Schematic diagram illustrating the split kinesin assay. (A) Three constructs are expressed together: a vesicle marker labeled with GFP, a constitutively active axon-selective kinesin motor domain tagged with tdTomato and fused to FKBP (KIF5C⁵⁵⁹-tdTM-FKBP), and a myc-tagged kinesin tail domain fused to FRB. The kinesin tail domain binds its endogenous cargo vesicle, but co-assembles with the kinesin motor domain only after addition of the linker drug. (B) In the absence of linker drug, endogenous motor proteins transport GFP-labeled vesicles in dendrites, but they do not enter the axon. The constitutively active motor domain (black arrows) translocates toward the axonal tip, but does not bind cargo. After the linker drug is added, the split kinesin is assembled, but without interaction between the kinesin tail and the labeled vesicle population, this does not result in any changes in the transport behavior of the vesicles. (C) In case of a positive interaction between the tail and the vesicle, the tail is able to bind the vesicle population immediately after expression. After the addition of linker drug the split kinesin is assembled on the labeled vesicle. (D) Upon the addition of the linker drug, GFP-labeled vesicles that bind the expressed kinesin tail become attached to the constitutively active axonal KIF5C⁵⁵⁹-FKBP motor domain, which transports them into the axon.