Fig. 5: PI4P transport assay.
From: Coordination of transporter, cargo, and membrane properties during non-vesicular lipid transport
A Scheme of the assay. The amount of accessible PI4P (nPI4P(acc.)) exceeds the amount of Osh6. The FCCS read-out, Gcc, does not drop upon the Osh6 addition as the relative change of PI4P level in the PI4P donating LUVs is small and the biosensor’s response is saturated. B Scheme of the assay when also PI4P accepting LUVs are present in the system. The FCCS read-out, Gcc, drops as the PI4P accepting LUVs allow for the PI4P deposition and continuation of the transport. C Dependence of the FCCS read-out, Gcc, of SidC-Atto488 biosensor in our experimental system. The blue and red lines depict the PI4P concentration regions where the extraction and the transport can be observed, respectively. D PI4P transport to LUVs with competing ligand. Kinetics of the PI4P transport to the PI4P accepting LUVs composed of DOPC (red solid squares), DOPC/PS (red hollow squares), DOPC/diphytanoylPS (red solid circles). The experiment with no accepting LUVs is depicted by black solid squares. E Effect of charged lipids on the PI4P release. The PI4P accepting LUVs were composed of DOPC/PG (green) and DOPC/PA (orange). The data from the (D) (partially transparent) are shown for comparison. F Effect of fluidity on the PI4P release. The PI4P accepting LUVs were composed of POPC (green solid squares), POPC/DAG (green hollow squares), DOPC (red solid squares), DOPC/DAG (red hollow squares), and POPC/POPS (10 mol%)/cholesterol (25 mol%)—PM-like composition (blue solid squares). Experiment without the accepting LUVs is shown for comparison (gray squares). G PI4P transport rates for each composition of LUVs B. Composition of the experiment in 4D–F: cPI4P = 1.5 µM, cOsh6 = 250 nM, cSidC-Atto488 = 100 nM, ctotal lipids LUV A = 50 µM, ctotal lipids LUV B = 200 µM. All error bars represent the standard error of the mean, n = 10 measurements. The p-values were obtained from the two-sample t-test.
