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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Measuring Membrane Voltage with Microbial Rhodopsins
Membrane voltage (Vm) is a fundamental biological parameter that is essential for neuronal communication, cardiac activity, transmembrane transport, regulation of signaling, and bacterial motility. Optical measurements of Vm promise new insights into how voltage propagates within and between cells, but effective optical contrast agents have been lacking. Microbial rhodopsin-based fluorescent voltage indicators are exquisitely sensitive and fast, but very dim, necessitating careful attention to experimental procedures. This chapter describes how to make optical voltage measurements with microbial rhodopsins. (Source: Spring...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Live-Cell Imaging of Cytosolic NADH–NAD+ Redox State Using a Genetically Encoded Fluorescent Biosensor
NADH is an essential redox cofactor in numerous metabolic reactions, and the cytosolic NADH–NAD+ redox state is a key parameter in glycolysis. Conventional NADH measurements rely on chemical determination or autofluorescence imaging, which cannot assess NADH specifically in the cytosol of individual live cells. By combining a bacterial NADH-binding protein and a fluorescent protein variant, we have created a genetically encoded fluorescent biosensor of the cytosolic NADH–NAD+ redox state, named Peredox (Hung et al., Cell Metab 14:545–554, 2011). Here, we elaborate on imaging methods and technical consider...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Genetically Encoded Fluorescent Biosensors for Live Cell Imaging of Lipid Dynamics
Fluorescence imaging provides a powerful technique to visualize spatiotemporal dynamics of biomolecules in living cells, if fluorescent biosensors for the relevant biomolecules become available. Here I describe a fluorescent biosensor for a lipid second messenger, phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3). The biosensor overcomes limitations of existing methods for the lipid analysis and allows us to pinpoint that the PI(3,4,5)P3 concentrations are increasing and/or decreasing not only at the plasma membrane but also at organelle membranes, such as the Golgi apparatus membranes and endoplasmic reticulum membran...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Analysis of Compartmentalized cAMP: A Method to Compare Signals from Differently Targeted FRET Reporters
Förster resonance energy transfer (FRET)-based reporters are important tools to study the spatiotemporal compartmentalization of cyclic adenosine monophosphate (cAMP) in living cells. To increase the spatial resolution of cAMP detection, new reporters with specific intracellular targeting have been developed. Therefore it has become critical to be able to appropriately compare the signals revealed by the different sensors. Here we illustrate a protocol to calibrate the response detected by different targeted FRET reporters involving the generation of a dose–response curve to the cAMP raising agent forskolin. Thi...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Detecting cAMP with an Epac-Based FRET Sensor in Single Living Cells
Cyclic nucleotides such as cGMP and cAMP play pivotal roles as second messengers in many biological processes. Upon stimulation of appropriate signal transduction pathways, the levels of these messengers change rapidly. Such variations in second messenger level may also be spatially restricted within the cell. To detect dynamic and local changes in second messengers, we need to study them in living cells with high spatial and temporal resolution. Focusing on cAMP, here we describe how imaging of an EPAC-based FRET sensor in single cells provides that spatiotemporal resolution. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Quantitative Measurement of Ca2+ and Zn2+ in Mammalian Cells Using Genetically Encoded Fluorescent Biosensors
We describe important factors to consider when selecting a Ca2+ or Zn2+ biosensor, such as the sensor’s dissociation constant (K
d′) and its dynamic range. We also discuss the limits of quantitative measurement using these sensors and reasons why a sensor may perform differently in different biological systems or subcellular compartments. We outline protocols for (1) quickly confirming sensor functionality in a new biological system, (2) calibrating a sensor to convert a sensor’s FRET ratio to ion concentration, and (3) titrating a sensor in living cells to obtain its K
d&prime...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
An Introduction to Fluorescence Imaging Techniques Geared Towards Biosensor Applications
After providing a brief overview of the basics of fluorescence and FRET, this chapter discusses the most commonly used methods to record FRET. Emphasis is on microscopy methods that are widely used for biosensor imaging. We cover choice of instruments, describe various ways to detect FRET based on intensity as well as on donor lifetime, and provide some guidelines to match particular recording methods with specific scientific experiments. We end with an extensive discussion on further practical considerations that may greatly affect the success of the experiments. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
The Design and Application of Genetically Encodable Biosensors Based on Fluorescent Proteins
To track the activity of cellular signaling molecules within the endogenous cellular environment, researchers have developed a diverse set of genetically encodable fluorescent biosensors. These sensors, which can be targeted to specific subcellular regions to monitor specific pools of a given signaling molecule in real time, rely upon conformational changes in a sensor domain to alter the photophysical properties of green fluorescent protein (GFP) family members. In this introductory chapter, we first discuss the properties of GFP family members before turning our attention to the design and application of genetically enco...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Integrating Fluorescent Biosensor Data Using Computational Models
This book chapter provides a tutorial on how to construct computational models of signaling networks for the integration and interpretation of FRET-based biosensor data. A model of cAMP production and PKA activation is presented to provide an example of the model building process. The computational model is defined using hypothesized signaling network structure and measured kinetic parameters and then simulated in Virtual Cell software. Experimental acquisition and processing of FRET biosensor data is discussed in the context of model validation. This data is then used to fit parameters of the computational model such that...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
FRET and BRET-Based Biosensors in Live Cell Compound Screens
Live cell compound screening with genetically encoded fluorescence or bioluminescence-based biosensors offers a potentially powerful approach to identify novel regulators of a signaling event of interest. In particular, compound screening in living cells has the added benefit that the entire signaling network remains intact, and thus the screen is not just against a single molecule of interest but against any molecule within the signaling network that may modulate the distinct signaling event reported by the biosensor in use. Furthermore, only molecules that are cell permeable or act at cell surface receptors will be ident...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
A Multiparameter Live Cell Imaging Approach to Monitor Cyclic AMP and Protein Kinase A Dynamics in Parallel
Parallel detection of signaling activities allows us to correlate activity dynamics between signaling molecules. In this review, we detail a multiparameter live cell imaging method to monitor 3′,5′-cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activities in parallel. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Optical Calcium Imaging Using DNA-Encoded Fluorescence Sensors in Transgenic Fruit Flies, Drosophila melanogaster
The invention of protein-based fluorescent biosensors has paved the way to target specific cells with these probes and visualize intracellular processes not only in isolated cells or tissue cultures but also in transgenic animals. In particular, DNA-encoded fluorescence proteins sensitive to Ca2+ ions are often used to monitor changes in intracellular Ca2+ concentrations. This is of particular relevance in neuroscience since the dynamics of intracellular Ca2+ concentrations represents a faithful correlate for neuronal activity, and optical Ca2+ imaging is commonly used to monitor spatiotemporal activity across populations ...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Biosensor Imaging in Brain Slice Preparations
Cyclic-AMP dependent protein kinase (PKA) is present in most branches of the animal kingdom, and is an example in the nervous system where a kinase effector integrates the cellular effects of various neuromodulators. The recent development of FRET-based biosensors, such as AKAR, now allows the direct measurement of PKA activation in living cells by simply measuring the ratio between the fluorescence emission at the CFP and YFP wavelengths upon CFP excitation. This novel approach provides data with a temporal resolution of a few seconds at the cellular and even subcellular level, opening a new avenue of understanding the in...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Genetically Encoded Fluorescent Biosensors for Live-Cell Imaging of MT1-MMP Protease Activity
The proteolytic activity of Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is crucial for cancer cell invasion and metastasis. To visualize the protease activity of MT1-MMP with high spatiotemporal resolution at the extracellular plasma membrane surface of live cancer cells, a genetically encoded fluorescent biosensor of MT1-MMP has been developed. Here we describe the design principles of the MT1-MMP biosensor, the characterization of the MT1-MMP biosensor in vitro, and the live-cell imaging protocol used to visualize MT1-MMP activity in mammalian cells. We also provide brief guidelines for observing MT1-MMP subcellul...
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
Genetically Encoded FRET Indicators for Live-Cell Imaging of Histone Acetylation
Histone acetylation is dynamically and reversibly controlled by histone acetyltransferases and deacetylases during cellular events such as cell division and differentiation. However, the dynamics of histone modifications in living cells are poorly understood because of the lack of experimental tools to monitor them in a real-time fashion. Herein, we introduce Förster/fluorescence resonance energy transfer (FRET)-based indicators to visualize acetylation of histone H4, and describe a protocol for live-cell imaging with high spatiotemporal resolution. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - September 22, 2013 Category: Biochemistry Source Type: news
