I Like to Move It, Move It: The Role of Kif3B of 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 Function of Kinesin II in Primary Cilia Structure of Kif3B 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 3B6U.pbd structures by motor that travel along in the cilia. The Kinesin II Tip of primary cilium ATP binding motor 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 motility.

Inactive Inactive Kinesin II Microtubule 3B6U.pbd Function of Primary Cilia Dynein Kinesin II Cargo 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 Primary (nonmotile) cilia are structures found on almost every vertebrate cell that region involved in binding ATP is shown in purple and the neck region important function in cell signaling rather than the cell movement associated with motile cilia. Until Based on http://www.nature.com/nrm/journal/v12/n4/fig_tab/nrm3085_F2.html for movement along the microtubule is shown in deep pink. ADP is displayed in the 1990s, primary cilia were considered to be vestigial structures, but more recent yellow ball and stick format. Fig. 3: Kinesin II (blue) carries its cargo along the microtubule from the base toward research implicates these structures in development, proliferation, homeostasis and the tip of the primary cilium, while dynein (red), another motor protein, moves maintenance of the differentiated state. Malfunctioning primary cilia can result in Amino acid residues 96-104 are part of the ATP binding pocket in Kif3B. Binding materials in the opposite direction. diseases known as ciliopathies. 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 Primary cilia are structures used for communication between cells. Primary cilia Kif3A and for movement along the microtubule. do not have of their own, thus materials need to be transported into Structure of Primary Cilia and out of the cilia by motor proteins in order for primary cilia to grow and develop. A B microtubule doublet 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 Primary cilia involved in IFT. 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 Fig. 6: Knockdown of Kif3B in zebrafish A is accomplished by injecting embryos Kinesin II with Kif3B morpholino, a molecule that B.A. Link, unnpublished data http://www.kidneyresearchcenter.org/images/cysts_cilia_02-w197.jpg suppresses the expression of Kif3B ATP Fig. 1A: Electron micrograph showing Fig. 1B: Cross section of primary B protein. (A) Wild type embryo. (B) primary cilia protruding from the cell cilium displaying the ringed ADP Decreased Kif3B expression results in the curved body axis phenotype typical of membrane of neuroepithelial cells in arrangement of nine microtubule Inorganic P the brain of zebrafish embryos 30 pairs that make up the central cilia mutants, suggesting Kif3B is hours post fertilization (hpf). structural core (the axoneme). important for primary cilia formation. Motor domains

M.I. Willardsen, unpublished data Location of Primary Cilia Neck linker

A B C Microtubule Importance

Kif3B is vital in the proper functioning of the Kinesin II. Without Kinesin II, primary Fig. 4: Binding and hydrolysis of ATP (orange) and release of ADP (yellow) result in cilia would not be able to function in cell signaling. Some ciliopathes that are caused conformational changes in the motor protein domains (blue) and neck linker regions by malfunctioning primary cilia include Bardet-Biedl syndrome (BBS), Joubert (pink) of Kinesin II. The motor domains of Kinesin II (Kif3A and Kif3B) bind to the syndrome, and MORM (mental retardation, obesity, retinal dystrophy and micropenis) microtubule if ADP is not bound. Binding of ATP to the motor domain causes the syndrome. These diseases usually leave the individual with mental retardation, neck linker region to change orientation thus rotating the complex. Hydrolysis of ATP obesity problems, vision impairment, and other abnormalities. Understanding the results in the motor domain disconnecting from the microtubule. structure and mechanisms of Kif3B within Kinesin II is important in finding the M.I. Willardsen, unpublished data treatment for these diseases. Kinesin II is one of the motor protein complexes involved in IFT. Kinesin II is Fig. 2: Green fluorescent protein (GFP) labeling the microtubule axoneme of primary composed of three different subunits, Kif3B, Kif3A, and KAP3. Kif3B is one of the References: cilia in live zebrafish embryos 28 hpf. A) Primary cilia in the brain. B,C) Primary cilia in motor proteins in the Kinesin II complex. ATP hydrolysis activates Kif3B, enabling Gennerich, A and Vale, R. 2009. Walking the walk: how kinesin and dynein coordinate their steps. Current Opinion in Cell Biology 21:59-67. the otic (ear) vesicle. Figure 2C is a magnification of the area within the white box in the complex to “walk” along a microtubule and carry its cargo toward the end of the Goetz, S and Anderson, K. 2010. The primary cilium: a signalling centre during vertebrate development. Nature Reviews Genetics 11:331-344. 2B. In each view, the primary cilia are clearly seen protruding from the membrane of primary cilium. Singla, V and Reiter, JF. 2006. The primary cilium as the cell’s antenna: signaling at a sensory . Science 313:629-633. cells throughout the embryo. 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.

A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973). Yildiz, A, et al. 2008. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134:1030-1041.