2023 Posters
NMR Structural Characterization and Modeling of Spider Silk Assembly
Alexia de Loera
Using Silk-like PeptidesSpider silk continues to be studied due to its unique properties and potential applications in different fields, such as in biomedical applications as potential sutures and in material sciences due to its impressive mechanical characteristics and biocompatibility. The main technique used in our lab to better understand spider silk protein assembly is solution NMR. The protein sequence of the major ampullate spidroin 1 (MaSp1) of the latrodectus hesperus (black widow) spider has already been determined. It is known to be mainly made up a GGX repeating unit (where X is A, Q, Y or R) in between poly(A) runs. In this study, the sample analyzed is an isotope labeled 27 amino acid peptide that mimics the GGX domain that appears fourteen times in the MaSp1 protein sequence. The purpose is to use peptides as a minimalist approach to better understand the secondary and tertiary structure of MaSp1.
Study of filaments of the nematodes T. Aceti using various oils
Alyssa Agarie
Previous research indicates that Turbatrix aceti, commonly known as vinegar eels, can synchronize their body oscillations under favorable conditions, such as the droplet's contact angle ( θ > 68°) to produce metachronal waves. It is unknown whether nematodes can continue to produce fluid flow under various chemical compositions and viscosity. I prepared the experiment by combining 100 μl of nematode solution with oils containing various viscosities. Using tracking software imageJ, I analyzed the data to measure four components: the oscillation frequency inside and outside the oil, number of filament splits, density of nematodes per region, and how fast the filaments are advancing. I predicted the oils with low viscosity to have greater movement and spread out faster in comparison to the very viscous oils. My results confirm this statement. To further continue the research, I will create a program tracking nematodes to minimize data error.
Calibration and Applications of Optical Tweezers for Microrheological Investigation of Complex Fluids
Anna Krolik, Muping Wang
Optical tweezers have emerged as a powerful tool for manipulating microscale particles. This research project focuses on the construction and calibration of stiffness-based optical tweezers, augmented by its applications in microrheology. Through the use of lenses and mirrors, the laser path was fine-tuned to produce a functional optical trap. The trap stiffness was determined by analyzing power spectrum data from assessing displacement and velocity of a polypropylene microsphere during oscillatory motion. The calibrated optical tweezers were then used to investigate the microrheological properties of complex fluid systems. Specifically, the study concentrated on determining the viscosities of sodium caseinate and sodium carboxymethyl cellulose solutions through observation of the Brownian motion of microspheres, and quantifying these measurements by analyzing the power spectrum. Using optical tweezers to understand microrheology and fluid behavior paves the way for future work in material science, biomedical engineering, and soft matter physics.
Effects of Secondary Structure on Complex Coacervation
Anna Nguyen
The phase separation of oppositely charged macromolecules is primarily driven by electrostatic interactions, but other interactions such as pi-pi, pi-cation, and hydrophobicity can play a role. While the influence of these non-electrostatic interactions on phase separation is extensively studied, comprehending the impact of macromolecular structure remains incomplete. We hypothesize the conformation of biomacromolecules influences phase behavior from entropic contributions of internal flexibility and differences in charge density/spacing. To investigate this, we leverage DNA’s programmability to study single-strands, double-strands, and hairpins complexation with poly-l-lysine. Our work is summarized in a phase diagram with curves for each structure which highlights the similarities and distinctions in critical salt concentration and dense DNA phase. Ultimately, our research provides insight into how molecular structure modulates the composition and formation of biomolecular condensates.
Mechanisms of a DNA-translocating viral packaging motor: regulation of DNA gripping and friction between motor proteins and DNA
Brandon Rawson
Many viruses employ molecular motors during assembly to translocate their DNA genomes into viral procapsid shells. To investigate the interactions between motor proteins and DNA we developed a single-molecule DNA grip/slip assay with rapid solution exchange to probe effects of nucleotide binding/dissociation in phage lambda motors containing both the TerL and TerS subunits. Current models for motor function propose that ATP binding induces tight DNA gripping. Surprisingly we measure frequent DNA gripping and high motor-DNA friction even in the absence of nucleotide. This was not observed in phage T4 motors containing only TerL. We propose TerS plays an important role in increasing motor processivity by functioning as a "sliding clamp" to limit motor slipping and limit back slipping when TerL loses grip.. Additionally, we show that lambda has a "DNA end clamp" mechanism that prevents the viral genome from completely exiting the capsid once packaging has initiated.
Studying the Structure of Protein-Polymer Hybrid Materials using Small-Angle Neutron Scattering Techniques
Dong Le
Polymer-integrated protein crystals (PIXs) have captured attention as compelling materials that can exhibit the periodicity and structural order of crystals and the mechanical flexibility of polymeric networks, presenting the vast potential for applications in biomaterials, biocatalysts and drug delivery [1-2]. However, the structure and interaction of the polymer network within the protein crystal lattice remain significant challenges, thereby hindering the understanding required for further synthesis and control. Here, we presented two current studies addressing these gaps using Contrast Variation Small-Angle Neutron Scattering (CV-SANS) and Time-Resolved Small Angle Neutron Scattering (TR-SANS) techniques: The first project uses CV-SANS to investigate the nanostructure of polymers incorporated into protein crystals. By adjusting the hydrogen (H) and deuterium (D) ratio in the solvent, a series linear equation can be used to extrapolate the component scattering functions [3-4]. This approach will provide insights into the structure and arrangement of polymer within the crystal and pave the way for future research and optimization of these hybrid materials. The second project investigated the colloidal behavior of polymer-ferritin protein post-dissolution in D2O. This approach, renowned for the non-destructive analysis of SANS for biological macromolecules in solution, enables studying dynamic processes and the structural evolution of these macromolecules. By studying polymer-ferritin protein interactions in aqueous environments, we hope to contribute to the fundamental understanding of colloidal behavior, which has implications in fields like targeted drug delivery and bio-inspired nanomaterials development.
DNA-DNA sliding friction and non-equilibrium dynamics in viral genome ejection and comparison with polymer simulations
Doug Smith, Mounir Fizari
We introduce a new method to measure viral DNA ejection by using optical tweezers to pull single DNA molecules out from single virus capsids. We find that despite high driving pressure ejection from phage phi29 is initially very slow due to due to the very high DNA packing density, which restricts the mobility of the DNA biopolymer. Many polymer simulation studies have modeled this process and we confirm several of their predictions at a qualitative level. However, we show that these existing simulations fail to quantitatively predict the dynamics. We provide evidence that DNA-DNA friction not properly modeled by simulations greatly slows the initial ejection and that stochastic pausing observed during ejection is connected with the phenomenon of “clogging” exhibited by many soft matter systems. We present detailed comparisons of our experimental findings with polymer simulation studies and experimental and theoretical studies of clogging in soft matter system.
Utilizing Convolutional Neural Networks to Enhance Differential Dynamic Microscopy
Emmie Kao
Probing phosphate-induced liquid-liquid phase separation in spider silk proteins using solution NMR
Hannah Johnson
Multivalent anions such as phosphate have been shown to induce liquid-liquid phase separation (LLPS) in spider silk proteins before they have assembled into fibers. The LLPS process is now believed to be an intermediate pre-assembly step that leads to fibers with superior mechanical properties. This work seeks to understand the atomic level interactions that occur during phosphate exposure. Using solution NMR, spider silk glands were analyzed both in the presence and absence of potassium phosphate. Since LLPS has been shown in other protein systems to be stabilized by - and cation- interactions, a labeling scheme was developed to isotopically enrich arginine and tyrosine in native L. hespures (black widow) major ampullate silk proteins. We hypothesize that as silk proteins transition from a chaotropic to a kosmotropic environment in the spinning duct of the silk gland, phosphate facilitates more controlled nucleation and assembly of silk fibrils through pre-assembly of poly(Ala) regions.
Modeling Active Mechanics and Substrate Interaction of Cytoskeletal Protein Networks
Jonathan Michel
Mechanical process in cells, such as locomotion, cargo transport, and division, arise from complex interactions involving a multitude of constituents. The availability of purified cytoskeletal proteins has allowed a bottom-up study of cellular processes, with the aim of reproducing cellular behaviors with a minimal subset of ingredients. Recent advances in microfabrication and click chemistry offer the hope of constructing experiments to study how cells interact with substrates and obstacles. Moreover, there is hope that ingredients evolved over eons can be repurposed to make prototypes of soft machines. In this work, I will discuss agent-based modeling of the coalescence and active mechanics of bulk gels of actomyosin, which occurs naturally in the cell cortex. I will also discuss modeling of the interaction of actomyosin gels with a soft, passive substrate, in which an agent-based model is coupled to a finite element framework for elastodynamics.
Building Cytoskeleton Networks Around Cells and Hydrogels
Katarina Matic
Characterizing the Structure of Two Different Spider Silk Fibers Using Solid-State NMR.
Kevin Chalek
Major Ampullate (MA) silk is used by spiders in the formation of their webs and is the strongest type of spider silk. This strength comes, in part, from repetitive poly(Ala) and poly(Gly-Ala) regions that form nanocrystalline β-sheet domains in the silk fiber. The more disordered, non-β-sheet regions remain relatively unexplored. To further study the interactions that take place in these disordered regions, our lab has developed a new selective 1D 13C-13C spin-diffusion solid-state NMR (SSNMR) experiment. Aciniform (AC) silk is used by spiders to wrap their prey and is the toughest type of spider silk. AC silk is α-helical rich, exhibiting a coiled-coil conformation with a low β-sheet content (~15%). Despite the physical similarities of these two types of silk, their conformational and hierarchical differences in structure show how each has their own unique mechanical properties. SSNMR structure work is further supported by molecular dynamics simulations.
Pushing and pulling on the nucleus: the role of vimentin in nuclear shape
Maxx Swoger
The nucleus is the organelle of the cell responsible for controlling protein expression, which has direct effects on cell mechanics. Vimentin is a type of intermediate filament, highly expressed in motile cells, that forms a cage around the nucleus. Recent studies have shown that mechanical forces on the nucleus result in changes to what genes are expressed. The role of F-actin and microtubules in force transmission to the nucleus has been well studied, but the role of vimentin is still unclear. However, recent studies have shown the vimentin cage prevents nuclear rupture and DNA damage that cells may experience when moving. In this study we investigate how vimentin mediates force transmission to the nucleus by examining nuclear morphology and response to external stress created by compression or geometric constraints. These results will help determine how vimentin can be incorporated into a model of nuclear mechanosensing.
Engineering Adaptable Materials via Circadian Clock Proteins
Maya Nugent
Cyanobacteria utilize circadian clock proteins which oscillate between bound and unbound states over a period of 24 hours. The cyclic binding of the proteins is robust and can be manipulated into permanently bound or unbound states. These proteins, known as Kai proteins, can be exploited in order to crosslink synthetic microspheres. Over time, the system will oscillate between crosslinked and uncrosslinked states, creating a novel material that autonomously switches between gel-like and fluid-like states. Understanding and further developing this system will allow us to adapt this system to different conditions and create new crosslinked materials. Combining the rhythmic robustness of biological materials with adaptable and manipulable synthetic materials opens countless doors for pharmaceutical enhancement and biological infrastructure.
Thermoresponsive poly(N-isopropylacrylamide) Microgels with Cheerios Structures
Nathan Mermilliod
Thermoresponsive poly(N-isopropylacrylamide) (pNIPAm) microgels were synthesized by a three-step addition of monomer NIPAm within the first hour of the reaction. These microgels are designed to be larger and synthesized without any exogenous crosslinker making them ultra-low crosslinked in density. We found that the microgel particles are ~1.5 μm in diameter and form cheerios like hollowish structures. We also found that these cheerios like microgel particles are extremely robust and preserve their mechanical stability in a very high osmotic pressure. We hypothesize that these cheerios like microgels are formed during the subsequent addition of same monomers which effectively dominates the rate of the reaction at its critical growth phase. We are currently trying to understand how these particles are formed, shaped and stabilized during the reaction.
Heterogenous Population of Kinesin-Streptavidin Complex Revealed by Mass Photometry
Nathaniel Brown
The kinesin-streptavidin complex is widely used to drive filament-filament sliding in microtubule-based active matter studies. Although the stoichiometry of the kinesin-streptavidin complex is generally assumed to be 2:1, this assumption has not been experimentally verified. Here we employ mass photometry, a label-free single-molecule technique, to determine the mass of individual kinesin-streptavidin complexes in solution. We found that the complex population is heterogenous, although the relative abundance of different complexes depends sensitively on the kinesin:streptavidin incubation ratio. We identify an incubation ratio that maximizes the 2:1 complex stoichiometry optimal for filament-filament sliding in active matter studies.
Measuring the Hydrodynamics of Kinteoplast DNA with Microfluidics
Nick Cuomo
The discovery of graphene opened the door to studying many types of two-dimensional crystalline materials, but two-dimensional soft materials have also garnered interest. To find a suitable model for soft two-dimensional polymers, we have studied the properties of kinetoplast DNA (kDNA). Found in the mitochondria of the trypanosome parasite, kDNA are comprised of topologically interconnected small loops of DNA which create an effectively two-dimensional catenated network. Using microfluidics with non-uniform flow profiles, the hydrodynamic properties of the kDNA were measured. The key variable was the anisotropy of the kDNA as it flowed thought the microchannels, both with and without the presence of an external electric field. We found that the change in anisotropy as the kDNA moved through the microchannel was related to the size of the individual kDNA and the speed at which the kDNA were traveling.
Shapeshifing Chiral Liquid Crystals
Paco Navarro
Shapeshifting materials are of vast commercial interest in the world of materials science, due to their multifunctional nature and scalability. However, these are complex systems with many non-trivial parameters to control. Using Morpho, we simulated cholesteric liquid crystals to characterize the interactions between their material parameters and geometry. We found that in contrast to flat geometries, curved geometries have a much larger solution space. As a result, when simulating liquid crystal droplets we find many different valid geometries that can be shapeshifted into.
Pinch-off Dynamics of Particle-Stabilized Foam and Emulsion Filaments
Parisa Bazazi
Particle-stabilized foams and emulsions are ideal colloidal dispersions to be used as ink in drop-on-demand 3D printing systems. These unique systems provide the advantageous capability of encapsulating both hydrophilic and hydrophobic cargoes within the printed texture, offering versatile material properties. Emulsions and foams, in particular, enable interactive behavior between the printed frame and the surrounding media. In our study, we focus on investigating the pinch-off dynamics of Cellulose nanocrystals (CNC)-stabilized foams and emulsions within a surrounding liquid phase. We find that the viscoelastic properties of colloidal systems play a crucial role in controlling filament break-up, regardless of the nature of the dispersed droplets. The concentration of CNCs serves key role in the stability and printability for Pickering foams and emulsions. To facilitate practical implementation, we develop a comprehensive printability map that outlines the necessary conditions for CNC-based materials to be effectively utilized in drop-on-demand and direct ink-writing 3D printing technologies.
Microtubule organization in presence of active and passive crosslinkers
Prashali Chauhan
The microtubule cytoskeleton provides significant structural support for cells, much like bones in the human body. The formation of the mitotic spindle, which is a finite assembly of microtubules with many associated proteins, inspired us to investigate similar finite sized objects formed with a minimal system of microtubules and active and passive crosslinkers. Our study focuses on the interactions between MAP65, a plant-derived, antiparallel crosslinking protein, from the MAP65/PRC1/Ase1 family, and microtubules. MAP65 undergoes liquid-liquid phase separation and forms droplets of varying sizes. These droplets can act as nucleation centers for microtubules giving rise to different finite sized structures like tactoids, and asters. Especially these tactoids are reminiscent of the mitotic spindle, however unlike the spindle which is more liquid crystal like, they are rigid and thin. To introduce fluidity in the system, we also explore K401 as a dynamic microtubule crosslinker. We modulate the relative concentrations of crosslinking MAP65 and translocating K401 motors to determine how the competition between the two influences the formation and mechanics of these structures.
Unraveling the influence of vimentin on centrosome functioning and microtubule dynamics in polarized cell migration
Renita Saldanha
Cell migration is an important feature of migrating fibroblasts. For cell migration to occur, the cells must establish a polarity. The cytoskeletal filaments and the centrosome positioning play an important role in cell polarity. Intrinsically polarized microtubules are often associated with cell polarity. But recent studies show that loss of vimentin intermediate filaments (IF) leads to abnormally persistent cell migration in 3D microchannels. This result suggests vimentin also plays an important role in cell polarity. However, the interaction of vimentin IF with centrosome and microtubules to orchestrate cell polarity is unclear. To understand this, we investigate the effects of vimentin on the microtubule-nucleating activity of the cell centrosome and the dynamics of microtubule network using wild-type and vimentin-null mouse embryonic fibroblasts (mEF’s). Our results indicate, the presence of vimentin impacts the structure of the centrosome by increasing the area of the centrosome marked by Pericentriolar Matrix (PCM) proteins. Vimentin also promotes microtubule regrowth after nocodazole washout assay and enhances the stability of microtubules by increasing the presence of acetylated (post translational modification) microtubules. Our findings give a new insight to the role of vimentin in cell polarity by modulating centrosome structure and functioning.
Spatial Organization of Phase-separated DNA Droplets
Sam Wilken
Cells operate by compartmentalizing chemical reactions. Much recent work has shown that the spatiotemporal formation and control of membraneless compartments inside cells (liquid-liquid phase separation) is integral to cell function. Here, we investigate the long-range structures formed by a model phase-separating DNA system. We use DNA nanostars, a system of finite-valence particles, roughly 10nm in size, whose sequence is designed such that they self-assemble into liquid droplets on the micron scale via a binodal phase transition. We find that the structure is hyperuniform, corresponding to a disordered structure with anomalously small long range density fluctuations, characteristic of a spinodal decomposition that represents a perturbation that then relaxes to equilibrium via droplet Brownian motion. We hope that our work on near-equilibrium droplet assembly and structure provides a foundation to investigate droplet organizational mechanisms in driven/biological environments, or to implement droplet patterns as efficient biochemical reactors.
Balancing charge density and hydrophobicity to regulate cell behavior
Tran Truong
Cellular behaviors on artificial surfaces are crucial to advanced biomedical engineering. For artificial extracellular matrix (ECM), functional polymer surfaces offer great mechanical properties, various chemical species, and the ability to create varied topographic surfaces. Nevertheless, changes in these surfaces can trigger different physiological responses. Our study focuses on synthesizing dextran derivatives with anionic moieties, such as carboxylates and sulfonates. We examine how modified dextran influences cellular behavior by taking advantage of its distinct chemical structure and adjustable charge density. As a result of the 1H NMR technique, charge residues have been added to the structure of dextran. The influence of anionic charge density on cellular network shapes can be demonstrated using a Confocal Microscope. We aim to pinpoint essential anionic moieties affecting cell behaviors and create a novel synthetic dextran biomaterial category.
Machine Learning Applications in Biological Polymer Topology
Transito Gonzalez
Through studying DNA-based polymers with complex topologies, it is possible to obtain insight on the relationship between structure and dynamics. Nanopore technologies have allowed us to gather extensive data on a variety of such polymers. Now with the data in hand, the next step concerns its analysis. In this study, I present my results from the application of machine learning methods to linear and nonlinear DNA molecule data gathered via nanopore experiments. There were two main applications: principal component analysis in conjunction with k-means clustering and a stochastic gradient descent classification model. My goal is to utilize the power of machine learning to take on the analysis of DNA-based polymers to further our understanding of their complex topologies.
Advancements towards bulk production and purification of dsDNA for us in biomaterials
Wynter Paiva
Double-stranded DNA (dsDNA) exhibits many unique properties such as self-assembly, biocompatibility, rational design, and molecular recognition which allows it to behave as both a structural and functional moiety in hydrogels making it an ideal building block. Unfortunately, translation of these materials from the nanoscale to the macroscale has been challenging due to the time, cost, and waste associated with producing dsDNA on large scale. To overcome these challenges, we have developed new methodologies for gram scale production and purification of dsDNA that are scalable, accessible, and sustainable. This was accomplished using fed-batch fermentation of E. coli in a benchtop bioreactor coupled with alkaline lysis and anion-exchange chromatography for purification. We also report on bulk rheology of highly concentrated solutions of dsDNA up to 80 mg/mL, isolated using our approach, which is ~20x more concentrated than what has been reported in literature.
The endoplasmic reticulum as a dynamic liquid network
Zubenelgenubi Scott
The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While the molecular morphogens involved in shaping the ER have been identified, a quantitative model for its intricate large-scale network topology remains elusive. We develop a physical model of the ER as a ‘liquid network’, governed by a balance of tension-driven shrinking and new tubule growth. The simple two-parameter model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Intriguingly, certain geometric features of the liquid network model are parameter-independent and match the shape of the ER in COS7 cells. Using this model, we also explore the role of network dynamics in protein transport through the ER network.