IU Indianapolis ScholarWorks :: Browsing by Author "Day, Richard N."

Browsing by Author "Day, Richard N."

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Characterizing Novel Fluorescent Protein Pairings for Förster Resonance Energy Transfer (FRET)
(Office of the Vice Chancellor for Research, 2011-04-08) Ariss, Corey R.; Hays, Nicole; Day, Richard N.
Since it’s cloning, the sequence encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been engineered to produce fluorescent proteins (FPs) with emission in the blue to yellowish-green range of the visible spectrum. Furthermore, many FPs with homology to the Aequorea GFP have been cloned from marine organisms, providing new proteins that fluoresce into deep red spectrum. These new FPs expanded the repertoire of applications to include multi-color imaging of protein co-localization and behavior inside living cells. However, it is their use as donor and acceptor pairs for Förster Resonance Energy Transfer (FRET) microscopy that has generated the greatest interest. The most precise methods for measuring FRET detect the quenching of the donor by the acceptor, and Fluorescence Lifetime Imaging Microscopy (FLIM) can accurately measure the shorter donor lifetimes that result from FRET. Currently, the Aequorea GFP variants known as Cerulean (cyan) and Venus (yellow) are the most popular FRET pair. However, Venus has poor photostability, and the emission near 500 nm limits its utility as an acceptor. The objective of this study was to use FLIM to test the utility of different FP pairings for FRET studies with the goal to identify potentially improved FRET pairs.
Coronary artery disease in metabolic syndrome: a role for the sarcoplasmic reticulum Ca2+ ATPase
(2016-05-10) Rodenbeck, Stacey Dineen; Sturek, Michael S.; Day, Richard N.; Evans-Molina, Carmella; Mather, Kieren; Tune, Johnathan D.
Coronary artery disease (CAD) is a leading cause of death among Americans and is fueled by underlying metabolic syndrome (MetS). The prevalence and lethality of CAD necessitates rigorous investigations into its underlying mechanisms and to facilitate the development of effective treatment options. Coronary smooth muscle (CSM) phenotypic modulation from quiescent to synthetic, proliferative, and osteogenic phenotypes is a key area of investigation, with underlying mechanisms that remain poorly understood. Using a well-established pre-clinical model of CAD and MetS, the Ossabaw miniature swine, we established for the first time the time course of Ca2+ dysregulation during MetS-induced CAD progression. In particular, we used the fluorescent Ca2+ dye, fura-2, to examine alterations in CSM intracellular Ca2+ regulation during CAD progression, as perturbations in intracellular Ca2+ regulation are implicated in several cellular processes associated with CAD pathology, including CSM contractile responses and proliferative pathways. These studies revealed that the function of several CSM Ca2+ handling proteins is elevated in early CAD, followed by loss of function in severe atherosclerotic plaques. Decreased intracellular Ca2+ regulation occurred concurrently with reductions in CSM proliferation, measured with Ki-67 staining. In particular, alterations in sarcoplasmic reticulum (SR) Ca2+ store together with altered function of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were associated with induction of proliferation. Organ culture of coronary arterial segments revealed that culture-induced medial thickening was prevented by SERCA inhibition with cyclopiazonic acid (CPA). Activation of SERCA with the small molecule activator, CDN1163, increased CSM proliferation, which was attenuated by treatment with CPA, thus establishing upregulated SERCA function as a proximal inducer of CSM proliferation. Further, we demonstrated that in vitro treatment of CSM from lean Ossabaw swine with the glucagon-like peptide-1 (GLP-1) receptor agonist, exenatide, increased SERCA function. However, in vivo treatment of Ossabaw swine with MetS with the GLP-1 receptor agonist, AC3174, had no effect on CAD progression and in vitro examination revealed resistance of SERCA to GLP-1 receptor agonism in MetS. These findings further implicate SERCA in CAD progression. Collectively, these are the first data directly linking SERCA dysfunction to CSM proliferation and CAD progression, providing a key mechanistic step in CAD progression.
Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells
(PLOS, 2021-12-23) Day, Richard N.; Day, Kathleen H.; Pavalko, Fredrick M.; Anatomy, Cell Biology and Physiology, School of Medicine
Earlier, we proposed the "mechanosome" concept as a testable model for understanding how mechanical stimuli detected by cell surface adhesion molecules are transmitted to modulate gene expression inside cells. Here, for the first time we document a putative mechanosome involving Src, Pyk2 and MBD2 in MLO-Y4 osteocytes with high spatial resolution using FRET-FLIM. Src-Pyk2 complexes were concentrated at the periphery of focal adhesions and the peri-nuclear region. Pyk2-MBD2 complexes were located primarily in the nucleus and peri-nuclear region. Lifetime measurements indicated that Src and MBD2 did not interact directly. Finally, mechanical stimulation by fluid flow induced apparent accumulation of Src-Pyk2 protein complexes in the peri-nuclear/nuclear region, consistent with the proposed behavior of a mechanosome in response to a mechanical stimulus.
In vivo analysis of human LHX3 enhancer regulation
(2013-03) Park, Soyoung; Rhodes, Simon J.; Day, Richard N.; Harrington, Maureen A.; Herring, B. Paul; Skalnik, David Gordon
The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.
Inferring diffusion dynamics from FCS in heterogeneous nuclear environments
(Elsevier, 2015-07-07) Tsekouras, Konstantinos; Siegel, Amanda P.; Day, Richard N.; Pressé, Steve; Department of Physics, School of Science
Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.
Intravital microscopy of biosensor activities and intrinsic metabolic states
(Elsevier, 2017-09-01) Winfree, Seth; Hato, Takashi; Day, Richard N.; Department of Cellular & Integrative Physiology, Indiana University School of Medicine
Intravital microscopy (IVM) is an imaging tool that is capable of detecting subcellular signaling or metabolic events as they occur in tissues in the living animal. Imaging in highly scattering biological tissues, however, is challenging because of the attenuation of signal in images acquired at increasing depths. Depth-dependent signal attenuation is the major impediment to IVM, limiting the depth from which significant data can be obtained. Therefore, making quantitative measurements by IVM requires methods that use internal calibration, or alternatively, a completely different way of evaluating the signals. Here, we describe how ratiometric imaging of genetically encoded biosensor probes can be used to make quantitative measurements of changes in the activity of cell signaling pathways. Then, we describe how fluorescence lifetime imaging can be used for label-free measurements of the metabolic states of cells within the living animal.
IUPUI Imaging Research Council
(Office of the Vice Chancellor for Research, 2012-04-13) Hutchins, Gary D.; Wilson, Kathryn J.; Sturek, Michael S.; Du, Eliza Y.; Fletcher, James W.; Long, Eric C.; Molitoris, Bruce A.; Johnson, Daniel P.; Day, Richard N.; Barnett, William K.; Palakal, Mathew J.
Abstract The IUPUI Imaging Research Council was created by the IUPUI Vice Chancellor for Research to provide guidance and direction for expansion of research imaging initiatives across all Schools and Departments within IUPUI. The specific goals of the council are: • To encourage and coordinate collaboration among IUPUI researchers from different disciplines • To provide advice and guidance in the realization of highly competitive large grant proposals that will support and grow the IUPUI imaging efforts into major nationally and internationally recognized programs • To develop a strategic plan that will enable IUPUI to become nationally and internationally known as the place for imaging research and its applications • To determine strategic areas of strength and growth • To determine available and needed resources • To determine strategic external partnerships Activities organized by the council to date include sponsoring an IUPUI Imaging Research Workshop on November 17, 2011. This workshop consisted of invited presentations, a poster session, and working group breakout sessions. Working groups explored research opportunities and needs in four priority areas (neuroscience, cancer, cardiovascular disease, and remote sensing). The council has recently initiated a monthly seminar series and is actively developing an IUPUI research imaging strategic plan. For more information visit the IUPUI Imaging Research Initiative website at www.imaging.iupui.edu.
Kinetic vasculogenic analyses of endothelial colony forming cells exposed to intrauterine diabetes
(2017-05-11) Varberg, Kaela Margaret; Haneline, Laura S.; Clauss, Matthias A.; Day, Richard N.; Harrington, Maureen A.; Srour, Edward F.
Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.
Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy
(Elsevier, 2014-03-15) Day, Richard N.; Department of Cellular & Integrative Physiology, IU School of Medicine
The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster Resonance Energy Transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin.
Mechanisms of translational regulation in the pancreatic β cell stress response
(2014) Templin, Andrew Thomas; Mirmira, Raghavendra G.; Day, Richard N.; Fueger, Patrick T.; Harrington, Maureen A.; Wek, Ronald C.
The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.

Browsing by Author "Day, Richard N."

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