R-3 Repository :: Browsing by Author "Diehl, Michael R."

Browsing by Author "Diehl, Michael R."

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Analysis of Cooperative Behavior in Multiple Kinesins Motor Protein Transport by Varying Structural and Chemical Properties
(Springer, 2013) Uppulury, Karthik; Efremov, Artem K.; Driver, Jonathan W.; Jamison, D. Kenneth; Diehl, Michael R.; Kolomeisky, Anatoly B.
Intracellular transport is a fundamental biological process during which cellular materials are driven by enzymatic molecules called motor proteins. Recent optical trapping experiments and theoretical analysis have uncovered many features of cargo transport by multiple kinesin motor protein molecules under applied loads. These studies suggest that kinesins cooperate negatively under typical transport conditions, although some productive cooperation could be achieved under higher applied loads. However, the microscopic origins of this complex behavior are still not well understood. Using a discrete-state stochastic approach we analyze factors that affect the cooperativity among kinesin motors during cargo transport. Kinesin cooperation is shown to be largely unaffected by the structural and mechanical parameters of a multiple motor complex connected to a cargo, but much more sensitive to biochemical parameters affecting motor-filament affinities. While such behavior suggests the net negative cooperative responses of kinesins will persist across a relatively wide range of cargo types, it is also shown that the rates with which cargo velocities relax in time upon force perturbations are influenced by structural factors that affect the free energies of and load distributions within a multiple kinesin complex. The implications of these later results on transport phenomena where loads change temporally, as in the case of bidirectional transport, are discussed.
CLINICALLY TRANSLATABLE SOLUTIONS FOR MOLECULAR PHOTOACOUSTIC IMAGING
(2020-04-23) Han, Sangheon; Sokolov, Konstantin V.; Diehl, Michael R.
Detection of small numbers of cancer cells is one of the significant challenges in oncology, which prevents detecting a tumor in its early stages or onset of metastatic spreading of disease in cancer patients. Developing nanoparticles for molecular specific imaging, and therapy is a promising strategy to address this challenge. Many inorganic nanoparticles exhibit a bright signal for enhanced imaging contrast and exhibit unique size- dependent optical properties. Organic nanoparticles require to load dyes to serve as imaging agents and are able to degrade inside the host body to be excreted without long- term bodily accumulation. Photoacoustic imaging is a novel non-invasive imaging modality that can be used for highly sensitive detection of near-infrared (NIR) absorbing nanoparticles in tissue. NIR optical window is ideal for medical imaging due to the low tissue scattering and absorption, which in turn, provides longer tissue penetration depth. Therefore, nanoparticles with NIR absorption have great potential for photoacoustic imaging for highly sensitive cancer detection. In addition, nanoparticles can render molecular-specific toward cancer cells of interest via attachment of antibodies that target cancer biomarkers. During my doctoral study, I used ultra-small gold nanoparticles (AuNP) and organic nanoparticles loaded with indocyanine green (ICG) (Lipo-ICG and ICGJ@PEI Ps) that are conjugated with antibodies (anti-EGFR) in conjunction with photoacoustic imaging (PAI) for highly sensitive and specific cancer detection. Ultra-small AuNPs of 5 nm in diameter have two important advantages: 1) improved delivery in tissue due to their small size; and 2) an efficient renal clearance after administration. Renal clearance is a prerequisite for the approval of nanoparticles in clinics. As an alternative approach to inorganic AuNPs, organic nanoparticles such as liposomes and polymersomes were loaded with s (ICGJs), which displays a narrow peak in the NIR window, ideal for PA imaging. This approach provides the following advantages: 1) biodegradability to achieve renal clearance without long-term bodily accumulation, and 2) improved PAI sensitivity and molecular specificity. The main idea of my approach is based on the targeting of the epidermal growth factor receptor (EGFR). Individual 5 nm AuNPs do not exhibit NIR absorbance that is required for sensitive PAI. Attachment of anti-EGFR antibodies to 5 nm AuNPs renders their molecular specificity and receptor-mediated accumulation inside EGFR-expressing cancer cells; nanoparticle aggregation occurs in endosomal compartments after the accumulation, which leads to a dramatic increase of their absorption in the NIR spectrum. For organic nanoparticles, retention of ICGJs inside cancer cells after EGFR receptor-mediated accumulation can enable molecular PAI. Therefore, labeled cancer cells can be detected by the PAI with NIR excitation. In order to fully understand the contrast enhancement mechanism of ultra-small AuNPs, I carried out a series of experiments to study the endocytosis and aggregation of targeted 5 nm AuNPs using two-photon microscopy. Then, the feasibility of sensitive PAI was evaluated with the targeted 5 nm AuNPs at the cellular level. Liposomes and polymersomes loaded with ICGJs were tested as a new design of organic PA contrast agents for cancer detection. Future research directions can explore theranostic applications, including 1) radiosensitization by ultra-small AuNPs, and 2) longitudinal monitoring of molecular therapy using PAI with targeted organic nanoparticles loaded with ICG J aggregates. Future research directions can explore theranostic applications including: 1) radiosensitization by ultra-small AuNPs, and 2) longitudinal monitoring of molecular therapy using PAI with targeted organic nanoparticles loaded with ICG J aggregates.
Collective dynamics of processive cytoskeletal motors
(Royal Society of Chemistry, 2016) McLaughlin, R. Tyler; Diehl, Michael R.; Kolomeisky, Anatoly B.; Center for Theoretical Biological Physics
Major cellular processes are supported by various biomolecular motors that usually operate together as teams. We present an overview of the collective dynamics of processive cytokeletal motor proteins based on recent experimental and theoretical investigations. Experimental studies show that multiple motors function with different degrees of cooperativity, ranging from negative to positive. This effect depends on the mechanical properties of individual motors, the geometry of their connections, and the surrounding cellular environment. Theoretical models based on stochastic approaches underline the importance of intermolecular interactions, the properties of single motors, and couplings with cellular medium in predicting the collective dynamics. We discuss several features that specify the cooperativity in motor proteins. Based on this approach a general picture of collective dynamics of motor proteins is formulated, and the future directions and challenges are discussed.
Configuring robust DNA strand displacement reactions forﾠin situﾠmolecular analyses
(Oxford University Press, 2012) Duose, Dzifa Y.; Schweller, Ryan M.; Zimak, Jan; Rogers, Arthur R.; Hittelman, Walter N.; Diehl, Michael R.
The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterativein situﾠlabeling and erasing of markers on cells.
Development of Dynamic DNA Probes for High-Content in situ Proteomic Analyses
(2012-09-05) Schweller, Ryan; Diehl, Michael R.; Qutub, Amina A.; Farach-Carson, Cindy
Dynamic DNA complexes are able to undergo multiple hybridization and dissociation events through a process called strand displacement. This unique property has facilitated the creation of programmable molecular detection systems and chemical logic gates encoded by nucleotide sequence. This work examines whether the ability to selective exchange oligonucleotides among different thermodynamically-stable DNA complexes can be harnessed to create a new class of imaging probes that permit fluorescent reporters to be sequentially activated (“turned on”) and erased (“turned off”). Here, dynamic DNA complexes detect a specific DNA-conjugated antibody and undergo strand displacement to liberate a quencher strand and activate a fluorescent reporter. Subsequently, incubation with an erasing complex allows the fluorophore to be stripped from the target strand, quenched, and washed away. This simple capability therefore allows the same fluorescent dyes to be used multiple times to detect different markers within the same sample via sequential rounds of fluorescence imaging. We evaluated and optimized several DNA complex designs to function efficiently for in situ molecular analyses. We also applied our DNA probes to immunofluorescence imaging using DNA-conjugated antibodies and demonstrated the ability to at least double the number of detectable markers on a single sample. Finally, the probe complexes were reconfigured to act as AND-gates for the detection of co-localized proteins. Given the ability to visualize large numbers of cellular markers using dynamic DNA probe complexes, high-content proteomic analyses can be performed on a single sample, enhancing the power of fluorescence imaging techniques. Furthermore, dynamic DNA complexes offer new avenues to incorporate DNA-based computations and logic for in situ molecular imaging and analyses.
How the interplay between mechanical and non-mechanical interactions affects multiple kinesin dynamics
(American Chemical Society, 2012) Uppulury, Karthik; Efremov, Artem K.; Driver, Jonathan W.; Jamison, D. Kenneth; Diehl, Michael R.; Kolomeisky, Anatoly B.
Intracellular transport is supported by enzymes called motor proteins that are often coupled to the same cargo and function collectively. Recent experiments and theoretical advances have been able to explain certain behaviors of multiple motor systems by elucidating how unequal load sharing between coupled motors changes how they bind, step, and detach. However, non-mechanical interactions are typically overlooked despite several studies suggesting that microtubule-bound kinesins interact locally via short-range non-mechanical potentials. This work develops a new stochastic model to explore how these types of interactions influence multiple kinesin functions in addition to mechanical coupling. Non-mechanical interactions are assumed to affect kinesin mechanochemistry only when the motors are separated by less than three microtubule lattice sites, and it is shown that relatively weak interaction energies (~2 kBT) can have an appreciable influence over collective motor velocities and detachment rates. In agreement with optical trapping experiments on structurally-defined kinesin complexes, the model predicts that these effects primarily occur when cargos are transported against loads exceeding single-kinesin stalling forces. Overall, these results highlight the inter-dependent nature of factors influencing collective motor functions, namely, that the way the bound configuration of a multiple motor system evolves under load determines how local non-mechanical interactions influence motor cooperation.
Identification of a novel PRC2 recruiter in mammalian cells
(2013-04-18) Gou, Yufeng; Ma, Jianpeng; Diehl, Michael R.; Tao, Yizhi Jane; Wang, Qinghua
Polycomb repressive complex (PRC) 2 functions to repress thousands of target genes, and they are responsible for stem cell differentiation and carcinogenesis. However, how PRC2 are recruited to specific regions of their target genes remains elusive. In Drosophila, nine sequence-specific transcription factors including Zeste have been shown to act as PRC2 recruiters, but little is known about their homologues in mammalian cells, as a straightforward homology search failed to work in most cases. Aided by three-dimensional structure prediction and the use of the genomes of intermediate bridging species, we have identified a new protein, Zeste Homologue in humans (ZH), as the human homologue of Drosophila Zeste. Gel shift assays indicated that ZH binds to Zeste recognition sequence via its N-terminal DNA binding domain. ZH physically interacted with the components of PRC2 in GST-pull down assays. Chip-seq and Chip-qPCR experiments show the co-localization of ZH and PRC2 complex. Together, these findings revealed the critical function of ZH in recruiting PRC2 complexes to their target genes.
Mechanisms of Cooperation in Systems of Multiple Processive Motors
(2012) Driver, Jonathan; Diehl, Michael R.
The inside of a eukaryotic cell is a highly organized microscale factory that shuttles components that are created or obtained in one place for use or further modification in another. Diffusion cannot accomplish the feat of translocating an object in the cytoplasm to a particular location that is a micron or more away in a timely fashion, so cells rely instead on processive motor proteins. Microtubule motor proteins are enzymes that harness the chemical energy from ATP hydrolysis to produce force and carry vesicles, membrane-bound organelles, and other cargos along paths in the cell's microtubule filament network to their destinations in the cytoplasm. These proteins recognize the polarity of the microtubule, and different classes of motors walk in different directions with respect to this polarity, giving the cell control over the direction in which a cargo is carried. It has been observed experimentally that many cargos are carried by more than one motor simultaneously, and that these multiple-motor systems can consist both of motors of the same type and of varying numbers of motors of different types. Multiple-motor systems present the possibilities of both enhanced transport performance and of tunable behavior, where the number, type, and arrangement of motors on a group of cargos can be modulated by the cell like an analog-style control to induce those cargos to arrive at a particular distribution of locations in the cytoplasm. In order to resolve the mechanisms by which these things might occur, the combination of experimental and theoretical studies in this thesis focus on the relationship between the basic biophysical properties of the constituent motors in small multiple-motor systems and the degree and nature of the cooperation observed, from the standpoint of several relevant metrics. The results highlight the importance of both the mechanochemistry of the motors and the geometry of the system itself, and offer substantial new insights into why different classes of motors cooperate to different extents, with broad implications.
Molecular mechanisms of the interhead coordination by interhead tension in cytoplasmic dyneins
(National Academy of Sciences of the United States of America, 2018) Wang, Qian; Jana, Biman; Diehl, Michael R.; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José Nelson
Cytoplasmic dyneins play a major role in retrograde cellular transport by moving vesicles and organelles along microtubule filaments. Dyneins are multidomain motor proteins with two heads that coordinate their motion via their interhead tension. Compared with the leading head, the trailing head has a higher detachment rate from microtubules, facilitating the movement. However, the molecular mechanism of such coordination is unknown. To elucidate this mechanism, we performed molecular dynamics simulations on a cytoplasmic dynein with a structure-based coarse-grained model that probes the effect of the interhead tension on the structure. The tension creates a torque that influences the head rotating about its stalk. The conformation of the stalk switches from the α registry to the β registry during the rotation, weakening the binding affinity to microtubules. The directions of the tension and the torque of the leading head are opposite to those of the trailing head, breaking the structural symmetry between the heads. The leading head transitions less often to the β registry than the trailing head. The former thus has a greater binding affinity to the microtubule than the latter. We measured the moment arm of the torque from a dynein structure in the simulations to develop a phenomenological model that captures the influence of the head rotating about its stalk on the differential detachment rates of the two heads. Our study provides a consistent molecular picture for interhead coordination via interhead tension.
Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins
(National Academy of Sciences, 2017) Wang, Qian; Diehl, Michael R.; Jana, Biman; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José Nelson
Motor proteins are active enzymatic molecules that support important cellular processes by transforming chemical energy into mechanical work. Although the structures and chemomechanical cycles of motor proteins have been extensively investigated, the sensitivity of a motor’s velocity in response to a force is not well-understood. For kinesin, velocity is weakly influenced by a small to midrange external force (weak susceptibility) but is steeply reduced by a large force. Here, we utilize a structure-based molecular dynamic simulation to study the molecular origin of the weak susceptibility for a single kinesin. We show that the key step in controlling the velocity of a single kinesin under an external force is the ATP release from the microtubule-bound head. Only under large loading forces can the motor head release ATP at a fast rate, which significantly reduces the velocity of kinesin. It underpins the weak susceptibility that the velocity will not change at small to midrange forces. The molecular origin of this velocity reduction is that the neck linker of a kinesin only detaches from the motor head when pulled by a large force. This prompts the ATP binding site to adopt an open state, favoring ATP release and reducing the velocity. Furthermore, we show that two load-bearing kinesins are incapable of equally sharing the load unless they are very close to each other. As a consequence of the weak susceptibility, the trailing kinesin faces the challenge of catching up to the leading one, which accounts for experimentally observed weak cooperativity of kinesins motors.
Multiplexed and Reiterative Detection of Protein Markers in Cells using Dynamic Nucleic Acid Complexes
(2011) Duose, Dzifa Yawa; Diehl, Michael R.
The diagnosis, staging and clinical management of cancer and other diseases is becoming increasingly reliant upon the identification and quantification of molecular markers as well their spatial distribution in histological samples. Yet, due to spectral overlap of dyes and the inability to remove probes without affecting marker integrity, immunohistological methods are limited by the number of markers that can be examined on a single specimen resulting in loss of information that could be vital to diagnosis or treatment. This dissertation describes the development and characterization of an erasable multi-color imaging technology capable of detecting large numbers of molecular markers on a single biological sample. The system consists of (1) 'targets', which are single or partially hybridized DNA strands conjugated to a protein of interest for biomarker recognition in cells, and (2) multi-strand, fluorophore-containing DNA 'probe complexes' that react with the DNA portion of the target in a sequence dependent fashion to create fluorescent reporting complexes. The addition of a quencher-bearing ssDNA displaces the target's DNA strand to effectively remove the dye from the marker so that the sample can be re-imaged for other markers with minimal interference from prior iii rounds of labeling. Orthogonal DNA sequences and spectrally-separated dyes can be used to create multiple, unique target/probe pairs that associate specifically and can be imaged in parallel. The overall utility of this technology depends on high specificity of targets to respective probe complexes, highly efficient labeling and erasing to ensure that fluorescent signals can be used to fully quantify target abundance without the interference of signals from previous rounds of labeling, and short reaction times to allow for multiple rounds of processing on the same sample without loss of integrity. Based on the above criteria, three classes of probes were designed and their structure-function relationships elucidated to determine the contributions of complex size, free energy differences between intermediate states, and strand displacement on labeling and erasing kinetics and efficiencies on cells. A comparison of the kinetics of the labeling and erasing reactions for the three different constructs showed that reaction efficiencies depend less on calculated net free energy change than on the engineered state of the complex during the strand displacement reaction (i.e., the type of strand displacement reaction it participates in). This new paradigm in probe design allowed the system to meet its design goals, potentially increasing the diagnostic power of individual histological specimens and opening the door to more sophisticated analyses of cell phenotype and its functional relationship to disease.
Multiplexed In Situ Immunofluorescence Using Dynamic DNA Complexes
(Wiley, 2012) Schweller, Ryan M.; Zimak, Jan; Duose, Dzifa Y.; Qutub, Amina A.; Hittelman, Walter N.; Diehl, Michael R.
Negative interference in systems of coupled kinesin: A study of self-assembling complexes with defined structure
(2010) Rogers, Arthur Russell; Diehl, Michael R.
Intracellular transport is a crucial process that requires the work of motor proteins to distribute necessary cargos. Many times the motors must move over long distances and against high opposing forces than those generated by single motors. To accomplish this task motors appear to act in teams, as suggested by experiments that show enhanced force production and extended travel lengths. Many motors have been characterized individually, but experiments to study their collective mechanics rely on non-specific groupings where the copy number and geometric arrangement are not explicitly known. In order to resolve the true extent to which each motor contributes enhanced transport properties, a system must be developed that precisely controls the number of motors that are studied. Within this work, a convergent self-assembly approach is presented that allows structurally-defined complexes of kinesin-1 to be created. This approach also provides synthetic control over intermotor spacing and the elasticity of the mechanical motor linkages to rigorously characterize the effects of system structure on the interactions of exactly two motors. This synthetic coupled motor system was then used to examine the extent to which motor grouping enhances the transport properties of cargos. It was determined that the average velocity of coupled kinesin proteins was statistically indistinguishable from that of the single motor, while the average run lengths of the two-motor system were slightly longer (≈ 2X), but less than estimated for a system of non-interacting motors (≈ 4X). This study concludes that, under low loads, intermotor strain in coupled kinesin proteins increases the rate of motor detachment from the microtubule and decreases the rate at which additional motors rebind. The presence of negative interference in these complexes implies that groupings of kinesins preferentially travel in a single motor-attachment state, and that only a subset of cargo-bound motors are used during transport.
p38a/Mapkapk2a signaling regulates tristetraprolin in the yolk syncytial layer: A role for mRNA degradation in the morphogenesis of a novel embryonic structure in vertebrate development
(2014-04-02) Gomez de la Torre Canny, Sol; Wagner, Daniel S.; Bartel, Bonnie; Diehl, Michael R.; Gustin, Michael C.; Silberg, Jonathan J.
The yolk syncytial layer (YSL) is a novel embryonic structure that is unique to teleost fishes like the zebrafish. How existing genetic mechanisms can change to contribute to the generation of morphological novelties such as the YSL is a fundamental question of evolutionary biology. To address this question we examined the function of mapkapk2a (mk2a). Mk2a is required for YSL morphogenesis. To study the requirement of Mk2a signaling during embryogenesis, we analyzed the betty boop mutant (bbp). Bbp encodes Mk2a, the zebrafish homolog of mammalian MK2, a protein kinase activated by the p38 MAPK signaling pathway. bbp mutants display a striking lysis phenotype. bbp mutant embryos lose the expression of multiple YSL-specific genes. Thus, we examined the role of tristetraprolin (Ttp), a MK2 regulated mRNA-binding protein that promotes degradation of specific mRNA targets. Manipulation of the endogenous activity of Ttp showed that Ttp regulates the stability of YSL-specific mRNA molecules, most notably of mxtx2, which encodes for a zebrafish-specific transcription factor that activates a large proportion of YSL-specific genes. Specific activation of the Mk2a in the YSL inhibits Ttp activity in this cell layer, and prevents expression of Mxtx2 in other cells of the embryo. Expression of Mxtx2 or activation of the p38a /Mk2a pathway outside of the YSL results in dramatic defects in development. MK2 is not required for embryogenesis in mammals. Mutation of MK2 results in impaired inflammatory response and resistance to inflammatory diseases. The ability to manipulate the activity of the members of this conserved pathway in this novel context suggests that epiboly may be a useful platform to probe the molecular mechanism of TTP-dependent mRNA degradation that plays a crucial role in the regulation of the inflammatory response in mammals.
Peroxisome Biogenesis in Drosophila melanogaster: Protein Trafficking, Lipid Metabolism, and Muscle Function
(2013-12-02) Faust, Joseph; McNew, James A.; Bartel, Bonnie; Diehl, Michael R.; Stern, Michael; Bennett, Matthew R.
Peroxisomes are ubiquitous organelles required for many essential functions, such as fatty acid metabolism. Defects in peroxisome biogenesis cause a spectrum of human diseases known as peroxisome biogenesis disorders (PBDs). These devastating diseases lack effective therapies and it is unclear how peroxisome dysfunction causes the disease state. Animal models are needed to understand the connection between peroxisome biology and animal physiology. The fruit fly, Drosophila melanogaster, has recently become an important animal model in the study of peroxisomes. We have identified the major peroxisomal proteins and pathways in flies and examined peroxisomal protein trafficking. We have found that fruit fly peroxisomes share many features in common with higher animals, but display some important differences. Flies appear to have lost one of the pathways used in other organisms to target proteins to the peroxisomal matrix. Also some proteins are dually localized to peroxisomes and the cytoplasm likely through a weak interaction with the protein machinery that brings peroxisomal proteins into the organelle. We have also generated fly mutants with impaired peroxisome biogenesis and shown that peroxisomes are required for normal development and lipid metabolism. Flies with impaired peroxisome biogenesis also show defects in multiple processes that depend on muscle function, such as locomotion. PBD patients also display muscle defects, but it is thought to be a secondary effect of neuronal dysfunction. We propose that peroxisome loss in humans, like in flies, may directly affect muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in fly physiology and specifically in muscle cells may reveal novel aspects of PBD etiology.
Persistent tailoring of MSC activation through genetic priming
(Elsevier, 2024) Beauregard, Michael A.; Bedford, Guy C.; Brenner, Daniel A.; Sanchez Solis, Leonardo D.; Nishiguchi, Tomoki; Abhimanyu; Longlax, Santiago Carrero; Mahata, Barun; Veiseh, Omid; Wenzel, Pamela L.; DiNardo, Andrew R.; Hilton, Isaac B.; Diehl, Michael R.
Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce the expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor interferon response factor 1 (IRF1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFN-γ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN-γ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.
Programming in situ immunofluorescence intensities through interchangeable reactions of dynamic DNA complexes
(Wiley-VCH Verlag, 2012) Zimak, Jan; Schweller, Ryan M.; Duose, Dzifa Y.; Hittelman, Walter N.; Diehl, Michael R.
The regulation of antibody reporting intensities is critical to various in situ fluorescence imaging analyses. While such control is often necessary to visualize sparse molecular targets, the ability to tune marker intensities is also essential for highly multiplexed imaging strategies where marker reporting levels must be tuned to both optimize dynamic detection ranges and minimize crosstalk between different signals. Existing chemical amplification approaches generally lack such control. Here, we demonstrate that linear and branched DNA complexes can be designed to function as interchangeable building blocks that can be assembled into organized, fluorescence reporting complexes. We show that the ability to program DNA strand displacement reactions between these complexes offer new opportunities to deterministically tune the number of dyes that are coupled to individual antibodies in order to both increase and controllably balance marker levels within fixed cells.
Quantitative FLIM-FRET Measurement of Voltage Dependent Prestin Conformational Changes
(2013-09-16) Mooney, Chance; Raphael, Robert M.; Diehl, Michael R.; Mittleman, Daniel M.
The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.
Template-based Protein Structure Prediction and its Applications
(2013-08-26) Cheng, Yushao; Ma, Jianpeng; Diehl, Michael R.; Tao, Yizhi Jane
Protein structure prediction, also called protein folding, is one of the most significant and challenging research areas in computational biophysics and structural bioinformatics. With the rapid growth of PDB database, template-based modeling such as homology modeling and threading has become a popular method in protein structure prediction. However, it is still hard to detect good templates when the sequence identity is below 30%. In chapter 1, a profile-profile alignment method is proposed. It uses evolutionary and structural profiles to detect homologs, and a z-score-based method to rank templates. The performance of this method in the critical assessment of protein structure prediction experiments (CASP) was reported. In chapter 2, p53 mutations are studied as an application of protein structure prediction. The TP53 gene encodes a tumor suppressor protein called p53, and p53 mutations occur in about half of human cancers. Experimental studies showed that p53 cancer mutants can be reactivated by mutations on other sites. Machine learning technologies were used in this research. Multiple classifiers were built to predict whether a p53 mutant (single-point or multiple-point) would be transcriptionally active or not, based on features extracted from amino acid sequences and structures. The mutant structures were modeled using template-based protein structure prediction. Theses features were selected and analyzed using different feature selection methods, and classifiers were built under different learning settings, such as supervised learning and semi-supervised learning. The performances of these classifiers were analyzed and compared. Besides the study of single proteins, protein complexes in yeast are studied in chapter 3. Multiple classifiers were built to predict whether several given proteins can form a protein complex, based on features generated from amino acid sequences and protein-protein interaction network. Theses features were selected and analyzed using different feature selection methods. Also, these classifiers were built under different learning settings, such as supervised learning and active learning. The performances of these classifiers were analyzed and compared.
The biophysics of intracellular transport driven by structurally-defined systems of motor proteins
(2011) Jamison, Kenneth; Diehl, Michael R.
The number of motor proteins attached to cellular cargos is widely believed to influence intracellular transport processes and may play a role in transport regulation. However, to date, investigating the biophysics of multiple-motor dynamics has been challenging since the number of motors responsible for cargo motion is not easily characterized. This work examines the transport properties of structurally-defined motor complexes containing two kinesin-1 motors, from both an experimental and theoretical perspective. Motor complexes were synthesized using DNA as a molecular scaffold and engineered DNA-conjugated protein polymers as linkers to couple motors to scaffolds. After anchoring the motor complexes to a bead their dynamic properties were measured using an automated optical trapping instrument that could be used to perform both static (increasing load) and force-feedback (constant load) optical trapping experiments. Data from these experiments is compared to predictions from a microscopic transition rate model of multiple kinesin dynamics. Together, these studies uncovered that multiple kinesins typically cannot cooperate since the microtubule-bound configuration of a motor complex often prevents both kinesins from sharing cargo loads. Furthermore, multiple-motor behaviors are influenced by the fact that motor complexes display hysteretic force-velocity behaviors when applied loads change rapidly in time. Overall, such behaviors suggest the number of kinesins on a cargo will not be a key determinant of intracellular transport processes, and in turn, will not contribute appreciably to mechanisms that regulate cargo motion. However, this work also provides evidence that processive microtubule motors that are less efficient than kinesin (e.g., dynein) will cooperate productively, produce greater responses to motor number, and may therefore act as a regulator of cargo transport.

Browsing by Author "Diehl, Michael R."

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