Entries by Anette

FLIRT: fast local infrared thermogenetics for subcellular control of protein function

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 […]

Following Tubulin Dynamics during Spindle Formation using Photoconversion of mEos2

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 […]

Optogenetical Stimulation in Acute Brain Slices

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 […]

Cutting Actin Bundles in Single Cells

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 […]

Investigation of Protein Dynamics in Focal Adhesions Using FRAP and TIRF

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 […]