Biophysics Summer REU

Mentors: Hugo Sanabria, Ph.D., Department of Physics and Astronomy, and David Feliciano, Ph.D., Department of Biological Sciences.

Significance: The development of a balanced neural circuit requires the precisely and exquisitely orchestrated elaboration of dendrites to receive appropriate neural innervation (Zhang, 2014 No. 14). The establishment of dendrite architecture and of connectivity relies on subcellular regions at the distal tips of elaborating dendrites. The local translation of mRNAs within growing and mature dendrites, with near dynamic specializations called spines, allows neurons to create connections and respond to localized input required for normal neuronal function. The mammalian target of rapamycin (mTOR) is a serine/threonine-protein kinase and the catalytic component of two complexes (mTORC1 and mTORC2) that play a central role in anabolic growth, cellular metabolism, and survival (Iwata, 2016 No. 24). mTOR regulates neuron dendrite architecture including dendrite number, caliber, complexity, spine morphology, synaptogenesis and synaptic plasticity (Jaworski, 2006 No. 25). However, the mechanisms responsible are unclear. The participants in this project will use genetic techniques to modulate mTOR pathway activity and explore how mTOR regulates dendrite architecture.

Research plan: Two participants will collaborate to study how mTOR pathway impacts dendrite architecture. One participant will primarily work with the Feliciano Lab to study dendrite architecture in vivo in somatic genetic mosaic models and in vitro using primary cell culture. This participant will quantify the levels of mTOR substrates. To achieve this goal, the participant will perform confocal and multiphoton microscopy, western blot, and single dendrite arbor translational profiling. The second participant will perform quantitative super resolution imaging in the Sanabria Lab and will use Point Accumulation for Imaging in Nanoscale Topography probes to visualize the actin cytoskeleton and mTOR regulated transcript isoforms including Homer1a crucial for regulating dendrite morphology and was identified by the Feliciano Lab. Together, the participants will probe the role of mTOR in modulating dendro-architectonic topographic maps.

Equipment/techniques participants will learn and use: Confocal microscopy, super-resolution microscopy, and molecular and cell biology techniques including FPLC based polysome profiling.

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 biological 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, and 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 exposure to toxins (antibiotics, antimicrobials) affect the dielectric behavior of N. gruberi. One participant primarily engaged in mechanical and biological engineering research in the Martinez-Duarte Lab will develop an experimental platform to study the response of protist to various electric fields. This participant will use hydrodynamic and electrical forces to manipulate parasites of interest inside lab-on-a-chip and microfluidic systems selectively. The other participant, primarily doing research in the Morris Lab, will use molecular genetics approaches to generate amoebae that express distinct fluorescent markers according to specific stimuli. This participant will clone fluorescent protein genes into amoebae expression vectors, transfect them into cells, and score the expression of these proteins by flow cytometry and microscopy. Together, the participants will determine whether dielectric properties are specific enough to distinguish between subtle differences in the amoebae. Ultimately, this research will lead to better techniques for lysis, isolating low abundance individuals from potentially complex mixes of cells, or concentrations from large environmental samples.

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

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 (NPs) 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 better understand 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; and 3) hyperspectral imaging using Cytoviva. This project will be performed collaboratively between the two students and two faculty mentors. One student primarily working in Ramakrishna 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 Clemson 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 (Sun, 2021 No. 31; Xing, 2020 No. 32}. 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.