Quantitative FRET Analysis by Fast Acquisition Time Domain FLIM at High Spatial Resolution in Living Cells

Padilla‐Parra, Sergi; Audugé, Nicolas; Coppey-Moisan, Maïté; Tramier, Marc

doi:10.1529/biophysj.108.131276

Cited by 86 publications

(130 citation statements)

References 36 publications

Supporting

Mentioning

125

Contrasting

Order By: Relevance

“…Multifocal Multiphoton Fluorescence Lifetime Imaging Microscopy and Data Analysis-The FRET-FLIM system was described previously (27). Briefly, the FRET-FLIM apparatus combines multifocal multiphoton excitation (TriMscope, LaVision Biotec, Bielefeld, Germany) and a fast-gated CCD camera (Picostar, LaVision Biotec, Bielefeld, Germany).…”

Section: Journal Of Biological Chemistry 34247mentioning

confidence: 99%

“…Analysis of the data was done by using Image-J. Quantitative analysis was carried out as described previously (27). Briefly, the images from a timegated stack are first smoothed by a 3 ϫ 3 mask to decrease the noise.…”

Section: Journal Of Biological Chemistry 34247mentioning

confidence: 99%

“…To investigate the temporal and spatial association of amphiphysin 1 with N-WASP in Ser-W3 cells, FRET-FLIM was performed (27,32). First, we proved sensitivity of the method by demonstrating the homotypic interaction of amphiphysin 1 to form a homodimer.…”

Section: Amphiphysin 1 Directly Stimulates N-wasp-dependent Arp2/3mentioning

confidence: 99%

See 2 more Smart Citations

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

Yamada¹,

Padilla‐Parra²,

Park³

et al. 2011

Okayama I. Z.

Self Cite

View full text Add to dashboard Cite

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP-and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are requiredforthisstimulation.Acidicliposome-triggered,N-WASPdependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 colocalizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.The dynamic nature of the actin cytoskeleton is crucial for a variety of cellular events, including cell morphogenesis, cell migration, and intracellular membrane traffic (1). Actin polymerization is stimulated by a variety of actin regulatory proteins, prominent among which are WASP family proteins that function by triggering Arp2/3-mediated actin nucleation (2). Activation of WASP family proteins, in turn, is controlled by factors that bind these proteins and release an autoinhibitory intramolecular interaction that prevents their VCA domain from interacting with the Arp2/3 complex. As extensively shown for N-WASP, many such factors are proteins that bind to the N-WASP proline-rich region via the SH3 4 domain. Recently, we have found that the SH3 domain containing protein amphiphysin 1 stimulates actin polymerization during phagocytosis in testicular Sertoli cells, and this effect requires interactions of its C-terminal SH3 domain (3). Amphiphysin 1 is an endocytic adaptor present at high levels in brain at neuronal synapses but is also expressed at significant levels in Sertoli cells (4,5). In addition to a C-terminal SH3 domain, known to bind the GTPase dynamin and the phosphoinositide phosphatase synaptojanin (6), amphiphysin 1 contains an N-terminal BAR domain, a curved protein module that binds lipid bilayers and generates and senses curvature (7,8). It also contains binding motifs for clathrin and for the clathrin adaptor AP-2 (9). Hence, amphiphysin 1 was primarily studied as an endocytic protein capable of assembling at the neck of endocytic pits and of coupling clathrin-mediated budding to dynamin-mediated fission (10, 11). However, regulatory roles of amphiphysin 1 in actin cytoskeleton have also been suggested by studies of neuronal growth cones (12) and by the function of the amphiphysin homologue in yeast, . 81-86-235-7126; E-mail: kohji@md.okayama-u.ac.jp. 4 The abbrevi...

show abstract

Section: Journal Of Biological Chemistry 34247mentioning

confidence: 99%

Section: Journal Of Biological Chemistry 34247mentioning

confidence: 99%

See 1 more Smart Citation

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

Yamada¹,

Padilla‐Parra²,

Park³

et al. 2011

Okayama I. Z.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The benefits of the AMDI algorithm are not limited to this fitting method and may easily be extended to all the minimization algorithms classically used in time domain FLIM image analysis (such as the Newton trust region regression method, which is more robust to dispersive elements). Moreover, it should be noted that since the AMDI algorithm is an inflation of temporal fluorescence intensity decays, it is also compatible with all existing time domain FLIM image analysis strategies (28,(31)(32)(33)(34)(35).…”

Section: Discussionmentioning

confidence: 88%

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

et al. 2011

View full text Add to dashboard Cite

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress. To minimize exposure time, we have developed an original approach that statistically inflates the number of collected photons. Our approach, called Adaptive Monte Carlo Data Inflation (AMDI), combines the well-known bootstrap technique with an adaptive Parzen kernel. We here demonstrate using both Monte Carlo simulations and live cells that our robust method accurately estimate fluorescence lifetimes with exposure time reduced up to 50 times for monoexponential decays (corresponding to a minimum of 20 photons/pixel), and 10 times for biexponential decays (corresponding to a minimum of 5,000 photons/pixel), compared to standard fitting method. Thanks to AMDI, in Förster resonance energy transfer experiments, it is possible to estimate all fitting parameters accurately without constraining any parameters. By reducing the commonly used spatial binning factor, our technique also improves the spatial resolution of FLIM images. ' 2011 International Society for Advancement of Cytometry

show abstract

“…Recently, many efforts have been made to help simplify the results interpretation in classic FLIM experiments for nonspecialists (15)(16)(17). Among all these techniques, the polar plot or phasor (16,(18)(19)(20) is a promising approach that avoids complex fitting algorithm strategies and provides a fast, graphical representation of fluorescence lifetimes.…”

mentioning

confidence: 99%

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

et al. 2009

View full text Add to dashboard Cite

Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent. ' 2009 International Society for Advancement of Cytometry Key terms multispectral FLIM; SLIM; biological tissue; molecular interactions; FRET; phasor IN addition to intensity and wavelength, lifetime is a supplementary source of contrast in fluorescence microscopy, which greatly increases access to the biological information in living cells. Fluorescence Lifetime Imaging Microscopy (FLIM) has indeed been successfully applied to differentiate spectrally undistinguishable molecular species (1) and to map local modifications in labeled samples in terms of ion concentration (2), pH (3), and oxygen (4). FLIM has also been widely used to explore conformational change of proteins (5) or to separate interacting and non-interacting molecular fractions in Förster Resonance Energy Transfer (FRET) experiments (6,7).However, because of the low number of collected photons per pixel and the variability of the molecular environment inside a living cell, FLIM is usually limited to two lifetime components in the same pixel. This restriction makes it difficult to extract biologically relevant information from autofluorescent, heterogeneous, lightscattering samples such as biological tissue. Because of optical properties of tissue, a dedicated temporally and spectrally resolved system (called SLIM for Spectral and Lifetime Imaging Microscopy) is necessary for example to be able to explore molecular interactions on a nanometric scale (with FRET experiments).Temporal and spectral measurements can be performed using two approaches, either frequency domain (8,9) or time domain (10-12) measurements. In both cases, the determination of lifetimes and contributions of each molecular species are mainly achieved by fitting the collected data at each pixel using equations with multiple

show abstract

Quantitative FRET Analysis by Fast Acquisition Time Domain FLIM at High Spatial Resolution in Living Cells

Cited by 86 publications

References 36 publications

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

Contact Info

Product

Resources

About