Workshop 6: Transport in a cell

(April 12,2010 - April 15,2010 )

Organizers


Michael Diehl
Department of Bioengineering, Rice University
Anatoly Kolomeisky
Department of Chemistry, Rice University

Accepted Speakers

Sibylle Brenner
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine
Charles Brooks
Department of Chemistry, University of Michigan
Debashish Chowdhury
Department of Physics, Indian Institute of Technology Kanpur
John Cooper
Cell Biology and Physiology, Washington University School of Medicine
Michael Fisher
Institute for Physical Science and Technology, College of Business and Management
Valdimir Gelfand
Department of Cell and Molecular Biology, J. L. Kellogg Graduate School of Management
Arne Gennerich
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine
William Guilford
Department of Biomedical Engineering, University of Virginia
Alan Hunt
Department of Biomedical Engineering, University of Michigan
Alexei Kornyshev
Department of Chemistry, Imperial CollegeLondon
Jed Macosko
Physics, Wake Forest University
Liviu Movileanu
Department of Physics, Syracuse University
Alexander Nemukhin
Chemistry, M.V.Lomonosov Moscow State University
David Odde
Biomedical Engineering, University of Minnesota
Erwin Peterman
Dept. of Physics & Astronomy, Vrije University
Sarah Rice
Department of Cell Biology, J. L. Kellogg Graduate School of Management
Paul Selvin
Department of Physics, University of Illinois at Urbana-Champaign
Joanna Sulkowska
Center for Theoretical Biological Physics (CTBP)&Department of Physics, University of California at San Diego
Sean Sun
Dept. of Chemical and Biomolecular Engineering, Johns Hopkins University
Peter Vekilov
Depts of Chemical and Biomolecular Engineering and Chemistry, University of Houston
Kristen Verhey
Cell and Developmental Biology, University of Michigan Medical School
Monday, April 12, 2010
Time Session
09:00 AM
09:50 AM
Michael Fisher - How Kinesin Walks on a Microtubule: A View of the Story So Far
Progress in the description and understanding of the stochastic dynamics of the homodimeric motor protein kinesin is discussed on the basis of theory [1] and experiment [2]. A single molecule of kinesin, powered by ATP, may haul a cargo towards the plus end of a microtubule attaining speeds up to 900 nm/s while taking hundreds of discrete ~ 8 nm steps before detaching. Notable issues include: the presence (or absence) of substeps, the variation of processivity and randomness under controlled assisting and resisting loads, including backstepping under superstall conditions, the value of the stall force, the nature - "inch-worm" or "hand-over-hand" - of successive steps, their odd-even symmetry, evidenced by the "limping" seen in recent experiments on short-tethered constructs, and its dependence on the vectorial character of the loading force. Adequate accounts are provided by 'minimal' 2N-state periodic sequential kinetic models (with N=2) with both forward and reverse rates that, while possibly extended by diffusion-time or dwell-time distributions embodied in 'mechanicities', depend appropriately on the imposed mechanical force vector in a way reflecting an underlying 'free-energy landscape' picture.

1) Molecular Motors: A Theorist's Perspective, A.B. Kolomeisky and M. E. Fisher, Annu. Rev. Phys. Chem. 58 (2007) 675-95.

2) Kinesin Motor Mechanics: Binding, Stepping, Tracking, Gating and Limping, S.M. Block, Biophys. J. 92 (2007) 2986-95.
10:10 AM
11:00 AM
Joanna Sulkowska - Joanna Sulkowska's Lecture
Joanna Sulkowska's Lecture
11:20 AM
12:10 PM
John Cooper - The Function of Dynein in Budding Yeast: Mechanism, Regulation and Checkpoint Activity
I will discuss work from our lab and other labs on how dynein functions to position the mitotic spindle and the nucleus in budding yeast. Interactions of cytoplasmic microtubules with the cortex control spindle position, and dynein represents one of the major pathways by which spindle position is controlled. Dynein functions by an "offloading" mechanism in which dynein targeted to dynamic microtubule plus ends is transferred to the cortex, where it is anchored and activated for minus-end directed motor activity, which pulls the spindle into the neck between mother and bud. Dynein is a multi-subunit complex, and dynein requires the function of dynactin, another multisubunit complex. Other factors are important for dynein targeting, both to the plus end and to the cortex. Dynein appears to be regulated spatially and temporally, during the course of its action. Finally, loss of dynein function and failure to properly position the spindle leads to activation of a cell-cycle checkpoint, which delays the progression of the cell cycle until alternative mechanisms are able to move the spindle into the mother / bud neck. The mechanism of this checkpoint relies on feedback information from cytoplasmic microtubules, based on new laser-cutting experiments.
02:00 PM
02:50 PM
Paul Selvin - FIONA looks at individual molecular motors walk and run
The standard diffraction limit of light is about 250 nm, meaning that you cannot "resolve" objects closer than this distance. Despite this, we have come up with a method to measure 1.5 nm in x-y plane, with 1-500 msec, using a technique we call Fluorescence Imaging with One Nanometer Accuracy (FIONA). FIONA also has the advantage that it looks at individual, or single, proteins. We have chosen to study molecular motors, both in vitro and in vivo. We find that all tested motors walk in a hand-over-hand fashion. We also find evidence that in vivo, two of the same motors carry cargo simultaneously-but not cooperatively. Because of limited signal-to-noise, this uses a new Hidden Markov Method technique. We also see passing of cargo from one type of motor to another. Finally, we find that we can extend FIONA to two-photon microscopy using quantum dots. Time permitting, we shall also discuss recent efforts where we can see individual ion channels, proteins which pass ions (such as sodium and potassium) between the inside and outside of the cell. This has enormous clinical ramifications, such as the study of strokes, Alzheimer's, and nicotine-addiction. Interesting mathematical tricks to improve the signal-to-noise will be discussed.
03:10 PM
04:00 PM
Sarah Rice - The conserved L5 loop establishes the pre-powerstroke conformation of the kinesin-5 motor, Eg5
Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, to L5, and to the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of STLC, an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of nucleotide state.

Work done in collaboration with Adam G. Larson, Nariman Naber, Roger Cooke, and Edward Pate.
Tuesday, April 13, 2010
Time Session
09:00 AM
09:50 AM
Valdimir Gelfand - Dynamic instability of organelle transport
Dynamic instability of organelle transport
10:10 AM
11:00 AM
Arne Gennerich - Toward a unified walking model for cytoplasmic dynein
Cytoplasmic dynein is a two-headed motor protein that generates microtubule (MT) minus-end-directed motility in eukaryotic cells. It contains four AAA (ATPase associated with various cellular activities) domains per head that can bind ATP, and has the ability to take hundreds of steps along MTs before it dissociates and diffuses away. Such continuous movement requires coordination of the mechanochemical cycles of both heads so that the front head remains bound to the track while the rear head detaches. However, the molecular mechanism that underlies the coordination of dynein's head domains remains unknown. Here, using mutagenesis studies and optical tweezers-based unbinding-force measurements, we show that single S. cerevisiae cytoplasmic dynein heads exhibit nucleotide and loading-direction dependent strong and weak MT-binding states and a pronounced directional instability of MT-binding that promotes rear head detachment. By measuring the MT-binding strength of mutant dynein heads incapable of nucleotide binding and/or hydrolysis by one or two of the four AAA domains as a function of nucleotide, we discover unique roles of dynein's AAA domains in regulating MT-binding affinity. We find that ATP binding to AAA1 causes rapid head dissociation under forward load, while ADP binding to AAA3 decreases dynein's MT-binding affinity under backward load. Comparison of our data with the previously reported force-induced bidirectional stepping of individual two-headed dynein motors allows us to assign nucleotide states to dynein's leading and trailing head domains, and to derive the first complete mechanochemical walking model for cytoplasmic dynein.
11:20 AM
12:10 PM
Sean Sun - Mechanics of Actomyosin Interaction and the Role of Substrate Stiffness on Actin Network Dynamics
Myosin is a major molecular force generator in the cell. The essential features of myosin interaction with actin filaments are understood. In the cell, the interactions of myosin with F-actin, substrate adhesions and other actin-associated proteins are less clear. We will borrow ideas from mathematical models of skeletal muscle to develop simple models for integrin focal adhesions, actin cross-linking proteins and non-muscle myosin-II. When these components are combined, a dynamical picture of the actin network emerges. In this talk, we will focus on the role of cell-substrate stiffness on the actin dynamics. Possible implications for mechanical sensing by cells are discussed.
02:00 PM
02:50 PM
Charles Brooks - Functional mechanical deformations in Natures machines
In this talk I will present findings from our studies of the functionally important mechanical processes in the ribosome and a AAA+ helicase. I will examine a number of coarse-graining approaches to decomposing the functional components of motion in these systems using a combination of elastic deformation theory, Go-type structure centric folding models and Brownian motion. From our studies we see that functional motions associated with tRNA translocation in the ribosome are largely attributed to the lowest energy for deformation eigenvector directions from elastic theory. We also find that the decomposition of the elements of translocation of the AAA+ helicase along single stranded DNA into conformational change, substrate coupling and asymmetry in substrate binding lead to a simple picture of the essential features of functioning in this molecular motor.
03:10 PM
04:00 PM
Kristen Verhey - Road signs for kinesin transport
We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particles in the time range 1 millisecond - 10 seconds in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
04:20 PM
05:10 PM
Michael Diehl - Collective behaviors of multiple interacting kinesin motors: Can kinesin number regulate intracellular transport?
Collective behaviors of multiple interacting kinesin motors: Can kinesin number regulate intracellular transport?
Wednesday, April 14, 2010
Time Session
09:00 AM
09:50 AM
Debashish Chowdhury - Motoring along a nucleic acid strand: template-dictated polymerization of macromolecules of life
Polymerases and ribosome are molecular machines which perform three important biological functions. Like cytoskeletal motors, each of these moves along a track using chemical energy for performing mechanical work. Moreover, it decodes genetic information chemically-encoded in the sequence of the subunits of the track. Furthermore, it polymerizes a macromolecule (DNA, RNA or protein) using the required subunits in a sequence that is dictated by the sequence of subunits of a template which serves also as its track for translocation. Enormous progress has been made in the last decade in understanding the structure and dynamics of these molecular machines using a combination of X-ray crystallography, cryo-electron microscopy, single-molecule imaging and manipulation. In recent years, we have developed models of these machines capturing the key features of their structure and dynamics to gain a quantitative understanding of their operational mechanism. We have also investigated the traffic-like collective movement of ribosomes simultaneously on the same mRNA track (and similar traffic of RNA polymerases on a DNA). We have also suggested new experiments for testing our theoretical predictions on the stochastic translocation-and-pause kinetics of a single motor as well as on their collective spatio-temporal organization.
10:10 AM
11:00 AM
Alexei Kornyshev - DNA chasing DNA: physics of homology recognition and the secret of perfect match
DNA chasing DNA: physics of homology recognition and the secret of perfect match
11:20 AM
12:10 AM
Alexander Nemukhin - Modeling adrenosine triphosphate (ATP) hydrolysis in myosin

Myosin is a prototypical molecular motor that utilizes adenosine triphosphate (ATP). Coupling between the chemical reaction of ATP hydrolysis in myosin and conformational transitions of the protein is crucial for its function. Despite multiple efforts to dissect the reaction mechanism and the corresponding 'mechanochemical' coupling, a clear mechanistic picture is still under examination. The combined quantum mechanical - molecular mechanical (QM/MM) approaches are applied to construct the reaction energy profiles for the ATP hydrolysis in the active site of the protein and to specify reaction products and possible intermediates. Molecular dynamics simulations coordinated with the QM/MM studies assist to conceive the events occurring immediately after the intrinsic chemical reaction.

02:00 PM
02:50 PM
Alan Hunt - Spindle Pole Mechanics: Architecture, Compliance, and Force Distribution
The mitotic spindle is a macromolecular ensemble of microtubules, motors, and non-motor microtubule-associated proteins responsible for chromosome movements and segregation. Chromosome segregation is an inherently mechanical process, but little is known about spindle mechanics or the mechanical roles of specific spindle proteins. Here, we assemble spindle poles in a cell-free mitotic extract, and apply optical trapping to directly assess the mechanics of microtubules emanating from these poles. Silica beads are anchored to microtubules through biotin-neutravidin linkages, and microtubule movements are assessed with nanometer precision as the optical trap applies a constant antipoleward force on the microtubule-associated bead. We observe that microtubules exhibit bidirectional movements reaching nm/s, and attenuated in the presence of AMP-PNP, indicative of molecular motor activity. Microtubule linkages to spindle poles are remarkably compliant, with a mean stiffness of just 0.025 pN/nm, and are apparently mediated by only a handful of crosslinkers. Depletion of the homotetrameric, bipolar motor Eg5 does not alter the mean microtubule velocity, but increases the amplitude and speed of short-time-scale (seconds) excursions and significantly reduces the stiffness of microtubule attachments to poles. The large compliance of microtubule linkage to spindle poles provides a robust, non-brittle mechanical architecture capable of accommodating microtubule movements associated with poleward microtubule flux without coordinated rearrangement of crosslinks. Furthermore compliance helps distribute strain amongst microtubules focused at the pole, thereby integrating even antagonistic spindle forces such as those resulting from poleward and antipoleward chromosome movements without disrupting the overall spindle architecture.
03:10 PM
04:00 PM
William Guilford - Pushmi-pullyu: The peculiar dynamics of microtubule-based motors in Chlamydomonas

Molecular motors in living cells are involved in cell movement and developmental shape changes, and are responsible for intraflagellar transport (IFT) of organelles and other cargo. Chlamydomonas, a unicellular alga, is an ideal organism for studying motor function. Kinesin-2 and dynein-2, the motors for anterograde and retrograde transport in Chlamydomonas respectively, are responsible for both IFT and the flagellar membrane transport of extracellular cargo. Kinesin and dynein bind to a flagellar transmembrane protein, FMG-1, which in turn binds to extracellular cargo, allowing for the movement of extracellular cargos (e.g. microbeads, bacteria, or accumulated debris) along the length of the flagellum. Thus Chlamydomonas offers a unique circumstance for measuring motor biophysics, coordination, and regulation inside the cell from outside the cell. We capture microspheres in a laser trap, present them to immobilized Chlamydomonas flagella, and through flagellar membrane transport record molecular motor protein function as the extracellular bead is moved as cargo. Our early work suggested that oppositely directed molecular motors are reciprocally coordinated rather than operating in a tug-of-war fashion. Further, many motors (> 10) can engage simultaneously to move the extracellular cargo. Our recent work shows that the velocities of transport are quantal (multiples of a fundamental velocity), and routinely travel at much higher velocities than those expected or allowed by our current understanding of processive motors. Further, the force generated on extracellular cargo is not proportional to the contact area with the membrane. These data suggest motor dynamics and mechanics that are unique to the intracellular environment.

04:20 PM
05:10 PM
Jed Macosko - Multiple motors cooperating in vivo and in vitro: What have we learned?

Since the landmark gliding experiments by Toshio Yanagida and coworkers on myosin in the 1980's and a seminal 1989 Nature paper by Joe Howard, Jim Hudspeth and Ron Vale on kinesin, motor cooperativity in most people's minds was solved. Motors that only occasionally touched their tracks, like myosin, could cooperate to acheive higher speeds. Motors that were attached persistantly to their rails, like kinesin, could not. However, Howard, Hudspeth, and Vale's conclusion that increasing kinesin did not result in higher microtubule gliding speeds applied only to a very special opposing force regime. Namely, their experiment was performed in the absence of any appreciable load. Newer studies in vitro, including Joe Howard's own elegant work with Alan Hunt and Fred Gittes in 1994, show that the number of kinesins matters a great deal when they jointly pull a significant load. More recent work in vivo by Paul Selvin, Volodya Gelfand and coworkers, as well as our own work, conclude that kinesin and dynein cooperativity is important in fast vesicle transport in an intracellular environment of high viscous drag. These conclusions have been met with criticism, which we will present along with rebuttals. A synthesis of our in vivo and in vitro work will be documented, and our minimal load-sharing model will be introduced. This model has potential to qualitatively explain all multimotor results to-date, though measurements of significantly higher motor speeds in vivo than in vitro are still puzzling.

Thursday, April 15, 2010
Time Session
09:00 AM
09:50 AM
David Odde - 1D versus 2D models for microtubule self-assembly: New tests at the nanoscale

Microtubules (MTs) are stabilized by lateral and longitudinal bonds between ??-tubulin heterodimers, and so self-assembly can potentially be treated as a 2D problem along the axial and circumferential directions. Despite the 2D structure, microtubule assembly data have consistently been interpreted in the context of the 1D model of Oosawa (J. Theor. Biol., 1970). I will discuss the implications of this assumption, and how considering a 2D model (VanBuren et al., PNAS, 2002) leads us to question the conclusions drawn from the 1D analysis. In addition, I will present unpublished nanoscale fluorescence microscopy and optical tweezers data demonstrating the limitations of the 1D model, and confirming the 2D model predictions. We conclude that the kinetics of MT assembly have been consistently underestimated in the MT literature by an order-of-magnitude, and that tubulin addition-loss occurs with near kHz dynamics. These findings have important implications for the regulation of MT assembly in vivo, and point to a model where infrequent and weak alterations of the tubulin-tubulin bonds and tubulin mechanical properties via MT-associated proteins and therapeutic drugs mediate control of self-assembly.

10:10 AM
11:00 AM
Peter Vekilov - Viscoelasticity in homogeneous protein solutions
We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particles in the time range 1 millisecond - 10 seconds in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
11:20 AM
12:10 PM
Erwin Peterman - Illuminating the way Kinesin-1 walks using FRET between the motor domains

Kinesin-1 is a motor protein that walks processively along microtubules in a hand-over-hand manner driving intracellular transport of vesicles and organelles. Each step of 8 nm requires the hydrolysis of one ATP and takes about 10 ms at cellular ATP concentrations. Key aspects of kinesin's walking mechanism are not fully understood. One important question concerns the configuration of the two motor domains during processive motion. Here, we use a novel assay based on single-molecule confocal fluorescence microscopy to characterize Kinesin-1's stepping mechanism in vitro. A key advantage of our approach over conventional wide-field methods is that our time resolution is far better, less than 0.1 ms. We apply this approach to kinesin constructs that are labeled with a donor fluorophore on the one motor domain and an acceptor on the other. We follow the distance between the motor domains during stepping with F?rster Resonance Energy Transfer. We use four different homodimeric kinesin constructs with dye molecules attached to different sites of the motor domain. With this approach, we can identify an intermediate state in the stepping process that lasts 2-3 ms at saturating ATP concentration. In this intermediate state one motor domain is bound to the microtubule and the other is rotated and substantially less than 8 nm away.

02:00 PM
02:50 PM
Liviu Movileanu - Single-molecule Science with A Nanopore: Inspiration from Nature

A nanopore may act as an amazingly versatile single-molecule probe that can be employed to reveal several important features of nucleic acids and proteins. The underlying principle of nanopore probe techniques is simple: the application of a voltage bias across an electrically insulated membrane enables the measurement of a tiny picoamp-scale transmembrane current through a single hole of nanometer size, called a nanopore. Each molecule, translocating through the nanopore, produces a distinctive current blockade, the nature of which depends on its biophysical properties as well as the molecule-nanopore interaction.


Such an approach proves to be quite powerful, because single small molecules and biopolymers are examined at very high spatial and temporal resolutions. I will discuss our recent work that provided a mechanistic understanding of the forces that drive protein translocation through a nanopore. These measurements facilitate the detection and exploration of the conformational fluctuations of single molecules and the energetic requirements for their transition from one state to another.


I will also describe our recent strategies for engineering new functional nanopores, in organic and silicon-based materials, with properties that are not encountered in nature. From a practical point of view, this methodology shows promise for the integration of engineered nanopores into nanofluidic devices, which would provide a new generation of research tools in nanomedicine and high-throughput applications for molecular biomedical diagnosis.

03:10 PM
04:00 PM
Anatoly Kolomeisky - Channel-Facilitated Molecular Transport Across Cellular Membranes

N/A

Name Affiliation
Badamdorj, Dorjsuren dbadamdo@tnstate.edu Mathematics and Physics, Tennessee State University
Brenner, Sibylle sibylle@gennerichlab.org Department of Anatomy and Structural Biology, Albert Einstein College of Medicine
Brooks, Charles apeters@umich.edu Department of Chemistry, University of Michigan
Brown, Anthony brown.230@osu.edu Neuroscience, The Ohio State University
Bundschuh, Ralf bundschuh@mbi.osu.edu Departments of Physics and Biochemistry, The Ohio State University
Chan, Michael chan@chemistry.ohio-state.edu Biochemistry, The Ohio State University
Chowdhury, Debashish debch@iitk.ac.in Department of Physics, Indian Institute of Technology Kanpur
Cooper, John jcooper11@gmail.com Cell Biology and Physiology, Washington University School of Medicine
Diehl, Michael diehl@rice.edu Department of Bioengineering, Rice University
Driver, Jonathan jdriver@rice.edu Department of Bioengineering, Rice University
Fisher, Michael xpectnil@ipst.umd.edu Institute for Physical Science and Technology, College of Business and Management
Gelfand, Valdimir vgelfand@northwestern.edu Department of Cell and Molecular Biology, J. L. Kellogg Graduate School of Management
Gennerich, Arne gennerich@gmail.com Department of Anatomy and Structural Biology, Albert Einstein College of Medicine
Guilford, William whg2n@virginia.edu Department of Biomedical Engineering, University of Virginia
Hendricks, Adam adamhe@mail.med.upenn.edu Physiology, University of Pennsylvania
Herman, Paul herman.81@osu.edu Molecular Genetics, The Ohio State University
Holzwarth, George gholz@wfu.edu Department of Physics, Wake Forest University
Hunt, Alan ajhunt@umich.edu Department of Biomedical Engineering, University of Michigan
Jamison, Kenneth dkj4958@rice.edu Department of Bioengineering, Rice University
Joo, Jaewook jjoo1@utk.edu Physics, University of Tennessee
Kiani, Narsis Aftab narsis.kiani@bioquant.uni-heidelberg.de Bioquant, Heidelberg University
Kolomeisky, Anatoly tolya@rice.edu Department of Chemistry, Rice University
Kornyshev, Alexei A.Kornyshev@imperial.ac.uk Department of Chemistry, Imperial CollegeLondon
Landahl, Eric elandahl@depaul.edu Physics, DePaul University
Li, Ye liyethutju@gmail.com Dept. of Chemical and Biomolecular Engineering, University of Houston
Macosko, Jed macoskjc@wfu.edu Physics, Wake Forest University
McKinley, Scott mckinley@math.duke.edu Mathematics Department, Duke University
Meier, Iris meier.56@osu.edu MG/PCMB, The Ohio State University
Movileanu, Liviu lmovilea@physics.syr.edu Department of Physics, Syracuse University
Murthy, Abhishek amurthy@es.sunysb.edu Computer Science, Stony Brook University (SUNY)
Nemukhin , Alexander anem@lcc.chem.msu.ru Chemistry, M.V.Lomonosov Moscow State University
Odde, David oddex002@umn.edu Biomedical Engineering, University of Minnesota
Peterman, Erwin erwinp@nat.vu.nl Dept. of Physics & Astronomy, Vrije University
Rice, Sarah redheadsarah@gmail.com Department of Cell Biology, J. L. Kellogg Graduate School of Management
Salama, Samir salama.3@buckeyemail.osu.edu MCDB, The Ohio State University
Selvin, Paul shunk@illinois.edu Department of Physics, University of Illinois at Urbana-Champaign
Sodhi, Puneet sodhi.8@buckeyemail.osu.edu NGSP, The Ohio State University
Sulkowska, Joanna jsulkow@physics.ucsd.edu Center for Theoretical Biological Physics (CTBP)&Department of Physics, University of California at San Diego
Sun, Sean ssun@jhu.edu Dept. of Chemical and Biomolecular Engineering, Johns Hopkins University
Vekilov, Peter vekilov@uh.edu Depts of Chemical and Biomolecular Engineering and Chemistry, University of Houston
Verhey, Kristen kjverhey@umich.edu Cell and Developmental Biology, University of Michigan Medical School
Wang, Lina wang.1095@buckeyemail.osu.edu Neuroscience, The Ohio State University
Wu, Jian-Qiu wu.620@osu.edu Molecular Genetics and MCB, The Ohio State University
Yamada, Richard yryamada@umich.edu Mathematics, University of Michigan
Ye, Fangfu fye@illinois.edu Department of Physics, University of Illinois at Urbana-Champaign
Yu, Xiaoxiao yu.375@buckeyemail.osu.edu Biomedical Engineering, The Ohio State University
Zhang, Yunxin yxzhang@umd.edu Inst. for Physical Science and Technology, College of Business and Management
Functional mechanical deformations in Natures machines
In this talk I will present findings from our studies of the functionally important mechanical processes in the ribosome and a AAA+ helicase. I will examine a number of coarse-graining approaches to decomposing the functional components of motion in these systems using a combination of elastic deformation theory, Go-type structure centric folding models and Brownian motion. From our studies we see that functional motions associated with tRNA translocation in the ribosome are largely attributed to the lowest energy for deformation eigenvector directions from elastic theory. We also find that the decomposition of the elements of translocation of the AAA+ helicase along single stranded DNA into conformational change, substrate coupling and asymmetry in substrate binding lead to a simple picture of the essential features of functioning in this molecular motor.
Motoring along a nucleic acid strand: template-dictated polymerization of macromolecules of life
Polymerases and ribosome are molecular machines which perform three important biological functions. Like cytoskeletal motors, each of these moves along a track using chemical energy for performing mechanical work. Moreover, it decodes genetic information chemically-encoded in the sequence of the subunits of the track. Furthermore, it polymerizes a macromolecule (DNA, RNA or protein) using the required subunits in a sequence that is dictated by the sequence of subunits of a template which serves also as its track for translocation. Enormous progress has been made in the last decade in understanding the structure and dynamics of these molecular machines using a combination of X-ray crystallography, cryo-electron microscopy, single-molecule imaging and manipulation. In recent years, we have developed models of these machines capturing the key features of their structure and dynamics to gain a quantitative understanding of their operational mechanism. We have also investigated the traffic-like collective movement of ribosomes simultaneously on the same mRNA track (and similar traffic of RNA polymerases on a DNA). We have also suggested new experiments for testing our theoretical predictions on the stochastic translocation-and-pause kinetics of a single motor as well as on their collective spatio-temporal organization.
The Function of Dynein in Budding Yeast: Mechanism, Regulation and Checkpoint Activity
I will discuss work from our lab and other labs on how dynein functions to position the mitotic spindle and the nucleus in budding yeast. Interactions of cytoplasmic microtubules with the cortex control spindle position, and dynein represents one of the major pathways by which spindle position is controlled. Dynein functions by an "offloading" mechanism in which dynein targeted to dynamic microtubule plus ends is transferred to the cortex, where it is anchored and activated for minus-end directed motor activity, which pulls the spindle into the neck between mother and bud. Dynein is a multi-subunit complex, and dynein requires the function of dynactin, another multisubunit complex. Other factors are important for dynein targeting, both to the plus end and to the cortex. Dynein appears to be regulated spatially and temporally, during the course of its action. Finally, loss of dynein function and failure to properly position the spindle leads to activation of a cell-cycle checkpoint, which delays the progression of the cell cycle until alternative mechanisms are able to move the spindle into the mother / bud neck. The mechanism of this checkpoint relies on feedback information from cytoplasmic microtubules, based on new laser-cutting experiments.
Collective behaviors of multiple interacting kinesin motors: Can kinesin number regulate intracellular transport?
Collective behaviors of multiple interacting kinesin motors: Can kinesin number regulate intracellular transport?
How Kinesin Walks on a Microtubule: A View of the Story So Far
Progress in the description and understanding of the stochastic dynamics of the homodimeric motor protein kinesin is discussed on the basis of theory [1] and experiment [2]. A single molecule of kinesin, powered by ATP, may haul a cargo towards the plus end of a microtubule attaining speeds up to 900 nm/s while taking hundreds of discrete ~ 8 nm steps before detaching. Notable issues include: the presence (or absence) of substeps, the variation of processivity and randomness under controlled assisting and resisting loads, including backstepping under superstall conditions, the value of the stall force, the nature - "inch-worm" or "hand-over-hand" - of successive steps, their odd-even symmetry, evidenced by the "limping" seen in recent experiments on short-tethered constructs, and its dependence on the vectorial character of the loading force. Adequate accounts are provided by 'minimal' 2N-state periodic sequential kinetic models (with N=2) with both forward and reverse rates that, while possibly extended by diffusion-time or dwell-time distributions embodied in 'mechanicities', depend appropriately on the imposed mechanical force vector in a way reflecting an underlying 'free-energy landscape' picture.

1) Molecular Motors: A Theorist's Perspective, A.B. Kolomeisky and M. E. Fisher, Annu. Rev. Phys. Chem. 58 (2007) 675-95.

2) Kinesin Motor Mechanics: Binding, Stepping, Tracking, Gating and Limping, S.M. Block, Biophys. J. 92 (2007) 2986-95.
Dynamic instability of organelle transport
Dynamic instability of organelle transport
Toward a unified walking model for cytoplasmic dynein
Cytoplasmic dynein is a two-headed motor protein that generates microtubule (MT) minus-end-directed motility in eukaryotic cells. It contains four AAA (ATPase associated with various cellular activities) domains per head that can bind ATP, and has the ability to take hundreds of steps along MTs before it dissociates and diffuses away. Such continuous movement requires coordination of the mechanochemical cycles of both heads so that the front head remains bound to the track while the rear head detaches. However, the molecular mechanism that underlies the coordination of dynein's head domains remains unknown. Here, using mutagenesis studies and optical tweezers-based unbinding-force measurements, we show that single S. cerevisiae cytoplasmic dynein heads exhibit nucleotide and loading-direction dependent strong and weak MT-binding states and a pronounced directional instability of MT-binding that promotes rear head detachment. By measuring the MT-binding strength of mutant dynein heads incapable of nucleotide binding and/or hydrolysis by one or two of the four AAA domains as a function of nucleotide, we discover unique roles of dynein's AAA domains in regulating MT-binding affinity. We find that ATP binding to AAA1 causes rapid head dissociation under forward load, while ADP binding to AAA3 decreases dynein's MT-binding affinity under backward load. Comparison of our data with the previously reported force-induced bidirectional stepping of individual two-headed dynein motors allows us to assign nucleotide states to dynein's leading and trailing head domains, and to derive the first complete mechanochemical walking model for cytoplasmic dynein.
Pushmi-pullyu: The peculiar dynamics of microtubule-based motors in Chlamydomonas

Molecular motors in living cells are involved in cell movement and developmental shape changes, and are responsible for intraflagellar transport (IFT) of organelles and other cargo. Chlamydomonas, a unicellular alga, is an ideal organism for studying motor function. Kinesin-2 and dynein-2, the motors for anterograde and retrograde transport in Chlamydomonas respectively, are responsible for both IFT and the flagellar membrane transport of extracellular cargo. Kinesin and dynein bind to a flagellar transmembrane protein, FMG-1, which in turn binds to extracellular cargo, allowing for the movement of extracellular cargos (e.g. microbeads, bacteria, or accumulated debris) along the length of the flagellum. Thus Chlamydomonas offers a unique circumstance for measuring motor biophysics, coordination, and regulation inside the cell from outside the cell. We capture microspheres in a laser trap, present them to immobilized Chlamydomonas flagella, and through flagellar membrane transport record molecular motor protein function as the extracellular bead is moved as cargo. Our early work suggested that oppositely directed molecular motors are reciprocally coordinated rather than operating in a tug-of-war fashion. Further, many motors (> 10) can engage simultaneously to move the extracellular cargo. Our recent work shows that the velocities of transport are quantal (multiples of a fundamental velocity), and routinely travel at much higher velocities than those expected or allowed by our current understanding of processive motors. Further, the force generated on extracellular cargo is not proportional to the contact area with the membrane. These data suggest motor dynamics and mechanics that are unique to the intracellular environment.

Spindle Pole Mechanics: Architecture, Compliance, and Force Distribution
The mitotic spindle is a macromolecular ensemble of microtubules, motors, and non-motor microtubule-associated proteins responsible for chromosome movements and segregation. Chromosome segregation is an inherently mechanical process, but little is known about spindle mechanics or the mechanical roles of specific spindle proteins. Here, we assemble spindle poles in a cell-free mitotic extract, and apply optical trapping to directly assess the mechanics of microtubules emanating from these poles. Silica beads are anchored to microtubules through biotin-neutravidin linkages, and microtubule movements are assessed with nanometer precision as the optical trap applies a constant antipoleward force on the microtubule-associated bead. We observe that microtubules exhibit bidirectional movements reaching nm/s, and attenuated in the presence of AMP-PNP, indicative of molecular motor activity. Microtubule linkages to spindle poles are remarkably compliant, with a mean stiffness of just 0.025 pN/nm, and are apparently mediated by only a handful of crosslinkers. Depletion of the homotetrameric, bipolar motor Eg5 does not alter the mean microtubule velocity, but increases the amplitude and speed of short-time-scale (seconds) excursions and significantly reduces the stiffness of microtubule attachments to poles. The large compliance of microtubule linkage to spindle poles provides a robust, non-brittle mechanical architecture capable of accommodating microtubule movements associated with poleward microtubule flux without coordinated rearrangement of crosslinks. Furthermore compliance helps distribute strain amongst microtubules focused at the pole, thereby integrating even antagonistic spindle forces such as those resulting from poleward and antipoleward chromosome movements without disrupting the overall spindle architecture.
Channel-Facilitated Molecular Transport Across Cellular Membranes

N/A

DNA chasing DNA: physics of homology recognition and the secret of perfect match
DNA chasing DNA: physics of homology recognition and the secret of perfect match
Multiple motors cooperating in vivo and in vitro: What have we learned?

Since the landmark gliding experiments by Toshio Yanagida and coworkers on myosin in the 1980's and a seminal 1989 Nature paper by Joe Howard, Jim Hudspeth and Ron Vale on kinesin, motor cooperativity in most people's minds was solved. Motors that only occasionally touched their tracks, like myosin, could cooperate to acheive higher speeds. Motors that were attached persistantly to their rails, like kinesin, could not. However, Howard, Hudspeth, and Vale's conclusion that increasing kinesin did not result in higher microtubule gliding speeds applied only to a very special opposing force regime. Namely, their experiment was performed in the absence of any appreciable load. Newer studies in vitro, including Joe Howard's own elegant work with Alan Hunt and Fred Gittes in 1994, show that the number of kinesins matters a great deal when they jointly pull a significant load. More recent work in vivo by Paul Selvin, Volodya Gelfand and coworkers, as well as our own work, conclude that kinesin and dynein cooperativity is important in fast vesicle transport in an intracellular environment of high viscous drag. These conclusions have been met with criticism, which we will present along with rebuttals. A synthesis of our in vivo and in vitro work will be documented, and our minimal load-sharing model will be introduced. This model has potential to qualitatively explain all multimotor results to-date, though measurements of significantly higher motor speeds in vivo than in vitro are still puzzling.

Single-molecule Science with A Nanopore: Inspiration from Nature

A nanopore may act as an amazingly versatile single-molecule probe that can be employed to reveal several important features of nucleic acids and proteins. The underlying principle of nanopore probe techniques is simple: the application of a voltage bias across an electrically insulated membrane enables the measurement of a tiny picoamp-scale transmembrane current through a single hole of nanometer size, called a nanopore. Each molecule, translocating through the nanopore, produces a distinctive current blockade, the nature of which depends on its biophysical properties as well as the molecule-nanopore interaction.


Such an approach proves to be quite powerful, because single small molecules and biopolymers are examined at very high spatial and temporal resolutions. I will discuss our recent work that provided a mechanistic understanding of the forces that drive protein translocation through a nanopore. These measurements facilitate the detection and exploration of the conformational fluctuations of single molecules and the energetic requirements for their transition from one state to another.


I will also describe our recent strategies for engineering new functional nanopores, in organic and silicon-based materials, with properties that are not encountered in nature. From a practical point of view, this methodology shows promise for the integration of engineered nanopores into nanofluidic devices, which would provide a new generation of research tools in nanomedicine and high-throughput applications for molecular biomedical diagnosis.

Modeling adrenosine triphosphate (ATP) hydrolysis in myosin

Myosin is a prototypical molecular motor that utilizes adenosine triphosphate (ATP). Coupling between the chemical reaction of ATP hydrolysis in myosin and conformational transitions of the protein is crucial for its function. Despite multiple efforts to dissect the reaction mechanism and the corresponding 'mechanochemical' coupling, a clear mechanistic picture is still under examination. The combined quantum mechanical - molecular mechanical (QM/MM) approaches are applied to construct the reaction energy profiles for the ATP hydrolysis in the active site of the protein and to specify reaction products and possible intermediates. Molecular dynamics simulations coordinated with the QM/MM studies assist to conceive the events occurring immediately after the intrinsic chemical reaction.

1D versus 2D models for microtubule self-assembly: New tests at the nanoscale

Microtubules (MTs) are stabilized by lateral and longitudinal bonds between ??-tubulin heterodimers, and so self-assembly can potentially be treated as a 2D problem along the axial and circumferential directions. Despite the 2D structure, microtubule assembly data have consistently been interpreted in the context of the 1D model of Oosawa (J. Theor. Biol., 1970). I will discuss the implications of this assumption, and how considering a 2D model (VanBuren et al., PNAS, 2002) leads us to question the conclusions drawn from the 1D analysis. In addition, I will present unpublished nanoscale fluorescence microscopy and optical tweezers data demonstrating the limitations of the 1D model, and confirming the 2D model predictions. We conclude that the kinetics of MT assembly have been consistently underestimated in the MT literature by an order-of-magnitude, and that tubulin addition-loss occurs with near kHz dynamics. These findings have important implications for the regulation of MT assembly in vivo, and point to a model where infrequent and weak alterations of the tubulin-tubulin bonds and tubulin mechanical properties via MT-associated proteins and therapeutic drugs mediate control of self-assembly.

Illuminating the way Kinesin-1 walks using FRET between the motor domains

Kinesin-1 is a motor protein that walks processively along microtubules in a hand-over-hand manner driving intracellular transport of vesicles and organelles. Each step of 8 nm requires the hydrolysis of one ATP and takes about 10 ms at cellular ATP concentrations. Key aspects of kinesin's walking mechanism are not fully understood. One important question concerns the configuration of the two motor domains during processive motion. Here, we use a novel assay based on single-molecule confocal fluorescence microscopy to characterize Kinesin-1's stepping mechanism in vitro. A key advantage of our approach over conventional wide-field methods is that our time resolution is far better, less than 0.1 ms. We apply this approach to kinesin constructs that are labeled with a donor fluorophore on the one motor domain and an acceptor on the other. We follow the distance between the motor domains during stepping with F?rster Resonance Energy Transfer. We use four different homodimeric kinesin constructs with dye molecules attached to different sites of the motor domain. With this approach, we can identify an intermediate state in the stepping process that lasts 2-3 ms at saturating ATP concentration. In this intermediate state one motor domain is bound to the microtubule and the other is rotated and substantially less than 8 nm away.

The conserved L5 loop establishes the pre-powerstroke conformation of the kinesin-5 motor, Eg5
Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, to L5, and to the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of STLC, an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of nucleotide state.

Work done in collaboration with Adam G. Larson, Nariman Naber, Roger Cooke, and Edward Pate.
FIONA looks at individual molecular motors walk and run
The standard diffraction limit of light is about 250 nm, meaning that you cannot "resolve" objects closer than this distance. Despite this, we have come up with a method to measure 1.5 nm in x-y plane, with 1-500 msec, using a technique we call Fluorescence Imaging with One Nanometer Accuracy (FIONA). FIONA also has the advantage that it looks at individual, or single, proteins. We have chosen to study molecular motors, both in vitro and in vivo. We find that all tested motors walk in a hand-over-hand fashion. We also find evidence that in vivo, two of the same motors carry cargo simultaneously-but not cooperatively. Because of limited signal-to-noise, this uses a new Hidden Markov Method technique. We also see passing of cargo from one type of motor to another. Finally, we find that we can extend FIONA to two-photon microscopy using quantum dots. Time permitting, we shall also discuss recent efforts where we can see individual ion channels, proteins which pass ions (such as sodium and potassium) between the inside and outside of the cell. This has enormous clinical ramifications, such as the study of strokes, Alzheimer's, and nicotine-addiction. Interesting mathematical tricks to improve the signal-to-noise will be discussed.
Joanna Sulkowska's Lecture
Joanna Sulkowska's Lecture
Mechanics of Actomyosin Interaction and the Role of Substrate Stiffness on Actin Network Dynamics
Myosin is a major molecular force generator in the cell. The essential features of myosin interaction with actin filaments are understood. In the cell, the interactions of myosin with F-actin, substrate adhesions and other actin-associated proteins are less clear. We will borrow ideas from mathematical models of skeletal muscle to develop simple models for integrin focal adhesions, actin cross-linking proteins and non-muscle myosin-II. When these components are combined, a dynamical picture of the actin network emerges. In this talk, we will focus on the role of cell-substrate stiffness on the actin dynamics. Possible implications for mechanical sensing by cells are discussed.
Viscoelasticity in homogeneous protein solutions
We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particles in the time range 1 millisecond - 10 seconds in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
Road signs for kinesin transport
We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particles in the time range 1 millisecond - 10 seconds in solutions of a model protein. The tested solutions exhibit significant elasticity at high frequencies, while at low frequencies, they are purely viscous. We attribute this viscoelasticity to a dense network of weakly-bound chains of protein molecules with characteristic lifetime of 10-100 ms. The found intrinsic viscoelasticity of protein solutions should be considered in biochemical kinetics models.
video image

The Function of Dynein in Budding Yeast: Mechanism, Regulation and Checkpoint Activity
John Cooper I will discuss work from our lab and other labs on how dynein functions to position the mitotic spindle and the nucleus in budding yeast. Interactions of cytoplasmic microtubules with the cortex control spindle position, and dynein represents one of

video image

The conserved L5 loop establishes the pre-powerstroke conformation of the kinesin-5 motor, Eg5
Sarah Rice Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resona

video image

Mechanics of Actomyosin Interaction and the Role of Substrate Stiffness on Actin Network Dynamics
Sean Sun Myosin is a major molecular force generator in the cell. The essential features of myosin interaction with actin filaments are understood. In the cell, the interactions of myosin with F-actin, substrate adhesions and other actin-associated proteins a

video image

Road signs for kinesin transport
Kristen Verhey We probe the transport properties in protein solutions stable with respect to any, solid or liquid, phase separation as a step in the understanding of transport in the cytosol of live cells. We determine the mean squared displacement of probe particl

video image

Motoring along a nucleic acid strand: template-dictated polymerization of macromolecules of life
Debashish Chowdhury Polymerases and ribosome are molecular machines which perform three important biological functions. Like cytoskeletal motors, each of these moves along a track using chemical energy for performing mechanical work. Moreover, it decodes genetic informa

video image

DNA chasing DNA: physics of homology recognition and the secret of perfect match
Alexei Kornyshev DNA chasing DNA: physics of homology recognition and the secret of perfect match