnormalities. Understanding the
structure and mechanisms of Kif3B within Kinesin II is important in finding the
treatment for these diseases.
References:
Gennerich, A and Vale, R. 2009. Walking the walk: how kinesin and dynein coordinate their steps. Current Opinion in Cell Biology 21:59-67.
Goetz, S and Anderson, K. 2010. The primary cilium: a signalling centre during vertebrate development. Nature Reviews Genetics 11:331-344.
Singla, V and Reiter, JF. 2006. The primary cilium as the cell’s antenna: signaling at a sensory organelle. Science 313:629-633.
Yamazaki, H, et al. 1995. KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for
membrane organelle transport. Journal of Cell Biology 130(6):1387-1399.
Yildiz, A, et al. 2008. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134:1030-1041.