Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy

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

A Southern Progressive: M. A. Cassidy and the Lexington Schools, 1886-1928

The 42-year career of M. A. Cassidy exemplifies the transition of public school leadership in Kentucky from non-educators who held religious-political ideologies to professional progressive educators who sought to make Kentucky schools more efficient through expertise and scientific management. This concept was fully adopted in Section 183 of the Kentucky Constitution (1891) which required the General Assembly to “provide for an efficient system of common schools throughout the state.” Confident that professional educators were best suited to devise solutions to social problems, and justified by the twin notions of equality of educational opportunity and meritocracy, Cassidy was part of a new breed of progressive educators who joined with the business community to declare that a modest amount of schooling would prepare all for a life of equality, not by restructuring society, but by making each individual better. Cassidy belonged to that class of Southern accommodationist progressive educators who saw themselves as the teachers and guardians of subordinate African Americans in whom they would cultivate “some measure of collaboration and consent.” Cassidy presents as a Southern-style reformer working in a border state, but one who held conservative and progressive ideals in equal measure. Like most white Southerners, Cassidy believed that blacks were inherently inferior to whites. But unlike his Southern peers--and despite being part of a community that did not embrace social mobility for blacks--Cassidy was an early adopter of educational equality that included blacks, albeit, in a separate system under Jim Crow. His attention to physical and operational improvements to black schools, including enhanced teacher training and the addition of innovative programs for students, was remarkable for its place and time. But apparent philosophical conflicts fall into place once we see Cassidy for who he was: a public official who necessarily had to work closely with his constituency to achieve his goals; a change agent who used the bully pulpit to extol the virtues of literacy and a proper education; a Christian in the Social Gospel tradition who saw a duty to the least among us; and a personable superintendent who used his sense of Southern gentility to attract more citizens to the enterprise

Intravital microscopy of biosensor activities and intrinsic metabolic states

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

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example—first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM

A Persistent Quandary: The Rural School Improvement Project, 1953-1957

Berea College\u27s Rural School Improvement Project worked directly with more than 5,000 children and 63 teaching fellows in 39 different schools over 13 counties, and one independent school district, involving 10 county school supervisors. Project estimates claimed an indirect impact on approximately 45,000 children within the RSIP school districts. The RSIP represented the thinking of national leaders of rural education in the 1950s who promoted improved administration of the schools combined with an active community engagement program based on “full respect for human personality” and “shared judgments.” Following so many decades of poverty and isolation, it is no easy task to gauge the impact of a program like the Rural School Improvement Project on the children and communities of southeastern Kentucky. Like the progress-minded projects that came before it - and those that would come after - the seemingly intractable challenges attending the Appalachian region kept educational equity at bay. Indeed, it would take another thirty-two years before the landmark Kentucky Supreme Court decision in Rose v Council for Better Education would declare the state’s inequitable and inadequate school system to be unconstitutional. “Sixty-six” property poor, and mostly rural, school districts had sued the General Assembly citing the abiding fiscal inequities which had grown to as much as an 8:1 ratio when compared to one urban district. With the passage of the Kentucky Education Reform Act, in 1990, the General Assembly provided substantial funding equity to rural schools - before returning to its historical pattern of periodic attention amid chronic neglect

Attitudes and Preferences of ESL Students to Error Correction

This article presents the findings of a survey of ESL students' attitudes toward and preferences for the correction of spoken errors by native speaker friends. The 418 subjects reported generally positive attitudes toward error correction, and claimed to prefer even more correction than their friends did. They saw correcting errors as facilitating--even being necessary--for the improvement of their oral English

Error Correction in Native-Nonnative Conversation

The purpose of this paper is to report the results of an investigation into how native speakers (NSs) of English in social settings correct the errors committed by their friends who are nonnative speakers (NNSs) of English. While there are a number of studies which describe error correction in the second/foreign language classroom (e.g., Allwright 1975; Fanselow 1977; Holley and King 1971), little is known about what NSs do when their NNS friends commit errors. Gaskill (1980) studied sample conversations of one NNS with several NSs. He concluded that when, and if, NS correction occurs, it is usually modulated in form to show NS uncertainty. However, as Cathcart and Olsen (1976) noted, personalities involved in the interaction affect the amount and type of correction supplied. Since Gaskill had only one NNS as his source of data, his results may not be representative. The NSs in our data used two strategies tocorrect NNS errors: on-record corrections off-record corrections. After describing the subjects and the methods used in collecting the data, we discuss in detail these two strategies. We also present, by way of contrast, several noncorrective discourse strategies which NSs used in order to clear up conversational difficulties. A model of error correction is proposed, which shows that most NS error corrections were given at transition points and not as interruptions. The paper concludes with implications of the results for the classroom and with suggestions for future research

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail

A Versatile, Portable Intravital Microscopy Platform for Studying Beta-cell Biology In Vivo

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo. We have developed several adaptable tools to create a versatile platform to interrogate β-cell function in vivo. Specifically, we developed β-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo β-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study β-cell structure and signaling in vivo, and its portability for use in virtually any mouse model will enable meaningful studies of β-cell physiology in the endogenous islet niche