Fig. 4. Multi-round reversible control of cellular cargo transport and study of kinesin-cargo specificity using cRGT-based SNACIP.

a Schematic view of how cRGT in combination with TMP inhibitor reversibly controls KIF5B-mediated cellular transport of peroxisomes. In this process, attachment of the N-terminal motor region (1–560) of KIF5B, i.e. KIF5B^N, to an appropriate cargo, e.g. peroxisome, can activate KIF5B^N and subsequently stimulate the anterograde transport of cargo toward the plus end of microtubules, usually located at the periphery region of a cell. b Representative confocal images of live HeLa cells co-expressing PEX3-mCherry-eDHFR (peroxisome localization) and KIF5B^N-EGFP before adding cRGT (Pre), after adding 24 μM cRGT for 1.5 h, after adding 10 μM TMP for 10 min and after washing out TMP (10 min). c Zoomed-in images of (b, cyan squares) with details of individual peroxisomes displayed. d Statistical PCC analysis between red and green channels (n = 21 cells for Pre, n = 22 cells for cRGT, n = 20 for cells TMP, n = 21 cells for Wash); one-sided Student’s t-test was used; see Methods for description of box plots. e Line profile analysis of the images in b. f Representative confocal images of live HeLa cells co-expressing mCherry-eDHFR-Rab5a (early endosome localizer) and KIF5B^N-EGFP before addition of cRGT (Pre), after adding 24 μM cRGT for 1.5 h, after adding 10 μM TMP for 10 min, and after washing out TMP (10 min). g Zoomed-in images of (f, cyan squares) with more details displayed. h Statistical PCC analysis between red and green channels (n = 22 cells for Pre, n = 30 cells for cRGT, n = 26 cells for TMP, n = 14 cells for Wash); one-sided Student’s t-test was used; see Methods for description of box plots. i Line profile analysis of the confocal images in (f) which revealed that colocalization was induced but no efficient anterograde transport of early endosomes to the very periphery of cells occurred. All scale bars: 10 μm.