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We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in(More)
The extracellular signal-regulated protein kinase ERK1/2 is a crucial effector linking extracellular stimuli to cellular responses: upon phosphorylation ERK [also known as mitogen-activated protein kinase P42/P44 (MAPK)] concentrates in the nucleus where it activates specific programs of gene expression. Notwithstanding the importance of this process,(More)
We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise(More)
We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible(More)
Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide-tagged cargoes, although some in vitro studies have suggested the(More)
Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to(More)
Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic(More)
A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and(More)
Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells(More)
Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable to living-cell studies such as sequential photolabeling of distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing to the relevance of fluorescent proteins from Aequorea victoria (AFPs) for cell biology,(More)