RESUMO
We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIE-lsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells.
Assuntos
Espectrometria de Fluorescência/métodos , Linhagem Celular Tumoral , Humanos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/ß-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/ß-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.
Assuntos
Proteínas do Citoesqueleto/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , beta Catenina/genética , Animais , Comunicação Autócrina/genética , Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Comunicação Parácrina/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Via de Sinalização Wnt/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.
