Quantitative Molecular Imaging in Living Cells via FLIM

Chang, Ching-Wei; Mycek, Mary Ann

doi:10.1007/978-1-4419-9828-6_8

Cited by 7 publications

(3 citation statements)

References 73 publications

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“…Consistent with our previous studies, SF irradiation led to retraction of the severed ends of the targeted SF (top row), and inspection of the donor and FRET intensity (middle rows) revealed that the VinTS localizes to FAs as expected. Because the FRET intensity also depends on factors unrelated to donoracceptor separation (Chang and Mycek, 2012;Chang et al, 2007;Chang et al, 2009;Zhong et al, 2007), such as the amount of localized VinTS, we created FRET ratio maps in which we masked the FRET intensity to show only FAs and normalized this intensity by the donor intensity (bottom row). In these maps, an increasing FRET ratio corresponds to an increasing FRET signal (actual degree of energy transfer) or decreasing tension, which can in turn be tracked on an FA-by-FA basis.…”

Section: Mapping Tension Distributions Of Single Stress Fibersmentioning

confidence: 99%

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Chang

Kumar

2013

Journal of Cell Science

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SummaryActomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and highresolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

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Section: Mapping Tension Distributions Of Single Stress Fibersmentioning

confidence: 99%

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Chang

Kumar

2013

Journal of Cell Science

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“…Genetically encoded fluorophores, most likely fluorescent proteins, are commonly used for live‐cell FRET applications. Fluorescent‐protein FRET probes can be made to detect the conformational change of a full‐length protein (Hao and Macara, ); protease cleavage (Buranachai et al, ; Ouyang et al, ); binding of a protein sensory domain with its ligand (Kalab et al, ; Nguyen and Daugherty, ; Shimozono et al, ; Kolossov et al, ); kinase and small GTPase activity (Miyawaki et al, ; Nakamura et al, ; Zhang and Allen, ; Zhong et al, ; Nakaya et al, ; Chang et al, ; Machacek et al, ; Chang and Mycek, ); and even the movement of a protein sensory domain (Sakai et al, ; Tsutsui et al, ) (Some of these applications are reviewed by Kalab and Soderholm ()). FRET probes with an elastic linker connecting the donor and accepter have recently made it possible to study molecular tension in the context of cell mechanics and mechano‐transduction with high spatial and temporal resolution (Meng et al, ; Grashoff et al, ; Meng and Sachs, ; Murakoshi et al, ; Verma et al, ; Chang and Kumar, ; Conway et al, ).…”

Section: Fluorescence Resonance Energy Transfer Microscopymentioning

confidence: 99%

“…Figure adapted and reprinted with permission from Kuchibhotla et al (2009Kuchibhotla et al ( ). et al, 2008; kinase and small GTPase activity (Miyawaki et al, 1997;Nakamura et al, 2006;Zhang and Allen, 2007;Zhong et al, 2007;Nakaya et al, 2008;Chang et al, 2009;Machacek et al, 2009;Chang and Mycek, 2012a); and even the movement of a protein sensory domain (Sakai et al, 2001;Tsutsui et al, 2008) (Some of these applications are reviewed by Kalab and Soderholm (2010)). FRET probes with an elastic linker connecting the donor and accepter have recently made it possible to study molecular tension in the context of cell mechanics and mechanotransduction with high spatial and temporal resolution (Meng et al, 2008;Grashoff et al, 2010;Meng and Sachs, 2011;Murakoshi et al, 2011;Verma et al, 2012;Chang and Kumar, 2013;Conway et al, 2013).…”

Section: Fluorescence Resonance Energy Transfer Microscopy the Theorymentioning

confidence: 99%

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

Santos

Chang

Mycek

et al. 2015

Molecular Reproduction Devel

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The combination of fluorescent-probe technology plus modern optical microscopes allows investigators to monitor dynamic events in living cells with exquisite temporal and spatial resolution. Fluorescence recovery after photobleaching (FRAP), for example, has long been used to monitor molecular dynamics both within cells and on cellular surfaces. Although bound by the diffraction limit imposed on all optical microscopes, the combination of digital cameras and the application of fluorescence intensity information on large-pixel arrays have allowed such dynamic information to be monitored and quantified. Fluorescence lifetime imaging microscopy (FLIM), on the other hand, utilizes the information from an ensemble of fluorophores to probe changes in the local environment. Using either fluorescence-intensity or lifetime approaches, fluorescence resonance energy transfer (FRET) microscopy provides information about molecular interactions, with Ångstrom resolution. In this review, we summarize the theoretical framework underlying these methods and illustrate their utility in addressing important problems in reproductive and developmental systems.

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Brief History ofICTMolecules

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Intramolecular Charge Transfer

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Quantitative Molecular Imaging in Living Cells via FLIM

Cited by 7 publications

References 73 publications

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy

Brief History ofICTMolecules

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