Biology subjects

Valenta, H.

Publications and source records attributed to Valenta, H..

3 recordsLinked to original sources

bioRxiv2023

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the usable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/538742v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f49247org.highwire.dtl.DTLVardef@1af85c2org.highwire.dtl.DTLVardef@768a92org.highwire.dtl.DTLVardef@83b81b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

Yellow fluorescent proteins (YFP) are widely used as optical reporters in Forster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. Here, we characterize the yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol-1.L.cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with sub-diffraction resolution using STED nanoscopy and is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP is valuable FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live cell imaging including FRET experiments at acidic pH.

biophysics↗

bioRxiv2021

Consequences of the constitutive NOX2 activity in living cells: cytosol acidification, apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2*-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called Trimera, composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy. HighlightsO_LITrimera is a tool to trigger a continuous ROS production in living cells C_LIO_LIContinuous NOX2 activity causes cytosol acidification and apoptosis C_LIO_LIROS overproduction leads to localized oxidation of the membrane lipids C_LIO_LITrimera tends to clusterize in the plasma membrane of COSNOX and COS-7 cells C_LI

biophysics↗