Overview

The workshop aims to unite researchers and practitioners who work towards the understanding of biological structures and dynamics from the molecular to the ecological scale and apply these insights to the engineering of novel materials, devices and systems.


Location

The workshop will take place in Columbia University in the City of New York.


Organizer

Prof. Henry Hess

 

Department of Biomedical Engineering, Columbia University

hhess@columbia.edu

Co-organizers

 

Dr. Gadiel Saper

Department of Biomedical Engineering, Columbia University

   
Registration

 

To register, please send an email to hhess@columbia.edu

 

Schedule

 

June 12th: 7:00-9:00 pm warm-up

June 13th June 14th June 15th
 
 
9:00 - 9:30 Welcome (Hess) Walter Paxton Ashutosh Agarwal
 
9:30 - 10:00 Ayusman Sen Tania Patino Allie Obermeyer
 
10:00 - 10:30 Amit Sitt Michael Shelley Dirk Trauner
 
10:30 - 11:00 Coffee Break Coffee Break Coffee Break
 
11:00 - 11:30 Zev Bryant Samuel Sanchez Akira Kakugo
 
11:30 - 12:00 Arne Gennerich Camelia Prodan Erik Schaeffer
 
12:00 - 12:30 Isabella Guido Ying Yang Kazuhiro Oiwa
 
12:30 - 2:00 Lunch (on your own) Lunch (on your own) Lunch (on your own)
 
2:00 - 2:30 Orit Shefi Jinglin Fu Stanislav Tsitkov/Hanie Yousefi
 
2:30 - 3:00 Milan Stojanovic Alf Mansson Aarat Kalra/Gadiel Saper
 
3:00 - 3:30 Helen Lu Loai Abdelmohsen Discussion
 
3:30 - 4:00 Coffee Break Coffee Break Coffee Break
 
4:00 - 4:30 Kyle Bishop Erin Barnhart
 
4:30 - 5:00 Takahiro Nitta Parag Katira
 
5:00 - 5:30 Discussion Discussion
 
5:30 - 6:00 Break Break
 
6:00 - 8:00 Dinner Poster Session
 
 

 

Invited Speakers (Confirmed)

 

Prof. Loai Abdelmohsen

TU Eindhoven

Exploiting Dynamicity to Induce Motility: Motion of Membranized Coacervate Motors

Understanding how different building blocks can be assembled into synthetic systems displaying cell-like architectures and functions is a major scientific challenge. Scientists have considered this multidisciplinary challenge as a way to provide insight into the fundamental processes of living systems, concurrently developing diverse potential applications of cell-mimicking constructs. In fact, the area of protocellular research has attracted growing interest in the last few years, and has showed significant progress in the design of biomimetic micro- and nano-compartments that mimic some structural and functional aspects of natural cells. Noteworthy, compartmentalization, or separation of materials from the external environment by a physical boundary (membrane) is a key hallmark for the origin of life – lipid cellular membranes are essential for hosting vital biochemical processes and maintaining the integrity of living cells. Following a bottom-up approach, several synthetic strategies for the creation of artificial compartments at different length scales have been developed, such us the assembly of polymeric vesicles (polymersomes), lipid vesicles (liposomes), virus capsids, colloidosomes, and coacervates. Remarkably, these compartments have been used for the reconstitution of certain cellular functions such as protein expression, metabolite synthesis, enzymatic cycles, transmembrane transport, and motion. Autonomous motion has been an important source of inspiration for scientists who, over the years, have created a variety of synthetic motor systems, imitating biological motility. Notwithstanding, there is a fundamental difference in the way movement is regulated in synthetic and natural systems. Cellular autonomous motion (e.g., vesicular transport and motility), displays adaptive features as a result of random dynamic processes, which are governed by enzyme-mediated energy input and consumption, and molecular interactions. Mimicking dynamic behaviors in synthetic systems has recently drawn much attention from the scientific community. In this presentation I will show how we couple motility of coacervate compartments to a dynamic process, which is maintained by stochastic events and how we compartmentalize such coacervates in giant liposomes and investigate their motion in confinement.

 

Prof. Ashutosh Agarwal

University of Miami

Human Organoids on a Chip

Organs on Chips are being designed as accurate models of healthy and diseased human organs. These collaborative ventures seek to replace the current paradigm of high quantity, low quality data with limited applicability to the human patient with mid-quantity, high quality data that will eventually be patient specific. However, multiple daunting, yet scientifically fertile roadblocks will need to be addressed along the way: identifying a robust source of primary or stem cell derived human tissues; recapitulation of organ-specific microenvironments and architectures; development of a universal culture media for multiple cell/tissue types; agile deployment of multiple chips through a network of pumps, valves, switches, collection ports, and sampling ports; integration of on-chip sensors and analytics for real-time evaluation of function; extrapolation of in-vitro results to in vivo outcomes; allometric scaling laws of organ chips with respect to each other; and finally, translating these devices in a scalable manner into the laboratories of basic scientists and pharmaceutical companies. This seminar will cover our contributions in each of these thrust areas.

 

Prof. Erin Barnhart

Columbia University

Dendrite architecture determines mitochondrial localization patterns in vivo

Reliable distribution of mitochondria throughout complex neuronal architectures is a challenging problem. By quantifying in vivo mitochondrial transport and localization patterns in the dendrites of Drosophila visual system neurons, we show that mitochondria make up a dynamic system at steady-state, with significant transport of individual mitochondria within a stable global pattern. Mitochondrial motility patterns are unaffected by visual input, suggesting that neuronal activity does not regulate mitochondrial localization in vivo. Instead, we present a mathematical model in which four simple scaling rules enable the robust self-organization of the mitochondrial population. Experimental measurements of dendrite morphology validate key model predictions: to maintain equitable distribution of mitochondria across asymmetrically branched subtrees, dendritic branch points obey a parent-daughter power law that preserves cross-sectional area, and thicker trunks support proportionally bushier subtrees. Altogether, we propose that “housekeeping” requirements, including the need to maintain steady-state mitochondrial distributions, impose important constraints on neuronal architecture.

 

Prof. Kyle Bishop

Columbia University

Quincke oscillators: Dynamics, synchronization, and assembly of self-oscillating colloids

Dielectric particles in weakly conducting fluids rotate spontaneously when subject to strong electric fields. Such Quincke rotation near a plane electrode leads to particle translation that enables physical models of active matter. We show that Quincke rollers can also exhibit oscillatory dynamics, whereby particles move back and forth about a fixed location. We explain how oscillations arise for micron-scale particles commensurate with the thickness of a field-induced boundary layer in the nonpolar electrolyte. Collections of Quincke oscillators assemble to form low-density lattices and close-packed crystals that exhibit varying degrees of phase synchronization and orientational alignment. We discuss how hydrodynamic and electric interactions can lead to attraction or repulsion depending on the relative position, phase, and orientation of neighboring oscillators. This system provides an experimental model for active matter based on many self-oscillating units.

 

Prof. Zev Bryant

Stanford University

Making and measuring macromolecular machines

Molecular machines lie at the heart of biological processes ranging from DNA replication to cell migration. We use single-molecule tracking and manipulation to characterize the structural dynamics of these nanoscale assemblies, and further challenge our understanding by designing and testing structural variants with novel properties that expand the functional range of known biomolecular machines. In the process, we are developing an engineering capacity for molecular motors with tunable and dynamically controllable physical properties, providing a toolkit for precise perturbations of mechanical functions. We have recently developed a new generation of light-responsive myosin motors, enabling precise control of fast and processive molecular transport in vitro and in living cells. I will describe our ongoing efforts to augment and diversify engineered cytoskeletal motors, including newly developed light-responsive filamentous myosins for control of contractility. I will further discuss our measurements of dynamics and mechanics in CRISPR endonucleases. In the latter work, we have used high-resolution multimodal single-molecule methods to study the DNA interrogation process, observing intermediate steps in target recognition and probing important effects of DNA torsion on the dynamics and specificity of these nucleoprotein machines.

 

Prof. Jinglin Fu

Rutgers University

Bio-mimetic Multienzyme Assembly by Nucleic Acids Nanostructures

Cellular functions rely on a series of organized and regulated multienzyme cascade reactions. The catalytic efficiency of multienzyme complexes depends on the spatial organization of composite components which are precisely controlled to facilitate substrate transport and regulate activities. If these cellular mechanisms can be mimicked and translated to a non-living artificial system, it can be useful in a broad range of applications that will bring significant scientific and economic impact. Self-assembled DNA nanostructures are promising to organize biomolecular components into prescribed, multi-dimensional patterns. Here, we described a robust strategy for DNA-scaffolded assembly and confinement of biochemical reactions. DNA nanostructures are exploited to organize spatial arrangements of multienzyme cascades with control over their relative distance, substrate channeling paths, compartmentalization, local confinement of ligands, as well as the construction of smart and biomimetic reactors. The combination of addressable DNA assembly and multienzyme cascades promises to deliver breakthroughs toward the engineering of novel biomimetic reactors, which have great potential for broad applications from chemical synthesis, functional biomaterials and biofuel production to therapeutics and diagnosis.

 

Prof. Arne Gennerich

Albert Einstein College of Medicine

Single-Molecule Studies of KIF1A Motion and Force Generation

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced the V8M and Y89D disease mutations. Both mutations dramatically reduce the force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates. In this talk, I will present these findings and discuss our newest structure-function and single-molecule optical tweezers studies that provide new insights into the coordination and force generation of the superprocessive KIF1A.

 

Prof. Isabella Guido

Max Planck Institute for Dynamics and Self-Organization

Exploring instabilities in 3D active matter

Networks of biopolymers and motor proteins are out-of-equilibrium systems useful for the understanding of emergent behaviour of active matter. The continuous supply of energy at the molecular scale enables the active constituents to generate internal forces and stresses that drive spontaneous motion. An interesting class of such systems are active nematics, fluids composed of self-organising elongated particles that in-vitro assemble into dynamic structures at length scales several orders of magnitude larger than those of their components. Here we present an experimental study on 3D active nematics made of microtubules, kinesin-1 motor proteins and a depleting agent. The network is subjected to the force exerted by the motors that crosslink the filaments and let them slide against each other. This intrinsic activity leads the system into diverse dynamical states that are sensitive to boundary condition or initial component concentration. In this way, the system evolves toward a flattened and contracted 2D sheet that undergoes a wrinkling instability in the third dimension and subsequently transitions to an active disordered state. We observe that the wrinkle wavelength is independent of the ATP concentration. A theoretical model describes its relation with the appearance time and a numerical simulation confirms the key role of kinesin motors in the contraction and extension of the network. Finally, we show how motor concentration and environmental cues influence the network properties.

 

Prof. Akira Kakugo

Hokkaido University

Cooperative cargo transportation by a swarm of biomolecular motors

Cooperation is a strategy that has been adopted by groups of organisms to execute complex tasks more efficiently than single entities. Cooperation increases the robustness and flexibility of the working groups and permits sharing of the workload among individuals. Here, we demonstrate molecular transportation through the cooperative action of a large number of artificial molecular machines, photoresponsive DNA-conjugated microtubules driven by kinesin motor proteins. Mechanical communication via conjugated photoresponsive DNA enables these microtubules to organize into groups upon photoirradiation. The groups of transporters load and transport cargo, and cargo unloading is achieved by dissociating the groups into single microtubules. The group formation permits the loading and transport of cargoes with larger sizes and in larger numbers over long distances compared with single transporters. We also demonstrate that cargo can be collected at user-determined locations defined by ultraviolet light exposure.

 

Dr. Aarat Kalra

Princeton University

Light at the End of a Nanotunnel: Microtubules as Optical Devices

The next generation of nanotechnological devices must successfully integrate biochemistry with electronics. Microtubules, tubulin protein polymers found ubiquitously in eukaryotic cells, are ideal candidates for this task. The protein α, β tubulin is stacked linearly in 13 columns (called protofilaments) which associate laterally forming the cylindrical microtubule. The regular, repeating arrangement of tubulin confers mechanical strength to microtubules, allowing their role as structural supports in a variety of nanodevices. Short distances between aromatic amino acids in tubulin and the ordered packing of tubulin in a microtubule may also allow them to act as devices that transport electronic energy outside the cell. Here, we studied tubulin autofluorescence to examine electronic energy transfer between tryptophan residues in microtubules. We measured tryptophan fluorescence lifetimes in the presence of quenching agent to determine photoexcitation diffusion length in microtubules. We found that our observations could not be sufficiently explained by Förster theory, with diffusion lengths resembling those in photosynthetic complexes. While the presence of etomidate and isoflurane influenced tryptophan photophysics, changing the number of protofilaments (13 versus 14) did not have any significant effect. Our studies show that microtubules may act as long-range electronic energy transport channels working outside physiological conditions, integrating biological complexes with engineered materials.

 

Prof. Parag Katira

San Diego State University

Acto-myosin contractility: From individual motor stepping to emergent phenomena during cell migration

Numerous models describing force generation by myosin motors interacting with actin filaments exist, particularly in the context of muscle cell function and contractility, but also in the context of cell-substrate interactions mediated via actin-myosin stress fibers and focal adhesions. The point of these models is to explain biological phenomena and gain insight into the inner workings of cells. Recently we encountered cell migration phenomena displayed by highly metastatic cancer cells that could not be explained by existing models. This led us to revisit and revamp some of the existing models of actin-myosin driven contractility, formation, growth, and dissolution of focal adhesion complexes. In my talk, I will describe these revamped models and discuss applications and insights gained from them.

 

Prof. Helen H. Lu

Columbia University

 

Prof. Alf Mansson

Linnaeus University

Single molecule fluorescence data suggest multistep substrate binding and product release in ATP turnover by myosin, of potential significance in energy transduction

An optimized TIRF microscopy based assay was developed to study ATP turnover by myosin motor fragments on glass surfaces. After eliminating various artefacts through aggressive surface cleaning and use of several triple state quenchers and redox agents we found that the distributions of ATP binding dwell times on myosin are best described by the sum of at least 2 to 3 exponential processes. This applies with and without actin. Two of the processes are due to ATP turnover by myosin and actomyosin, respectively. A remaining process (rate constant of 0.2-0.5 s-1) is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests a role in accelerated ATP transport to the active site. Studies of conditions with no sliding between actin and myosin, as in isometrically contracting muscle, reveals heterogeneity in the ATP turnover kinetics, previously predicted by modelling. We also found that the amplitudes of all exponential processes were inhibited by addition of orthophosphate (Pi) in the 0.1 – 40 mM range suggesting that Pi competitively inhibits non-specific binding of ATP to myosin. We interpret the latter effect as reflecting one aspect of multistep release of Pi from the active site. Other aspects of this mechanism as well as its functional significance will be discussed during my talk.

 

Prof. Takahiro Nitta

Gifu University

A Printable Actuator by Engineered Kinesin Motors

Animal muscles have given inspirations to engineers in designing and developing actuators, where myosin motors are assembled in a highly organized manner to scale up their molecular scale actions into macroscopic ones. Here, we present a soft actuator by engineered kinesin motors which can drive millimeter scale objects and is compatible with printing technology. Upon illumination of UV, the engineered kinesins self-assembled into kinesin filaments, which subsequently induced contraction of the actuator. Using spatially patterned illuminations, the actuators were formed in designated locations, enabling efficient integrations of the actuators into mechanical components. The force generated by the actuators reached micronewton range, which is sufficient to actuate mechanical parts of millimeter scale. Computer simulations revealed contraction behaviors of the actuators, and gave physiological implications on structural basis of skeletal muscles. Having a large force generation and being compatible with printing technology, we believe this actuator by engineered kinesin motors would find applications in micro- and soft robotics.

 

Prof. Allie Obermeyer

Columbia University

Protein complex coacervates as models of biomolecular condensates

Compartmentalization of cellular components helps facilitate the complex combination of bioprocesses that must occur simultaneously to maintain life. While traditionally cellular compartments have been defined by the presence of a lipid bilayer, it has recently been appreciated that many cellular compartments are not bounded by a semi-permeable membrane. These compartments, termed biomolecular condensates, arise by phase separation and/or percolation of biomacromolecules. Some of these biomolecular condensates appear to have similar properties as complex coacervates, or liquid-liquid phase separated mixtures of oppositely charged polyions. Despite these similarities, protein polyions differ significantly from the synthetic polyelectrolytes most often used to create complex coacervates. Here we develop protein-based complex coacervates as models of biomolecular condensates. We first developed design rules for the formation and stability of protein coacervates in vitro and in cells and now look to impart out-of-equilibrium behavior to these otherwise static materials.wo

 

Prof. Kazuhiro Oiwa

ICT Research Institute

Creation of novel molecular motors and chemo-sensors on the basis of motor proteins’ function and bacterial chemotaxis behavior

Applying biological materials and functions to artificial devices paves the way to development of advanced smart systems. We have used protein motors and bacterial chemotaxis to create novel molecular motor and sensors. Molecular motors found in creatures, such as myosins, kinesins, and dyneins, are ATPases and so unique that they convert the chemical energy coupled with ATP hydrolysis directly into force and movement. Despite their nanometer sizes, they move unidirectionally on protein filaments under stormy thermal agitation. The decades of intensive studies, however, have not revealed the essence of this directional motion: we do not know how the structural change of motors and the asymmetric structures of the motor-filament interface contribute to the directional motion. Understanding the essence of directional motion has been hampered by the limitation that neither motors nor cytoskeletal protein filaments can be rationally re-designed in order to address key questions. To overcome the limitation, we took the bottom-up approaches, in which we designed new molecular motors and tracks and created them using protein- and DNA-building blocks [1]. We constructed hybrid motors of dynein and DNA-binding proteins. DNA was used as a track instead of cytoskeletal filaments because of its stability and abilities of synthesis and self-organization. In in vitro motility assays, these hybrid motors moved 10-helix DNA nanotubes at the mean velocity of 8 nms-1. Furthermore, the ensemble of hybrid motors transported single DNA origami cargoes along immobilized DNA nanotubes. The hybrid motors are shown to recognize the specific DNA sequence that is periodically incorporated into the DNA nanotube. Therefore, our strategy has paved the way into systematic approaches to the motor mechanism and to nanotechnological applications using large repertories of DNA-based molecular tools. In addition, we have developed a biosensor system on the basis of bacterial chemotaxis [2]. Chemo-sensing is of vital importance in survival of any organism. In bacterial chemotaxis, multiple receptors sense chemicals to regulate a single signalling system controlling the direction (counter clockwise or clockwise) of flagellar rotation. Such an integrated system with binary output seems better suited to judging chemicals either favourable or unfavourable but not to detecting their identity. Here we developed a setup to monitor behaviours of multiple cells stimulated simultaneously as well as a statistical framework based on Bayesian inferences. Although responses of individual cells varied substantially, ensemble averaging of their time courses appeared characteristic to attractant species, indicating the bacterium retains information of input chemical species. These results provide a basis for novel bio-inspired sensors that could install other cell types as well to sense wider ranges of chemicals.
1. R Ibusuki, et al., (2022) Programmable molecular transport achieved by engineering protein motors to move on DNA nanotubes. Science 375, 1159-1164. https://doi.org /10.1126/science.abj5170
2. H Tanaka, et al., (2022) Bayesian-based decipherment of in-depth information in bacterial chemical sensing beyond pleasant/unpleasant responses. Scientific reports 12, 2965, https://doi.org/10.1038/s41598-022-06732-4

 

Prof. Tania Patino

TU Eindhoven

Bioengineering motile life-like nanosystems powered by enzymes

 

Prof. Walter Paxton

Brigham Young University

A Tale of Two Systems: Catalysts for Renewable Energy and for Morphogenesis of pH-Responsive Assemblies

Spatially-confined reaction sites can be exploited to drive mechanical actuation, pattern surfaces with nanoscale precision, and create and dissipate chemical potential gradients, and mimic biological processes. In this talk, I will discuss two recent examples of catalysis from my lab for (i) the electrocatalytic oxidation of carbohydrates to produce electrical energy, and (ii) for the enzyme-mediated reorganization of pH-switchable amphiphiles to produce responsive micelles, gels, and giant vesicles.

 

Prof. Samuel Sanchez

Institute for Bioengineering of Catalonia

Enzyme machines powering swarms of enzyme-nanobots going in vivo

One of the dreams in nanotechnology is to engineer small vehicles and machines, called here nanobots, which can eventually be applied in vivo for medical purposes. Yet, reaching that fascinating goal is not a trivial thing and several challenges need to be addressed. First, researchers need to incorporate efficient but also bio-friendly propulsion mechanisms into the nanobots. Our strategy comprises the use of biocatalysts such enzymes for converting biologically available fuels into a propulsive force. Secondly, nanoparticles’ chassis should be generally recognized as safe (GRAS) material, biocompatible and/or biodegradable. In my talk, I will present how we bioengineer hybrid nanobots combining the best from the two worlds: biology (enzymes) and (nano)technology (nano- micro-particles) providing swimming capabilities, biocompatibility, imaging, multifunctionality and actuation. Besides the understanding of fundamental aspects (1), and controlling the performance of micro-nanobots (2) I will present some of the proof-of-concept applications of biocompatible nanobots such as the efficient transport of drugs into cancer cells (3) and 3D spheroids (4), sensing capabilities (5), antibactericidal applications (6) and the use of molecular imaging techniques like PET-CT (7) or Photoacoustic (8) for the tracking and localization of swarms of nanobots both in vitro and in vivo in confined spaces like mice bladder.

(1) Arqué et al. Nat. Commun. 2019. 10, (1) 1-12.; Patino et al. Acc. Chem. Res. 2018, 51, (11) 2662-2671 (2) Patino et al. J. Am. Chem.Soc. 2018, 140 (25) 7896-7903 (3) Hortelao et al. Adv. Funct. Mat 2018, 28, 1705086 (4) Hortelao et al. ACS Nano 2019, 13, (1), 429-439 (5) Patino et al. NanoLett. 2019, 19, (6), 3440-3447 (6) Arqué et al. ACS Nano 2022, 16, 5, 7547–7558 (7) Hortelao et al. Sci. Robotics. 2021, 6, (52), eabd2823. (8) D. Xu et al. ACS Nano, 2021, 15 (7), 11543-11554

 

Gadiel Saper, PhD

Columbia University

Robotic end-to-end fusion of microtubules powered by kinesin

The active assembly of molecules by nanorobots has advanced greatly since “molecular manufacturing”—that is, the use of nanoscale tools to build molecular structures—was proposed. In contrast to a catalyst, which accelerates a reaction by smoothing the potential energy surface along the reaction coordinate, molecular machines expend energy to accelerate a reaction relative to the baseline provided by thermal motion and forces. Here, we design a nanorobotics system to accelerate end-to-end microtubule assembly by using kinesin motors and a circular confining chamber. We show that the mechanical interaction of kinesin-propelled microtubules gliding on a surface with the walls of the confining chamber results in a nonequilibrium distribution of microtubules, which increases the number of end-to-end microtubule fusion events 20-fold compared with microtubules gliding on a plane. In contrast to earlier nanorobots, where a nonequilibrium distribution was built into the initial state and drove the process, our nanorobotic system creates and actively maintains the building blocks in the concentrated state responsible for accelerated assembly through the adenosine triphosphate–fueled generation of force by kinesin-1 motor proteins. This approach can be used in the future to develop biohybrid or bioinspired nanorobots that use molecular machines to access nonequilibrium states and accelerate nanoscale assembly.

 

Prof. Erik Schäffer

Universität Tübingen

Dissecting the Gait of Molecular Motors: Ultraresolution Optical Trapping Using Germanium Nanospheres

Simultaneously measuring the nanoscale motion and forces that molecular machines generate provides insights into how they work mechanically to fulfill their cellular function. To study these machines, we developed germanium nanospheres as probes for optical tweezers. With these high–refractive index nanospheres, we have improved the resolution of optical tweezers and discovered that the motor kinesin takes 4-nanometer substeps. Further, instead of detaching from their microtubule track under load, motors slid back on it, enabling rapid reengagement in transport. Germanium nanospheres are promising for bioimaging, sensing, optoelectronics, nanophotonics, and energy storage. For optical trapping, the nanospheres open a new temporal window by which to uncover hidden dynamics in molecular machines.

 

Prof. Ayusman Sen

Pennsylvania State University

Enzyme Catalysis-Induced Lateral Lipid Motility and Particle Transport on Membranes

The dynamic interplay between the composition of lipid membranes and the behavior of membrane-bound enzymes is critical to the understanding of cellular function and viability, and the design of membrane-based biosensing platforms. While there is a significant body of knowledge on how lipid composition and dynamics affect membrane-bound enzymes, little is known about how enzyme catalysis influences the motility and lateral transport in lipid membranes. Using enzyme-attached lipids in supported bilayers (SLB), we show catalysis-induced enhanced lateral diffusion of lipids in the bilayer. We also provide direct evidence of catalysis-induced fluctuations leading to the enhanced diffusion of passive tracers resting on the SLB. Additionally, by using active enzyme patches, we demonstrate the directional transport of tracers on SLBs. These are first steps in understanding diffusion and transport in lipid membranes due to active, out-of-equilibrium processes that are the hallmark of living systems. In general, our study demonstrates how active enzymes can be used to control diffusion and transport in confined 2-D environments.

 

Prof. Orit Shefi

Bar-Ilan University

Nano-engineered platforms for controlling neuronal growth

The ability to manipulate and direct cells in the nervous system has great implications in basic science and therapeutics. Physical mechanical forces, contact guidance cues and chemical cues play key roles in neuronal regeneration. In this talk I will present our recent studies of platforms for controlling neuronal growth and monitoring their activity that are based on interactions with nanometric elements and combine several of these cues. Such systems include 2D substrates embedded with nanotopographical cues, 3D hydrogel scaffolds with organized nano-structure, manipulations with magnetic nanoparticles that interact with neurons or embedded within them turning them into magnetic sensitive elements. I will present platforms that are based on several materials and include in vitro as well as in vivo assays. Our results demonstrate our ability to manipulate neuronal growth and affect neuronal regeneration.

 

Prof. Amit Sitt

Tel Aviv University

Polymeric Mesofibers Based Transformers: More than Meet the Eye

Shape-morphing active networks of mesoscale filaments are a common hierarchical feature in biology used for applying forces, transporting materials, and inducing motility with microscale resolution. Constructing synthetic morphing systems of similar dimensions and capabilities is challenging, yet holds potential for a range of technological applications, from micro-muscles to shape-morphing optical devices. In the first part of the talk, I will present a novel type of purely synthetic responsive 2D networks hierarchically constructed of thermoresponsive mesoscale polymeric fibers developed in my lab, which can exhibit morphing with microscale resolution. I will demonstrate that the morphing of such networks strongly depends on the physical attributes of the polymer itself and on on two intrinsic length scales - the fiber diameter and mesh size, which stems from network’s density. Depending on these parameters, such fiber-networks exhibit extremely different thermoresponsive driven morphing behaviors. However, the networks display memory and regain their original morphology upon shrinking. In the second part of the talk, I will discuss the self-fracturing of microfibers fabricated from custom-made amphiphilic tri-block copolymers. Such fibers spontaneously fracture periodically into relatively uniform microscale fragments whose length is dictated by the thickness of the fiber. Upon exposure to humid conditions, the amphiphilic nature of the copolymer promotes the adsorption of water and the swelling of the fragments, and can also result in healing of the fibers, returning them to their original, unfractured form.

 

Prof. Milan Stojanovic

Columbia University

An Update on Practical Applications of Molecular Computing in the Field of Cell Separation

 

Prof. Dirk Trauner

New York University

Optical Control of the Cytoskeleton Using Molecular Photoswitches

The tracks, nucleators, and molecular motors of the actin and tubulin cytoskeleton can be influenced with small molecules, which can be rendered photoswitchable. I will discuss the development and usefulness of molecular photoswitches that target F-actin, G-actin, microtubules, and the mitotic kinesin Eg5.

 

Stanislav Tsitkov, PhD

Massachusetts Institute of Technology

Computational approaches to identify ALS disease signatures from multi-omic data in a heterogeneous patient population

A challenge in the identification of disease-related signatures in heterogeneous patient populations is that standard differential analysis methods, which generally rely on location test statistics (i.e. sample means), are not effective. The application of signal deconvolution methods toward real clinical data could both identify these signatures and serve as a testbed to evaluate method efficacy in a clinical setting. One such setting is the study of amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disease that primarily affects motor neurons and exhibits significant clinical heterogeneity. The Answer ALS consortium has generated a comprehensive open-source multi-omic database to help identify biochemical mechanisms contributing to subtypes of ALS. The omics data, consisting of epigenomics (bulk ATAC-seq), transcriptomics (bulk RNA-seq), and bulk proteomics, are generated from iPSC-derived motor neuron cultures from ALS patients and controls. This combination of multiple modalities matched across individuals has enabled the testing of new multi-omics analysis methods to elucidate pathways underlying ALS. Here, we apply independent component analysis (ICA), non-negative matrix factorization (NMF), and co-expression analysis to identify both disease-related signatures shared across modalities and molecular signatures associated with differentiation and clinical covariates. These techniques reveal groups of chromatin regions, genes, and proteins that are significantly associated with ALS, but are overlooked by standard differential analysis approaches. These results are being used to generate experimentally-testable hypotheses of pathways contributing to ALS pathology, but further studies are needed to assess the utility of these methods in other clinical settings.

 

Prof. Ying Yang

University of Nevada Reno

Dithioacetal-Based Dynamic Polymers with Entropically Driven Ring-Chain Equilibrium

Traditional polymers rely on stable covalent bonds; however, biochemical systems depend on dynamic transformations of macromolecular structures to perform living functions. Dynamic covalent polymers based on numerous functional groups such as disulfides, thioesters, lactones, carbonates, and acetals have received increasing interest in the past decade. One dynamic covalent bond that has received only limited attention in the field of polymer chemistry but has been widely used in synthetic organic chemistry is dithioacetal. Although dithioacetals are relatively stable, they can be activated by using acid catalysts. Given their straightforward synthesis, tunability, and dynamics, we investigated dithioacetal as a reversible bond in developing recyclable polymers. We synthesized linear polydithioacetals which exhibited good mechanical strength. Ring-closing depolymerization of polydithioacetals in refluxing toluene with zinc(II) triflate catalyst yielded a mixture of cyclic dithioacetals of various ring sizes. Acid catalyzed ring-opening polymerization of these cyclic dithioacetal monomers at room temperature afforded the linear polydithioacetals. These results demonstrated polydithioacetals as promising candidates for use as polymers capable of chemical recycling to monomers. In this presentation, we will highlight the syntheses of the polydithioacetals, thermodynamics and kinetics of the reversible ring-closing and ring-opening processes, the effects of ring sizes, and the material properties.

Poster Session

 

Niladri Sekhar Mandal

The Pennsylvania State University

Enzyme chemotaxis: Kinetic asymmetry determines the evolution of chemical systems

Enzyme chemotaxis is the motion of enzyme molecules towards or away from a concentration gradient of their respective substrate and product molecules. Chemotaxis of enzymes has significant implications for the evolution of dissipative chemical systems and the emergence of organized life from simple biomolecules. Herein, we propose a mechanism for chemotaxis based on Brownian diffusion and reaction kinetics. We show that the direction of chemotaxis is dependent on two quantities: 1) Kinetic asymmetry, the difference between the unbinding rates of the substrate and the product and 2) diffusion asymmetry, the difference in the diffusivities of the unbound and the bound form of the enzyme. We employ trajectory thermodynamics to analyze the distribution of enzymes subjected to a gradient of substrate and product. The importance of kinetic and diffusion asymmetry is examined using trajectory thermodynamics and is then confirmed by solving mass balance equations. It has been suggested that in the evolution of matter to life-like structures the most favored state is the one with the maximum rate of dissipation. In contrast, we are able to show that the rate of dissipation does not play any role in the determining the direction of chemotaxis. Thus, we conclude that kinetic and diffusion asymmetry constitute the key driving forces behind enzyme chemotaxis and further suggest may play a role in the evolution of simple matter into complex systems.

 

Oz Mualem

Bar-Ilan University

Neuronal cell manipulations and analysis at the single cell level

 

Juan Bautista Rodriguez

Columbia University

Mechanochemistry (not severing enzymes or MAP unbinding) accounts for microtubule breaking

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

 

Joseph Rubin

Columbia University

Towards Characterization of Glycosylation Patterns of Single IgA Molecules

IgA1 nephropathy, the most common form of primary nephropathy, is triggered by the damage to glomeruli from deposition of complexes formed between polyclonal IgA1 antibody proteins that are “galactose-deficient” (GD-IgA1) and antibodies directed to this GD-IgA1. Glycosylation patterns on IgA1’s are highly variable and are globally shifted towards those structures that have more GD-forms in IgA1 patients. We are working towards creating an assay to characterize these shifting patterns at the level of single IgA molecules using single molecule fluorescence measurements. Similar measurements, when applied to patient samples, may be used for early detection and understanding of the etiology of IgA1 nephropathy. Alexa Flour 647 labelled IgA1 in HBS was deposited on a borosilicate glass surface and rinsed out with HBS and blocking reagent. Alexa Fluor 488 labelled Jacalin was flowed in with various concentrations of Galactose. The flow cells were illuminated via Total Internal Reflection illumination by 488 nm and 647 nm lasers. Due to strong background emission from Jacalin in solution and Jacalin non-specifically binding to the glass, Fluorescence Resonance Energy Transfer (FRET) was used to observe Jacalin binding to IgA1. FRET images and IgA1 images were digitally overlayed, enabling us to record binding and unbinding events. Addition of the competitive binder Galactose to the solution led to an increase in the off times of Jacalin and IgA1 and a decrease in the on times in a concentration-dependent manner, yielding an increase in the dissociation constant demonstrating that the Jacalin is binding specifically to Glycans on IgA1.

     
       

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