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Magnetic Cytoskeleton Affinity (MICA) Purification for Fast Purification of Cargo-Attached Molecular Motors
262a
Monday, February 13, 2017
kinesin-2, and dynein and reconstituted their motility along microtubules (Hendricks et al., 2012). We find that tau biases bidirectional motility towards the minus end of the microtubule by preferentially inhibiting kinesin, such that the fraction of processive movements towards the minus end increases from 46% in the control case to 64% in the presence of 10 nM tau. Offaxis movements of dynein increase in the presence of tau, indicating dynein navigates around tau. Comparison of trajectories to a mathematical model suggests that tau biases bidirectional motility towards the minus end by increasing the unbinding rate of kinesin. Taken together, these results demonstrate that tau differentially inhibits teams of kinesin and dynein motors on a cargo to direct transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of kinesin and dynein driven motility. 1284-Pos Board B352 Ensemble Microscopy Reveals Nanoscale Patterns of Molecular Motors on Microtubules Louis Reese, Marian Baclayon, Nu`ria Taberner Carretero, Maurits Kok, Roland Dries, Eseng€ ul Yildirim, Andrea Martorana, Martin Depken, Marileen Dogterom. Bionanoscience, Delft University of Technology, Delft, Netherlands. The precise localization of proteins is of great importance to understand their biological function and to quantify molecular interactions. Here, we present an imaging strategy to obtain nanoscale localization in biochemical reconstitution experiments. This is achieved by imaging a large ensemble of molecules using a conventional total internal reflection fluorescence (TIRF) microscope with automated image acquisition in conjunction with post-acquisition alignment of the individual observations. The strategy bypasses typical limitations of time-lapse microscopy such as small sample size and bleaching. We apply our approach to study the localization of molecular motors on microtubules in a concentration range that spans from single molecule to macroscopic traffic jams. We find that motor proteins, dynein as well as kinesin, show stripe patterns on stabilized microtubules as well as strongly correlated intensity profiles on the nanoscale. The strategy of ensemble microscopy hence allows for spatial high-resolution imaging and is widely applicable in biochemical reconstitution experiments that probe interactions of proteins with the cytoskeleton or DNA for example. 1285-Pos Board B353 Mechanisms Underlying the Nucleation and Kinesin-Driven Assembly of Microtubule Rings George D. Bachand, Virginia VanDelinder, Christina Ting, Stephanie Brener. Sandia National Laboratories, Albuquerque, NM, USA. Active self-assembly processes rely on the conversion of chemical energy into mechanical work to overcome the limitations associated with diffusion-driven, passive self-assembly. For example, the transport of biotinylated microtubule filaments by surface-adsorbed kinesin motor proteins results in the actively assembly of bundles, rings, and spools in the presence of streptavidin. Observing the nucleation and assembly of these structures has been hindered by issues including controlled buffer exchange and photo-oxidative damage from fluorescent excitation. To address these issues, we developed a custom PDMS microfluidic device to characterize the earliest events involved in active assembly of microtubule rings and spools. Three distinct nucleation mechanisms were observed: pinning, collisions, and induced curvature. Pinning occurred when the leading tip of a microtubule encounters and stalls at an inactive motor. Experiments and numerical energy minimizations suggest that the diameter of ring/spools formed by pinning is strongly dependent on the surface density of kinesin motors. Collision-induced nucleation occurred when three (or more) microtubules simultaneous collide, forming a closed triangle that further evolved into a ring/spool. Collision accounted for the majority of nucleation events when photodamage was mitigated with our microfluidic device. The third nucleation mechanism, inducedcurvature, was only observed at long-time scale, and attributed to a frozenin curvature due to the binding of streptavidin-coated quantum dots to microtubules. We further showed that nucleation mechanisms affected both the diameter and rotation direction of the assembled rings/spools. Collectively, our studies provide fundamental insights on active assembly processes, which may be applied for regulating the morphology and functional properties of the resulting structures. Sandia National Laboratories is a multi-mission laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
1286-Pos Board B354 Magnetic Cytoskeleton Affinity (MICA) Purification for Fast Purification of Cargo-Attached Molecular Motors Marco Tjioe, Hyeon Ryoo, Pinghua Ge, Yuji Ishitsuka, Kevin Teng, Paul Selvin. Biophysics, University of Illinois at Urbana Champaign, Urbana, IL, USA. We have developed Magnetic Cytoskeleton Affinity (MiCA) purification method that is capable of isolating cargo attached molecular motors. We apply MiCA purification to the isolation of kinesin and compare with conventional microtubule affinity purification, which uses centrifugation to separate microtubules from the unbound or eluting kinesin. We show that MiCA purification generates purified kinesin yield comparable to that of the conventional microtubule affinity purification, and reduces the purification time from 1.5 hours to 0.5 hour, a 3 times improvement in speed. Using magnetic separation, we are now able to isolate quantum dot attached kinesin, which sediment together with microtubules upon centrifugation during conventional purification due to the dense quantum dot. This technique uses charge-charge interaction of the negatively charged microtubule and the positively charged magnetic beads to immobilize microtubule onto magnetic beads. Even though we have currently tested this MiCA purification technique only on cargo-attached kinesin, we foresee that the same protocol can be adjusted to purify cargo-attached dynein and other microtubule-associated proteins (MAPs). It also has the potential to purify cargo-attached myosin using the negatively charged actin instead of microtubule. 1287-Pos Board B355 Turning Around at the Tip: Single-Molecule Dynamics of Intraflagellar Transport in C. elegans Chemosensory Cilia Jona Mijalkovic, Jaap van Krugten, Felix Oswald, Seyda Acar, Erwin J. Peterman. Physics and Astronomy, Vrije Universiteit, Amsterdam, Netherlands. Bidirectional transport driven by motor proteins is essential for the proper distribution of cargo, and therefore vital for many cellular processes. Cilia are polar, microtubule-based cellular sensing hubs that rely on a process called intraflagellar transport (IFT) for their development, maintenance and function in signal-transduction. IFT trains, consisting of cargo and the IFTA and IFT-B protein complexes, are assembled at the ciliary base and driven co-operatively by kinesin-II and OSM-3 motors to the ciliary tip. The trains reverse direction at the tip and are transported back to the base by IFT dynein. The mechanism of IFT turnaround at the ciliary tip remains unknown. Here, we employ single-molecule fluorescence microscopy and single-particle tracking in the phasmid cilia of living C. elegans to probe IFT tip turnaround. Single-molecule trajectories reveal direct, pausing and diffusive turns in a sub-micrometer long turnaround region at the ciliary tip. Strikingly, while most IFT dyneins, OSM-3s and IFT-A particles turn almost instantaneously (within 600ms), IFT-B particles pause on average for 3 s before returning, with pauses lasting as long as 15 s. Further analysis reveals that IFT dynein and OSM-3 also exhibit diffusive behavior at the ciliary tip, whereas IFT-A and IFT-B are more spatially constrained. Our findings suggest that IFT trains dissociate at the tip and re-associate in retrograde moving trains. IFT-B, different than the other components, requires substantial remodeling while remaining spatially constrained at the tip, before it docks to an IFT-dynein driven retrograde train. Stochastic simulations of several tip turnaround scenarios support this model. Our data provides the first in vivo single-molecule quantification of IFT tip turnarounds, providing new insights into how bidirectional intracellular transport is organized and regulated. 1288-Pos Board B356 The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells Breane G. Budaitis1, Kristin I. Schimert2, Guido Scarabelli3, Barry J. Grant3, Kristen J. Verhey4. 1 Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA, 2Biophysics Program, University of Michigan, Ann Arbor, MI, USA, 3Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA, 4Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between b0 at the N-terminus (the coverstrand) and b9 in the first half of the necklinker. The CNB was shown to
Monday, February 13, 2017
kinesin-2, and dynein and reconstituted their motility along microtubules (Hendricks et al., 2012). We find that tau biases bidirectional motility towards the minus end of the microtubule by preferentially inhibiting kinesin, such that the fraction of processive movements towards the minus end increases from 46% in the control case to 64% in the presence of 10 nM tau. Offaxis movements of dynein increase in the presence of tau, indicating dynein navigates around tau. Comparison of trajectories to a mathematical model suggests that tau biases bidirectional motility towards the minus end by increasing the unbinding rate of kinesin. Taken together, these results demonstrate that tau differentially inhibits teams of kinesin and dynein motors on a cargo to direct transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of kinesin and dynein driven motility. 1284-Pos Board B352 Ensemble Microscopy Reveals Nanoscale Patterns of Molecular Motors on Microtubules Louis Reese, Marian Baclayon, Nu`ria Taberner Carretero, Maurits Kok, Roland Dries, Eseng€ ul Yildirim, Andrea Martorana, Martin Depken, Marileen Dogterom. Bionanoscience, Delft University of Technology, Delft, Netherlands. The precise localization of proteins is of great importance to understand their biological function and to quantify molecular interactions. Here, we present an imaging strategy to obtain nanoscale localization in biochemical reconstitution experiments. This is achieved by imaging a large ensemble of molecules using a conventional total internal reflection fluorescence (TIRF) microscope with automated image acquisition in conjunction with post-acquisition alignment of the individual observations. The strategy bypasses typical limitations of time-lapse microscopy such as small sample size and bleaching. We apply our approach to study the localization of molecular motors on microtubules in a concentration range that spans from single molecule to macroscopic traffic jams. We find that motor proteins, dynein as well as kinesin, show stripe patterns on stabilized microtubules as well as strongly correlated intensity profiles on the nanoscale. The strategy of ensemble microscopy hence allows for spatial high-resolution imaging and is widely applicable in biochemical reconstitution experiments that probe interactions of proteins with the cytoskeleton or DNA for example. 1285-Pos Board B353 Mechanisms Underlying the Nucleation and Kinesin-Driven Assembly of Microtubule Rings George D. Bachand, Virginia VanDelinder, Christina Ting, Stephanie Brener. Sandia National Laboratories, Albuquerque, NM, USA. Active self-assembly processes rely on the conversion of chemical energy into mechanical work to overcome the limitations associated with diffusion-driven, passive self-assembly. For example, the transport of biotinylated microtubule filaments by surface-adsorbed kinesin motor proteins results in the actively assembly of bundles, rings, and spools in the presence of streptavidin. Observing the nucleation and assembly of these structures has been hindered by issues including controlled buffer exchange and photo-oxidative damage from fluorescent excitation. To address these issues, we developed a custom PDMS microfluidic device to characterize the earliest events involved in active assembly of microtubule rings and spools. Three distinct nucleation mechanisms were observed: pinning, collisions, and induced curvature. Pinning occurred when the leading tip of a microtubule encounters and stalls at an inactive motor. Experiments and numerical energy minimizations suggest that the diameter of ring/spools formed by pinning is strongly dependent on the surface density of kinesin motors. Collision-induced nucleation occurred when three (or more) microtubules simultaneous collide, forming a closed triangle that further evolved into a ring/spool. Collision accounted for the majority of nucleation events when photodamage was mitigated with our microfluidic device. The third nucleation mechanism, inducedcurvature, was only observed at long-time scale, and attributed to a frozenin curvature due to the binding of streptavidin-coated quantum dots to microtubules. We further showed that nucleation mechanisms affected both the diameter and rotation direction of the assembled rings/spools. Collectively, our studies provide fundamental insights on active assembly processes, which may be applied for regulating the morphology and functional properties of the resulting structures. Sandia National Laboratories is a multi-mission laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
1286-Pos Board B354 Magnetic Cytoskeleton Affinity (MICA) Purification for Fast Purification of Cargo-Attached Molecular Motors Marco Tjioe, Hyeon Ryoo, Pinghua Ge, Yuji Ishitsuka, Kevin Teng, Paul Selvin. Biophysics, University of Illinois at Urbana Champaign, Urbana, IL, USA. We have developed Magnetic Cytoskeleton Affinity (MiCA) purification method that is capable of isolating cargo attached molecular motors. We apply MiCA purification to the isolation of kinesin and compare with conventional microtubule affinity purification, which uses centrifugation to separate microtubules from the unbound or eluting kinesin. We show that MiCA purification generates purified kinesin yield comparable to that of the conventional microtubule affinity purification, and reduces the purification time from 1.5 hours to 0.5 hour, a 3 times improvement in speed. Using magnetic separation, we are now able to isolate quantum dot attached kinesin, which sediment together with microtubules upon centrifugation during conventional purification due to the dense quantum dot. This technique uses charge-charge interaction of the negatively charged microtubule and the positively charged magnetic beads to immobilize microtubule onto magnetic beads. Even though we have currently tested this MiCA purification technique only on cargo-attached kinesin, we foresee that the same protocol can be adjusted to purify cargo-attached dynein and other microtubule-associated proteins (MAPs). It also has the potential to purify cargo-attached myosin using the negatively charged actin instead of microtubule. 1287-Pos Board B355 Turning Around at the Tip: Single-Molecule Dynamics of Intraflagellar Transport in C. elegans Chemosensory Cilia Jona Mijalkovic, Jaap van Krugten, Felix Oswald, Seyda Acar, Erwin J. Peterman. Physics and Astronomy, Vrije Universiteit, Amsterdam, Netherlands. Bidirectional transport driven by motor proteins is essential for the proper distribution of cargo, and therefore vital for many cellular processes. Cilia are polar, microtubule-based cellular sensing hubs that rely on a process called intraflagellar transport (IFT) for their development, maintenance and function in signal-transduction. IFT trains, consisting of cargo and the IFTA and IFT-B protein complexes, are assembled at the ciliary base and driven co-operatively by kinesin-II and OSM-3 motors to the ciliary tip. The trains reverse direction at the tip and are transported back to the base by IFT dynein. The mechanism of IFT turnaround at the ciliary tip remains unknown. Here, we employ single-molecule fluorescence microscopy and single-particle tracking in the phasmid cilia of living C. elegans to probe IFT tip turnaround. Single-molecule trajectories reveal direct, pausing and diffusive turns in a sub-micrometer long turnaround region at the ciliary tip. Strikingly, while most IFT dyneins, OSM-3s and IFT-A particles turn almost instantaneously (within 600ms), IFT-B particles pause on average for 3 s before returning, with pauses lasting as long as 15 s. Further analysis reveals that IFT dynein and OSM-3 also exhibit diffusive behavior at the ciliary tip, whereas IFT-A and IFT-B are more spatially constrained. Our findings suggest that IFT trains dissociate at the tip and re-associate in retrograde moving trains. IFT-B, different than the other components, requires substantial remodeling while remaining spatially constrained at the tip, before it docks to an IFT-dynein driven retrograde train. Stochastic simulations of several tip turnaround scenarios support this model. Our data provides the first in vivo single-molecule quantification of IFT tip turnarounds, providing new insights into how bidirectional intracellular transport is organized and regulated. 1288-Pos Board B356 The Role of the Cover-Neck Bundle in Multi-Motor Transport against Load in Cells Breane G. Budaitis1, Kristin I. Schimert2, Guido Scarabelli3, Barry J. Grant3, Kristen J. Verhey4. 1 Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA, 2Biophysics Program, University of Michigan, Ann Arbor, MI, USA, 3Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA, 4Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. Kinesin motor proteins share a highly-conserved catalytic motor domain responsible for coupling the chemical energy of ATP hydrolysis to force generation and microtubule-directed functions. Previous studies of kinesin-1 suggested that ATP binding to the motor domain induces the formation of the cover-neck bundle (CNB), a beta-sheet between b0 at the N-terminus (the coverstrand) and b9 in the first half of the necklinker. The CNB was shown to