2025 Biophysics Summer REU

Mentors: Hugo Sanabria, Ph.D., Department of Physics and Astronomy, and Meredith Morris, Ph.D., Department of Genetics and Biochemistry.

Significance: Compartmentalization of biological pathways into membrane-bounded organelles is a fundamental characteristic of eukaryotic cells. We are interested in resolving the mechanisms that regulate organelle formation, proliferation, and remodeling. As a model system, we use the protozoan parasite Trypanosoma brucei (T. brucei). This organism harbors small, membrane-enclosed organelles called glycosomes essential for survival. Experiments on parasites grown in culture reveal that glycosome composition, number and perhaps distribution change during development and in response to environmental nutrients such as glucose. However, it is unclear how individual glycosomes respond at the organelle level to developmental and environmental changes. Participants in this project will use several biophysical tools to measure glycosome composition, morphology and function and how glycosome changes in response to different stimuli.

Research plan: Two participants will collaborate to study how extracellular glucose influences glycosome dynamics. One participant will primarily work with the Morris Lab to monitor overall expression levels of different glycosome reporter proteins fused to fluorescent proteins via western blot and flow cytometry. The second participant will perform quantitative super-resolution microscopy in the Sanabria Lab via STED-FLIM to visualize the glycosome reporter proteins. Together, the participants will resolve the glucose-dependent glycosome landscape of African trypanosomes.

Equipment/techniques participants will learn and use: Confocal microscopy, super-resolution microscopy, and molecular and cell biology techniques.

Mentors: Delphine Dean, Ph.D., bioengineering, and Endre Takacs, Ph.D., Department of Physics and Astronomy.

Significance: Recent research suggests that low levels of radiation are not necessarily harmful to cells. However, the underlying physical, chemical and biological pathways that take place during low-dose irradiation remain largely unstudied. The participants will use combinations of computational and experimental approaches to characterize the effect of well-characterized ionizing radiation on cells and subcellular components. Together, they will contribute to the understanding of how low levels of radiation affect individual biological machinery in cells.

Research plan: Two participants will collaborate to study the interaction between low levels of monochromatic soft X-ray radiation and biological tissues. One student, primarily working on tissue engineering in the Dean Lab, will grow breast cells and characterize their properties in response to radiation and radiosensitizing materials using various cell and molecular biology assays. The other student, primarily performing radiation physics research in the Takacs Lab, will characterize the low-dose X-rays and model their effects on subcellular structures and components. Together, the participants will precisely quantify the radiation that cell cultures receive and use well-defined protocols to understand how low levels of radiation affect individual biological machinery in cells and cell cultures.

Equipment/techniques participants will learn and use: Mini-X-ray tubes, solid-state X-ray detectors, single-photon counters, spectral analysis, statistical analysis software, cell culture, optical microscopy and image analysis, cell proliferation and cycle analysis, cell function, and differentiation assays.

Mentors: Keisha Cook, Ph.D., School of Mathematical and Statistical Sciences, and Kimberly L. Weirich, Ph.D., Department of Materials Science and Engineering

Significance: Motor proteins walk through a complex network of cytoskeletal filaments to transport various cargo, including vesicles, organelles, mRNA and chromosomes, throughout the cell. The diverse array of cargo transport phenomena observed in cells arises from just a few families of motor proteins (dynein, kinesin and myosin) and filament networks (microtubules and microfilaments. However, it is poorly understood how cells use the ability to regulate the motor protein density on cargo, the length, pattern and topology of the filament networks, and the size and shape of the cargo to generate this diversity of intracellular transport phenomena.

Research plan: Two participants will collaborate to study how motor transport is influenced by cytoskeletal architecture in a reconstituted actin microfilament network. One participant, primarily conducting experiments in Kimberly L. Weirich’s lab, will use purified proteins to reconstitute actin cytoskeletal networks with various architectures through protein cross-linking and study motor dynamics using fluorescence microscopy. The second participant, primarily conducting theoretical and computational work with Keisha Cook, will use fluorescent microscopy to collect videos of the transporting motors, apply image processing algorithms to extract the transport data and analyze the data to quantify the properties of transport. Additionally, the second participant will employ mathematical modeling techniques to model the emergent properties of the motor transport. Together, the two participants will build and model a cellular process that gives insight into how motor proteins work together to facilitate cargo transport in an actin network.

Equipment/techniques participants will learn and use: fluorescent microscope; general biochemical and biophysical equipment including centrifuges, gel electrophoresis, and spectrophotometers; quantitative image analysis; and R, MATLAB, and Python programming languages.

Mentors: Rodrigo Martinez-Duarte, Ph.D., mechanical engineering, and Jim Morris, Ph.D., Department of Genetics and Biochemistry.

Significance: Nature’s machinery responses to electric fields vary in response to changes in life-cycle stage, environmental conditions, and exposure to toxic agents. Here, we will use a model eukaryote, the amoeba Naegleria gruberi, to score the impact of these changes on their responses to electric fields to establish a better understanding of how cellular changes impact the dielectric properties of cells. This understanding is the first step in advancing the use of electric fields as a means of robust on-chip sample preparation such as lysis and concentrating and characterizing cells from dilute systems for further study, which will offer researchers the ability to study rare or low-abundance cells in a population.

Research plan: Two participants will collaborate to study how growth, environmental factors, and toxin exposure impact the dielectric behavior of N. gruberi. One participant primarily focused on mechanical and biological engineering research in the Martinez-Duarte Lab will create an experimental platform using hydrodynamic and electrical forces within lab-on-a-chip and microfluidic systems to study protist responses to electric fields. The other participant, primarily from the Morris Lab, will employ molecular genetics to engineer amoebae expressing fluorescent markers in response to specific stimuli. They will assess protein expression using flow cytometry and microscopy. Together, they aim to determine if dielectric properties can distinguish subtle amoebae differences, potentially improving cell lysis and isolation techniques from complex environmental samples.

Equipment/techniques participants will learn and use: Molecular cloning, in vitro cell culture, transfection approaches, flow cytometry, microfluidics and dielectrophoresis.

Mentors: David Jacobson, Ph.D., Department of Chemistry, and Emil Alexov, Ph.D., Department of Physics and Astronomy.

Significance: Protein function depends on adopting a precise, three-dimensional folded structure that is a minimum of the free energy, the enthalpic component of which includes the interactions between all the amino acids of the protein and their environment. These interactions are particularly subtle in the case of membrane proteins. Mutation (substitution of one of the 20 amino acid species with another at a given position) alters the relative free energies of correctly and incorrectly folded conformations, which can lead to misfolding, aggregation and loss of function and can cause disease. In some cases, small-molecule pharmacological chaperones can preferentially bind to and stabilize the wildtype folded state. However, it is generally challenging to experimentally measure these changes in stability, especially in membrane proteins, and to predict these energetic effects via computational approaches.

Research plan: Two participants will collaborate to develop methods for predicting mutant energetic effects in membrane proteins grounded in precise experimental measurements. One participant, working with David Jacobson, will use advanced single-molecule force application methods to unfold human vasopressin receptor 2 and measure the change in unfolding free energy upon introduction of a mutation. Such measurements are not accessible using traditional biochemical techniques. The second participant will work with Emil Alexov to extend computational methods for predicting mutant energetic effects to this membrane-protein system. They will also explore the effects of known V2R pharmacological chaperones. Both participants will work with both mentors to refine the theory connecting the energies measured in the experimental and computational studies.

Equipment/techniques participants will learn and use: Protein expression and purification, atomic force microscopy, machine learning, energy calculations, programming, and quantitative data analysis.

Mentors: Terri Bruce, Ph.D., Department of Biological Sciences and director, Clemson Light Imaging Facility, and Ramakrishna Podila, Ph.D., Department of Physics and Astronomy.

Significance: Hyperspectral imaging provides a useful combination of spectrophotometry and microscopic imaging in the 400–1000nm range by accumulating reflectance spectrum for each pixel in a micrograph. HSI emerged as an excellent method for simultaneously imaging nanomaterial interactions with nature’s machinery, such as protein corona formation and cellular uptake. Previously, we explored the cellular uptake of silver (Ag) and gold (Au) nanoparticles with and without protein corona using HSI. Our studies provided new insights into the effects of protein corona on cellular uptake. Notwithstanding this progress, there is a need to understand better the relationship between the physical and biological properties that lead to the hyperspectral shifts/broadening, NP aggregation state and its microenvironment.

Research plan: The experimental focus of this project will include three parts:

1. Preparation and characterization of Ag/Au NPs of various sizes with and without protein corona.
2. In-vitro cell culture to study cellular uptake.
3. Hyperspectral imaging using cytoviva. This project will be performed collaboratively between the two students and two faculty mentors. One student primarily working in Podila’s laboratory will work on parts one and two, while the second assigned student to this project will focus on parts two and three under the guidance of Terri Bruce at the light imaging facility.

Equipment/techniques participants will learn and use: Cytovia hyperspectral imaging, electron microscopy, mammalian cell culture and spectroscopy.

Mentors: Feng Ding, Ph.D., Department of Physics and Astronomy, and Marc Birtwistle, Ph.D., Department of Chemical and Biomolecular Engineering.

Significance: Deposition of amyloid aggregation is the hallmark of Alzheimer’s disease and many other neurodegenerative diseases. Experimental evidence points to amyloid aggregation of beta-amyloid peptides, especially their small molecular weight oligomers populated during the aggregation process, as the culprit. Nonetheless, many antibody-based approaches aiming to clear amyloids have failed to yield an efficient cure. Therefore, there is a crucial need to further understand the process of amyloid aggregation that leads to cytotoxicity. The participants will seek to understand the molecular mechanisms of endogenous proteins and peptides as well as engineered nanomaterials with anti-amyloid properties.

Research plan: Building upon an ongoing and co-funded collaboration, two participants will collaborate to explore novel approaches to mitigate amyloid aggregation and cytotoxicity. One participant, primarily doing computer simulations in the Ding Lab, will perform multiscale molecular dynamics simulations to study amyloid aggregation in silico. The participant will investigate the molecular mechanisms of various anti-amyloid agents, including endogenous molecular chaperons, designed peptide inhibitors and engineered nanoparticles. The other participant will be primarily doing in vitro and in vivo experiments in the Birtwistle Lab. This participant will perform biophysical characterizations including ThT fluorescence assay and TEM imaging and cell viability/toxicity assay with high-throughput live-cell imaging to experimentally study the engineered anti-amyloid agents. Together, the participants will test computational predictions with experimental characterizations and validations and use simulations to provide molecular insights to mitigate amyloid aggregation and toxicity.

Equipment/techniques participants will learn and use: Linux systems, palmetto supercomputer programming, ThT fluorescence assay, TEM imaging, cell culture and high-throughput live cell imaging.

Mentors: Laura Finzi, Ph.D., and David Dunlap, Ph.D., Department of Physics and Astronomy Department, medical biophysics program, Clemson University.

Significance:DNA looping by proteins is a ubiquitous and fundamental level of gene regulation whereby the open or closed loop conformation acts as an on/off genetic switch. One such classic, looping gene regulator is the lac repressor which binds to specific sites at the beginning of and within the lactose operon to regulate expression of genes involved in lactose metabolism. However, looping is just one layer of gene regulation based on DNA topology. Indeed, the conformation and structure of DNA are dynamically changing throughout the cell cycle. Recent research has identified DNA sequences called flipons, located at transcription start sites and elsewhere, that easily flip from the “ground state,” right-handed helical form to a left-handed helix. This change is likely to affect protein binding to DNA. The importance of the right-to-left-handed (and vice versa) DNA transition is underscored by the fact that cells express proteins that recognize left-handed DNA and that these proteins are implicated in a variety of diseases and the immune response. In vitro experiments will be conducted to understand how protein binding responds to this transition in a variety of physiologically relevant environments and how loop-based transcriptional repression may be impacted by flipons. Single molecule techniques will be employed to avoid ensemble averaging and allow characterization of looping dynamics and energetics.

Research plan: The scope of this project accommodates two students. DNA constructs with and without flipons will be created using polymerase chain reactions and used to tether sub-micron diameter beads to microscope cover glasses for single particle tracking and manipulation. One set of measurements, using the tethered particle motion technique (TPM) and magnetic tweezers will probe the effect of flipons on DNA loop formation by the lac repressor. Another, using magnetic tweezers will reveal how the affinity of a left-handed DNA binding protein (Za) is affected by torsion in the DNA. Real-time tracking of tethered particles without (TPM) or with (MT) tension and torsion in the DNA reveals length changes in the tether associated with structural or topological transitions. Analysis of the lifetimes of various states across an ensemble of molecules reveals transition energetics that depend on sequence, electrostatics (salt concentrations), tension, torsion and protein binding.

Equipment/techniques that participants may learn and use: Students will use molecular and microbiologocal methods to create DNA constructs and learn to assemble tethered particles in microchambers for single molecule microscopy and manipulation. They will use MatLab-based custom software to analyze the motion of tethered particles to measure topological state lifetimes and calculate free energies of transitions. Interested students could develop code for such analyses using existing MatLab scripts as a guide.