engleski

Monitoring Rac Dynamics in Highly Motile Cells

Dictyostelium amoebae can switch their polarity within twenty seconds, and thus undergo the fastest polarization reversal among eukaryotic cells. Signalling by small Rho GTPases plays an essential role in this process. In particular, Rac1 GTPases play a dual role in the regulation of actin assembly, which is a key determining factor of cell polarity. At the cell front, Rac1 activates the Scar/WAVE complex and thereby stimulates the Arp2/3-mediated actin polymerization. At the cell back, Rac1 regulates stability of the cell cortex by initiating formation of a complex containing IQGAP-related protein DGAP1 and a heterodimer of actin-bundling proteins cortexillins. I will discuss possible consequences of Rac1 interactions with multiple partners on its dynamics in living motile cells and present result obtained by imaging and modelling approaches. In this context, I will describe in more detail characterization of a fluorescent probe that selectively binds to the active form of Rac1, and demonstrate its excellent properties for live cell imaging. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase, and was selected on the basis of yeast two-hybrid screen, GST pull-down assay, and FRET measurements by fluorescence lifetime imaging microscopy (FLIM). An important feature of DPAKa(GBD)-DYFP probe is its finely graded intensity distribution along the entire plasma membrane, which enables measurements of the Rac1 activity in different parts of the membrane. Importantly, ectopic expression of DPAKa(GBD)-DYFP does not affect cell motility, cytokinesis and growth, thus enabling long-term imaging with negligible photobleaching and phototoxicity.

Rac1 ; FLIM ; small GTPases ; fluorescent probe ; cell polarity

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