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Temperature-sensitive (ts) mutant proteins are great tools to analyze cell-regulative processes by temporally affected protein interactions. However, many observations are based on experiments where temperature shifts in the whole sample (e.g. embryos), and thus are missing spatial regulative aspects. In the study of S.M. Hirsch et al. (nature methods, 2018) they present FLIRT, which stands […]
To analyze the tubulin dynamics during spindle formation, Drosophila S2 cells were transfected with mEos2-labled tubulin. By local irradiation with UV laser light (405nm) the green fluorescent mEos2 photoconverts to a red fluorescent dye. In contrast to photobleaching, where the fluorescent signal is locally depleted, the photoswitching allowed the independent observation of two different tubulin […]
Channelrhodopsin-2 is a cation channel derived from algae that will open with millisecond precision upon illumination with blue light (excitation maximum around 470 nm) and depolarize neuronal membranes. This can be used to selectively activate neurons and neuronal fibers expressing this protein, replacing the need for unspecific extracellular electric stimulation. In a pilot study, we […]
As a proof of concept, experiments in acute brain slices were performed to characterize the uncaging efficiency of caged calcium in response to localized UV irradiation. By using Fluo4 as an indicator for
Actin bundles are the force generating part of a cell, and thus are major players during cell-mechanical processes like cell migration. By cutting individual actin fibers and quantifying the corresponding retraction, the underlying physical properties, like tension and internal forces can be determined. Additionally, by cutting force-generating stress-fibers during cell migration, cell responses, like loss […]
Mouse embryonic fibroblasts (MEF) were transfected with different mutations of the adaptor protein vinculin and FRAP experiments were performed to analyze the influence of the mutations on the incorporation of vinculin into focal adhesions (FAs). For the quantification of the real protein exchange dynamics within focal adhesions without overlying cytosolic diffusion artifacts, the FRAP technique […]