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Synthesis of Fluorescent-NTA and its Application to the Labeling of Photo-Controlled Kinesin Eg5
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Tuesday, March 1, 2016
into how these two motors adapt their enzymologies for their distinct functions. 2265-Pos Board B409 Regulation and Possible Physiological Role of BI-Directional Motility of the Kinesin-5 Cin8 Ofer Shapira1, Alina Goldstein1, Jawdat Al-Bassam2, Larisa Gheber1. 1 Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2 Molecular Cell Biology, UC Davis, Davis, CA, USA. The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclindependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear. Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its directionality from fast minus- to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end directionality. 2266-Pos Board B410 Trapping the Transition State of Kinesin-5 Produces a Different Multimotor Force Outcome than Inhibiting Product Release Minmin Luo, Edward Wojcik, Sunyoung Kim. Biochemistry and Molecular Biology, LSU School of Medicine and Health Sciences Center, New Orleans, LA, USA. Kinesin-5 (Eg5) is an essential mitotic motor that works in concert with multiple motors to form the bipolar spindle. Acute interest in the human kinesin-5 stems from the potential to target it for clinical therapy; many smallmolecule inhibitors bind to an allosteric loop-5 (L5) pocket, inhibit ADP release, and reduce Eg5 motion to a diffusive mode that does not require ATP hydrolysis. Herein, we show that not all L5-directed drugs have the same catalytic effect nor do they have equivalent impacts upon the motormicrotubule complex. Linear free energy relationships, derived from steady state kinetics measurements of wildtype and eight L5 mutant proteins, show that monastrol is a transition state inhibitor, uniquely through an allosteric mechanism, whereas STC and ispinesib are not. Unexpectedly, in vitro ‘‘tug of war’’ assays between human Eg5 and human Kinesin-1 (KHC) revealed that these two categories of small chemical inhibitors also elicit dramatically different effects on Eg5 motility. Inhibitors of product release allow diffusive microtubule sliding. In contrast, trapping the transition state reveals bimodal behaviour that ultimately causes Eg5 to function as a brake that resists microtubule sliding by KHC. We conclude that ADP state inhibition of Eg5 allows the other kinesin forces on the microtubule to be unchecked while inhibition at the transition state of Eg5 hinders the entire multi-motor ensemble. It is possible that these disparate force outcomes are the result of variation in how drug inhibition at different catalytic intermediates can affect inter-head communication both within Eg5 and between motors in the ensemble. Our work highlights that not all L5 directed drugs are equal, and suggests kinesin inhibitors trapping the transition state should be further explored for their therapeutic potential. 2267-Pos Board B411 Non-Canonical Microtubule Interaction by Yeast Kinesin-5, Cin8 Kayla M. Bell1, Hyokeun Cha2, Charles V. Sindelar3, Jared C. Cochran1. 1 Molecular and Cellular Biochemistry, Indiana University, Bloomington, Bloomington, IN, USA, 2Cell Biology, Yale University, New Haven, CT, USA, 3Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Kinesin-5’s were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 351 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural ˚ cryo-EM explanation for these biochemical observations by obtaining a 7A reconstruction of the microtubule-Cin8 complex in the ADPþAlFx state. Deletion of the large insert in Cin8’s loop L8 retains similar steadystate ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8’s microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching. 2268-Pos Board B412 High-Resolution Cryo-EM Studies on the Yeast Mitotic Kinesin-5 Hyo Keun Cha1, Kayla Bell2, Jared Cochran2, Charles Sindelar3. 1 Cell Biology, Yale University, New Haven, CT, USA, 2Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA, 3 Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. The kinesin-5 family is required for the assembly and maintenance of the bipolar organization of microtubules in the mitotic spindle. Recently, the yeast kinesin-5 (Cin8) was shown to exhibit unusual directional properties. Previously, all kinesins were thought to act exclusively as plus-end or minus-end directed, depending on the location of the motor domain. Despite possessing an N-terminal motor like conventional kinesin-1, the yeast kinesin5 Cin8 acts as a minus-end directed motor when working individually on single microtubules. Furthermore, Cin8 is the only known kinesin with the ability to switch directions when working in a team crosslinking two antiparallel microtubules. Thus, the accumulation of Cin8 at the spindle poles located at the minus-end is thought to trigger a directional switch to plusend directed motility, which is required for the bidirectional sliding of antiparallel microtubules. Deletion of a unique insert in loop 8 (L8) found only in Cin8 induces a strong bias towards minus-end directed motility, suggesting an important role in enforcing the switch to plus-end directed. To investigate the mechanistic basis for the unusual properties of Cin8, we are using cryoEM, which uniquely allows for high-resolution 3D reconstructions of Cin8 ˚ reconstruction when bound to microtubules. Initial efforts have yielded a ~7A in the ADP-AlFx state, which revealed Cin8-specific differences in the domain structure, such as the extended L8. We are pursuing higher resolution structures of this and other nucleotide states in order to reveal how Cin8 is able to achieve minus-end directed motility despite being an N-terminal kinesin. 2269-Pos Board B413 Synthesis of Fluorescent-NTA and its Application to the Labeling of PhotoControlled Kinesin Eg5 Yuki Tamura, Kei Sadakane, Kentaro Saido, Ryoma Yamamoto, Shinsaku Maruta. Dept.Bioinfo., Fac.Eng., Soka University, Hachioji, Japan. All members of the kinesin superfamily contain a structurally conserved loop L5 near the ATP-binding site. The length and the amino acids composition of L5 vary among the kinesin superfamily members. It is believed that L5 may be related to the enzymatic and motor function of kinesin. Interestingly, L5 of Eg5 is uniquely longer than that of other kinesins. Moreover, it was demonstrated that this loop undergoes conformational changes that are related to the nucleotide binding and neck linker docking. Previously we attempted to control Eg5 function photo-reversibly by incorporating photochromic
Tuesday, March 1, 2016 molecules into the L5. We prepared Eg5 mutants, E116C, E118C, T125C, W127C, D130C, which have a single cysteine residue in L5 in order to incorporate photochromic molecules specifically. The Eg5 mutants modified with azobenzene and spiropyran derivatives showed photo-reversible alteration of microtubules dependent ATPase activities. In this study, we synthesized a novel thiol reactive photochromic molecule, monoiodoacetyl-flugide (IAFG). Fulgimide performs photo-reversible isomerization between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light, respectively. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Although the modified Eg5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photo-reversible alteration upon UV and visible light irradiations. We utilized fluorescent probe bound NTA-Ni to label the His-tagged Eg5 modified with photochromic molecules. According to the methods of Soh et al., Dansyl-NTA and other fluorescent-NTA were synthesized and conjugated with Ni2þ. The Dansyl-NTA-Ni2þ bound to His tagged Eg5. Using the fluorescent NTA-Ni2þ, interaction of photo-regulated Eg5 with microtubules was monitored. 2270-Pos Board B414 Understanding the Sequence of Chemomechanical Transitions in Kinesin-5 Geng-Yuan Chen, William O. Hancock. Biomedical Engineering, State College, PA, USA. The kinesin-5 KSP/Eg5 pauses at microtubule plus-ends and enhances tubulin polymerization by increasing the growth rate and decreasing the catastrophe rate (Chen et al. 2015). The end pausing resembles that of the kinesin-8, kip3p, but has the opposite effect on microtubule dynamics. Upon initial binding to the microtubule lattice, kinesin-5 was reported to pause for 1-second pause before stepping (Krzysiak et al. 2008). However, this ‘‘initial pause’’ predicted from biochemical experiments have not been observed in our single-molecule motility experiments. Compared to transport kinesins, kinesin-5 is less processive but paradoxically less loaddependent, suggesting that kinesin-5 may use different gating mechanisms during processive walking. We measured a kcat of 12 s-1 for the kinesin-5 monomer ATPase and a K0.5 of 0.9 mM, which generates a kbiATPase of 13 mM-1 s-1. The bimolecular microtubule-encounter rate kbiADP was 9 mM-1 s-1 which predicts a monomer chemical processivity of kbi ratio ~1.4. This suggests that for each ATP turnover, kinesin-5 monomer readily detaches from the microtubule. The ADP off-rate of the bound head was 40 s-1, consistent with rate limiting hydrolysis. In half-site ADP release experiments with processive dimers, we measured maximal rates of 13 s-1 in ATP and 2.1 s-1 in ATPgS, which matched the single-molecule stepping rate in each nucleotide, suggesting that hydrolysis precedes attachment of the tethered head, consistent with recent work on kinesin-1. Hence, we propose that kinesin-5 processivity is gated by the race between detachment of the bound-head in the ADP-Pi state and attachment of the tethered-head. This study provides insights into gating mechanism with respect to processivity on the microtubule wall and in the possible strong-binding states at the microtubule plus-end. 2271-Pos Board B415 Three-Dimensional Motility of the Highly Processive Kinesin-8 Along the Microtubule Lattice Aniruddha Mitra1,2, Felix Ruhnow1,2, Salvatore Girardo3, Diez Stefan1,4. 1 B CUBE – Center for Molecular Bioengineering, Technische Universita¨t Dresden, Dresden, Germany, 2Center for Advancing Electronics Dresden, Technische Universita¨t Dresden, Dresden, Germany, 3BIOTEC, Biotechnology Center Technische Universia¨t Dresden, Dresden, Germany, 4 Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. During mitosis, kinesin-8 motors play a key role in regulating the spindle length based to their depolymerization activity at microtubule plus-ends. In order to reach the plus-ends of microtubules, kinesin-8 motors show superprocessive motility. However, the mechanism confering such high motor processivity even on crowded microtubules in the cytoplasm is not well understood. To gain insight into the stepping mechanism of kinesin-8, we explored the three-dimensional motility of yeast kinesin-8, Kip3, along the microtubule lattice in vitro. First, we performed microtubule gliding motility assays on surface coated with Kip3 motors and measured the rotations of gliding microtubules around their longitudinal axis using rhodamine speckled microtubules in combination with fluorescence-interference contrast microscopy [1]. We observed rotations with periodicities significantly smaller than the microtubule supertwist, indicating that the motors do not follow individual protofilaments but rather switch protofilaments stochastically in one direction [2]. Finally, we
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confirmed this hypothesis by 3D single-molecule stepping assays along freely suspended microtubules by 2D tracking of QDot-conjugated Kip3 motors in combination with parallax [3], a dual-focus imaging technique that provides nanometer information in z-direction. 1. Mitra A, Ruhnow F, Nitzsche B, Diez S. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces. PLoS One. 2015. 2. Bormuth V, Nitzsche B, Ruhnow F, Mitra A, Storch M, Rammner B, Howard J, Diez S. The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left. Biophys J. 2012. 3. Sun Y, McKenna JD, Murray JM, Ostap EM, Goldman YE. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 2009. 2272-Pos Board B416 Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends Benjamin C. Walker, Caleb A. Starr, Jared C. Cochran. Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10’s are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10’s work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (~21 mM) and physiologically relevant (~111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10’s in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFPtagged kinesin-10’s and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division. 2273-Pos Board B417 Kinetic Characterization of Novel Rice Plant Kinesin E11 Hironobu Taniguchi1, Naoto Inomoto2, Shinsaku Maruta1. 1 Div.Bioinfo., Soka Univ, Tokyo, Japan, 2Dep.Bioinfo., Soka Univ, Tokyo, Japan. Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures. In this study, we characterized biochemical and kinetic properties of another rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E.coli expression system and purified with Co-chelate column. The fluorescent ATP analogue, Mant-ATP was employed for the kinetic study. We observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters were analyzed by monitoring the FRET using stopped flow apparatus. The binding rate and dissociation rate were measured, and compared with those of other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are faster than those of other rice plant specific kinesin. We also examined the motility activity of E11 with in vitro microtubule gliding assay. The motility assay revealed that E11 has relatively higher motor activity than other rice kinesins.
Tuesday, March 1, 2016
into how these two motors adapt their enzymologies for their distinct functions. 2265-Pos Board B409 Regulation and Possible Physiological Role of BI-Directional Motility of the Kinesin-5 Cin8 Ofer Shapira1, Alina Goldstein1, Jawdat Al-Bassam2, Larisa Gheber1. 1 Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel, 2 Molecular Cell Biology, UC Davis, Davis, CA, USA. The homoterameric bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. S. cerevisiae cells express two kinesin-5s Cin8 and Kip1, which overlap in function. We have recently demonstrated that Cin8 and Kip1 are minus-end directed on the single-molecule level and can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). The mechanism of this directionality switch and its physiological significance remain unclear. We have also demonstrated that Cin8 is differentially phosphorylated during late anaphase at three cyclindependent kinase 1 (Cdk1) sites located in its motor domain. This phosphorylation regulates Cin8 activity during anaphase (Avunie-Masala et al., 2011), but its mechanism remains unclear. Here we examined the in vitro motile properties and in vivo functions of Cin8 by TIRF microscopy and live-cell imaging. We found that addition of negative charge in a phospho-mimic Cin8 mutant weakens the MT-motor interaction and regulates the motile properties and directionality of Cin8. We also found that of the three Cdk1 sites in the catalytic domain of Cin8, the S277 site contributes the most to regulation of Cin8 localization and function during anaphase. Finally, we found that in vitro under high ionic strength conditions, Cin8 not only moves to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 to reverse its directionality from fast minus- to slow plus-end directed motility. Clustering of Cin8 at the minus-end of the MTs serves as a primary site for capturing and antiparallel sliding of MTs. Based on these results, we propose a revised model for activity of Cin8 during mitosis and propose a physiological role for its minus-end directionality. 2266-Pos Board B410 Trapping the Transition State of Kinesin-5 Produces a Different Multimotor Force Outcome than Inhibiting Product Release Minmin Luo, Edward Wojcik, Sunyoung Kim. Biochemistry and Molecular Biology, LSU School of Medicine and Health Sciences Center, New Orleans, LA, USA. Kinesin-5 (Eg5) is an essential mitotic motor that works in concert with multiple motors to form the bipolar spindle. Acute interest in the human kinesin-5 stems from the potential to target it for clinical therapy; many smallmolecule inhibitors bind to an allosteric loop-5 (L5) pocket, inhibit ADP release, and reduce Eg5 motion to a diffusive mode that does not require ATP hydrolysis. Herein, we show that not all L5-directed drugs have the same catalytic effect nor do they have equivalent impacts upon the motormicrotubule complex. Linear free energy relationships, derived from steady state kinetics measurements of wildtype and eight L5 mutant proteins, show that monastrol is a transition state inhibitor, uniquely through an allosteric mechanism, whereas STC and ispinesib are not. Unexpectedly, in vitro ‘‘tug of war’’ assays between human Eg5 and human Kinesin-1 (KHC) revealed that these two categories of small chemical inhibitors also elicit dramatically different effects on Eg5 motility. Inhibitors of product release allow diffusive microtubule sliding. In contrast, trapping the transition state reveals bimodal behaviour that ultimately causes Eg5 to function as a brake that resists microtubule sliding by KHC. We conclude that ADP state inhibition of Eg5 allows the other kinesin forces on the microtubule to be unchecked while inhibition at the transition state of Eg5 hinders the entire multi-motor ensemble. It is possible that these disparate force outcomes are the result of variation in how drug inhibition at different catalytic intermediates can affect inter-head communication both within Eg5 and between motors in the ensemble. Our work highlights that not all L5 directed drugs are equal, and suggests kinesin inhibitors trapping the transition state should be further explored for their therapeutic potential. 2267-Pos Board B411 Non-Canonical Microtubule Interaction by Yeast Kinesin-5, Cin8 Kayla M. Bell1, Hyokeun Cha2, Charles V. Sindelar3, Jared C. Cochran1. 1 Molecular and Cellular Biochemistry, Indiana University, Bloomington, Bloomington, IN, USA, 2Cell Biology, Yale University, New Haven, CT, USA, 3Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Kinesin-5’s were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 351 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural ˚ cryo-EM explanation for these biochemical observations by obtaining a 7A reconstruction of the microtubule-Cin8 complex in the ADPþAlFx state. Deletion of the large insert in Cin8’s loop L8 retains similar steadystate ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8’s microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching. 2268-Pos Board B412 High-Resolution Cryo-EM Studies on the Yeast Mitotic Kinesin-5 Hyo Keun Cha1, Kayla Bell2, Jared Cochran2, Charles Sindelar3. 1 Cell Biology, Yale University, New Haven, CT, USA, 2Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA, 3 Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. The kinesin-5 family is required for the assembly and maintenance of the bipolar organization of microtubules in the mitotic spindle. Recently, the yeast kinesin-5 (Cin8) was shown to exhibit unusual directional properties. Previously, all kinesins were thought to act exclusively as plus-end or minus-end directed, depending on the location of the motor domain. Despite possessing an N-terminal motor like conventional kinesin-1, the yeast kinesin5 Cin8 acts as a minus-end directed motor when working individually on single microtubules. Furthermore, Cin8 is the only known kinesin with the ability to switch directions when working in a team crosslinking two antiparallel microtubules. Thus, the accumulation of Cin8 at the spindle poles located at the minus-end is thought to trigger a directional switch to plusend directed motility, which is required for the bidirectional sliding of antiparallel microtubules. Deletion of a unique insert in loop 8 (L8) found only in Cin8 induces a strong bias towards minus-end directed motility, suggesting an important role in enforcing the switch to plus-end directed. To investigate the mechanistic basis for the unusual properties of Cin8, we are using cryoEM, which uniquely allows for high-resolution 3D reconstructions of Cin8 ˚ reconstruction when bound to microtubules. Initial efforts have yielded a ~7A in the ADP-AlFx state, which revealed Cin8-specific differences in the domain structure, such as the extended L8. We are pursuing higher resolution structures of this and other nucleotide states in order to reveal how Cin8 is able to achieve minus-end directed motility despite being an N-terminal kinesin. 2269-Pos Board B413 Synthesis of Fluorescent-NTA and its Application to the Labeling of PhotoControlled Kinesin Eg5 Yuki Tamura, Kei Sadakane, Kentaro Saido, Ryoma Yamamoto, Shinsaku Maruta. Dept.Bioinfo., Fac.Eng., Soka University, Hachioji, Japan. All members of the kinesin superfamily contain a structurally conserved loop L5 near the ATP-binding site. The length and the amino acids composition of L5 vary among the kinesin superfamily members. It is believed that L5 may be related to the enzymatic and motor function of kinesin. Interestingly, L5 of Eg5 is uniquely longer than that of other kinesins. Moreover, it was demonstrated that this loop undergoes conformational changes that are related to the nucleotide binding and neck linker docking. Previously we attempted to control Eg5 function photo-reversibly by incorporating photochromic
Tuesday, March 1, 2016 molecules into the L5. We prepared Eg5 mutants, E116C, E118C, T125C, W127C, D130C, which have a single cysteine residue in L5 in order to incorporate photochromic molecules specifically. The Eg5 mutants modified with azobenzene and spiropyran derivatives showed photo-reversible alteration of microtubules dependent ATPase activities. In this study, we synthesized a novel thiol reactive photochromic molecule, monoiodoacetyl-flugide (IAFG). Fulgimide performs photo-reversible isomerization between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light, respectively. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Although the modified Eg5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photo-reversible alteration upon UV and visible light irradiations. We utilized fluorescent probe bound NTA-Ni to label the His-tagged Eg5 modified with photochromic molecules. According to the methods of Soh et al., Dansyl-NTA and other fluorescent-NTA were synthesized and conjugated with Ni2þ. The Dansyl-NTA-Ni2þ bound to His tagged Eg5. Using the fluorescent NTA-Ni2þ, interaction of photo-regulated Eg5 with microtubules was monitored. 2270-Pos Board B414 Understanding the Sequence of Chemomechanical Transitions in Kinesin-5 Geng-Yuan Chen, William O. Hancock. Biomedical Engineering, State College, PA, USA. The kinesin-5 KSP/Eg5 pauses at microtubule plus-ends and enhances tubulin polymerization by increasing the growth rate and decreasing the catastrophe rate (Chen et al. 2015). The end pausing resembles that of the kinesin-8, kip3p, but has the opposite effect on microtubule dynamics. Upon initial binding to the microtubule lattice, kinesin-5 was reported to pause for 1-second pause before stepping (Krzysiak et al. 2008). However, this ‘‘initial pause’’ predicted from biochemical experiments have not been observed in our single-molecule motility experiments. Compared to transport kinesins, kinesin-5 is less processive but paradoxically less loaddependent, suggesting that kinesin-5 may use different gating mechanisms during processive walking. We measured a kcat of 12 s-1 for the kinesin-5 monomer ATPase and a K0.5 of 0.9 mM, which generates a kbiATPase of 13 mM-1 s-1. The bimolecular microtubule-encounter rate kbiADP was 9 mM-1 s-1 which predicts a monomer chemical processivity of kbi ratio ~1.4. This suggests that for each ATP turnover, kinesin-5 monomer readily detaches from the microtubule. The ADP off-rate of the bound head was 40 s-1, consistent with rate limiting hydrolysis. In half-site ADP release experiments with processive dimers, we measured maximal rates of 13 s-1 in ATP and 2.1 s-1 in ATPgS, which matched the single-molecule stepping rate in each nucleotide, suggesting that hydrolysis precedes attachment of the tethered head, consistent with recent work on kinesin-1. Hence, we propose that kinesin-5 processivity is gated by the race between detachment of the bound-head in the ADP-Pi state and attachment of the tethered-head. This study provides insights into gating mechanism with respect to processivity on the microtubule wall and in the possible strong-binding states at the microtubule plus-end. 2271-Pos Board B415 Three-Dimensional Motility of the Highly Processive Kinesin-8 Along the Microtubule Lattice Aniruddha Mitra1,2, Felix Ruhnow1,2, Salvatore Girardo3, Diez Stefan1,4. 1 B CUBE – Center for Molecular Bioengineering, Technische Universita¨t Dresden, Dresden, Germany, 2Center for Advancing Electronics Dresden, Technische Universita¨t Dresden, Dresden, Germany, 3BIOTEC, Biotechnology Center Technische Universia¨t Dresden, Dresden, Germany, 4 Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. During mitosis, kinesin-8 motors play a key role in regulating the spindle length based to their depolymerization activity at microtubule plus-ends. In order to reach the plus-ends of microtubules, kinesin-8 motors show superprocessive motility. However, the mechanism confering such high motor processivity even on crowded microtubules in the cytoplasm is not well understood. To gain insight into the stepping mechanism of kinesin-8, we explored the three-dimensional motility of yeast kinesin-8, Kip3, along the microtubule lattice in vitro. First, we performed microtubule gliding motility assays on surface coated with Kip3 motors and measured the rotations of gliding microtubules around their longitudinal axis using rhodamine speckled microtubules in combination with fluorescence-interference contrast microscopy [1]. We observed rotations with periodicities significantly smaller than the microtubule supertwist, indicating that the motors do not follow individual protofilaments but rather switch protofilaments stochastically in one direction [2]. Finally, we
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confirmed this hypothesis by 3D single-molecule stepping assays along freely suspended microtubules by 2D tracking of QDot-conjugated Kip3 motors in combination with parallax [3], a dual-focus imaging technique that provides nanometer information in z-direction. 1. Mitra A, Ruhnow F, Nitzsche B, Diez S. Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces. PLoS One. 2015. 2. Bormuth V, Nitzsche B, Ruhnow F, Mitra A, Storch M, Rammner B, Howard J, Diez S. The highly processive kinesin-8, Kip3, switches microtubule protofilaments with a bias toward the left. Biophys J. 2012. 3. Sun Y, McKenna JD, Murray JM, Ostap EM, Goldman YE. Parallax: high accuracy three-dimensional single molecule tracking using split images. Nano Lett. 2009. 2272-Pos Board B416 Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends Benjamin C. Walker, Caleb A. Starr, Jared C. Cochran. Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA. Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10’s are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10’s work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (~21 mM) and physiologically relevant (~111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10’s in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFPtagged kinesin-10’s and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division. 2273-Pos Board B417 Kinetic Characterization of Novel Rice Plant Kinesin E11 Hironobu Taniguchi1, Naoto Inomoto2, Shinsaku Maruta1. 1 Div.Bioinfo., Soka Univ, Tokyo, Japan, 2Dep.Bioinfo., Soka Univ, Tokyo, Japan. Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures. In this study, we characterized biochemical and kinetic properties of another rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E.coli expression system and purified with Co-chelate column. The fluorescent ATP analogue, Mant-ATP was employed for the kinetic study. We observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters were analyzed by monitoring the FRET using stopped flow apparatus. The binding rate and dissociation rate were measured, and compared with those of other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are faster than those of other rice plant specific kinesin. We also examined the motility activity of E11 with in vitro microtubule gliding assay. The motility assay revealed that E11 has relatively higher motor activity than other rice kinesins.