Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

Baker, Bradley J.; Lee, H.; Pieribone, Vincent A.; Cohen, Lawrence B.; Isacoff, Ehud Y.; Knöpfel, Thomas; Kosmidis, Efstratios K.

doi:10.1016/j.jneumeth.2006.10.005

Cited by 105 publications

(94 citation statements)

References 22 publications

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“…2A). We note a membrane localization that is more distinct in the low temperature cultured cells, as quantitated by the autocorrelation plot in good spatial correlation between Y-NKCC-C and a membranelocalized voltage-sensitive dye (30).…”

Section: Resultsmentioning

confidence: 65%

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

Pedersen¹,

Carmosino

Forbush

2008

Journal of Biological Chemistry

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With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed cotemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

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Section: Resultsmentioning

confidence: 65%

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

Pedersen¹,

Carmosino

Forbush

2008

Journal of Biological Chemistry

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“…That project proved to be about a decade too early as those probes failed to work in mammalian cells. 6 Rather than being discouraged, Larry gathered the laboratories competing to develop these probes and convinced them to work together in order to avoid repeating the same failures over and over. One of those labs solved the trafficking problem by utilizing the newly discovered VSD from the Ciona VSP.…”

Section: Honoring Our Mentormentioning

confidence: 99%

Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors

et al. 2015

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Abstract. Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…The activation kinetics of this channel were significantly faster than that observed for FlaSh, however, the fractional change in fluorescence was low (~0.5% per 100 mV) in oocytes where the construct was overexpressed. In both cases, these constructs have not been especially useful for imaging activity in mammalian neurons, because the large fraction of intracellular versus plasma membrane FlaSh and SPARC makes signal detection difficult [13]. Because an important goal of ion channel-coupled sensors is imaging neural activity in real time, i.e.…”

Section: Channel-based Voltage Sensorsmentioning

confidence: 99%

Visualizing circuits and systems using transgenic reporters of neural activity

Barth

2007

Current Opinion in Neurobiology

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Genetically encoded sensors of neural activity enable visualization of circuit-level function in the central nervous system. Although our understanding of the molecular events that regulate neuronal firing, synaptic function, and plasticity has expanded rapidly over the past fifteen years, an appreciation for how cellular changes are functionally integrated at the circuit level has lagged. A new generation of tools that employ fluorescent sensors of neural activity promises unique opportunities to bridge the gap between cellular and system-levels analysis.This review will focus on genetically-encoded sensors. A primary advantage of these indicators is that they can be non-selectively introduced to large populations of cells using either transgenic or viral-mediated approaches. This ability removes the non-trivial obstacles of how to get chemical indicators into cells of interest, a problem that has dogged investigators who have been interested in mapping neural function in the intact CNS. Five different types of approaches and their relative utility will be reviewed here: 1) reporters of immediate-early gene (IEG) activation using promoters such as c-fos and arc, 2) voltage-based sensors, such as GFPcoupled Na + and K + channels, 3) Cl − based sensors, 4) Ca ++ -based sensors, such as Camgaroo and the troponin-based TN-L15, and 5) pH-based sensors, which have been particularly useful for examining synaptic activity of highly convergent afferents in sensory systems in vivo. Particular attention will be paid to reporters of IEG expression, since these tools employ the built-in threshold function that occurs with activation of gene expression, provoking new experimental questions by expanding the timescale of analysis for circuit and system-level functional mapping. Fluorescent reporters of inducible gene expressionImmediate-early gene expression (IEG) can be a reliable marker of elevated neuronal firing, reporting activity over time scales that range from minutes to hours compared to ms and seconds for voltage-or ion-based sensors. In ion-based approaches, indicators are constitutively present in the cell, and the fluorescent protein must be engineered to be a sensor with rapid onset and offset kinetics to achieve temporal fidelity with the process being measured. In contrast, monitoring IEG expression can be directly coupled to transcription of the reporter gene. However, because maturation of the fluorophore is required for detection, a temporal delay may be introduced into reporter detection. This delay in reporting activity is advantageous in that it expands the types of experimental questions that can be addressed. For example, do IEGexpressing cells remain more excitable after stimulus cessation? Are cells activated by two temporally distinct stimuli (i.e. training and recall in a memory task) overlapping or distinct?

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Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

Cited by 105 publications

References 22 publications

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors

Visualizing circuits and systems using transgenic reporters of neural activity

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