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. Author manuscript; available in PMC: 2020 Sep 8.
Published in final edited form as: Cell Rep. 2020 Jun 16;31(11):107775. doi: 10.1016/j.celrep.2020.107775

Figure 2. Reversible Association with Motor Proteins (RAMP) Distinguishes Axonal Lysosomes from SVPs.

Figure 2.

(A) Schematic representation of the coupling of lysosomes to the minus-end-directed kinesin KIFC1 using RAMP (Guardia et al., 2019).

(B) DIV5 rat hippocampal neurons were transfected with plasmids encoding LAMP1-SBP-RFP and Strep-KIFC1*-HA, together with SYG1-GFP (top and middle rows) or CD63-GFP (bottom row). Neurons were cultured in the presence of NeutrAvidin to remove biotin from the medium and thus enable the SBP-streptavidin interaction to take place. The following day, neurons were incubated for 1 h with (+) or without (−) biotin (in the absence of NeutrAvidin), as indicated in the figure. Neurons were then fixed and immunostained for HA and endogenous ankyrin G (ANKG) (to stain the AIS) and examined using confocal fluorescence microscopy. Scale bars: 20 μm. Arrowheads indicate the axon.

(C) Straightened and enlarged 50 mm segments of axons from (B) boxes showing the depletion of LAMP1-SBP-RFP and CD63-GFP, but not SYG1-GFP, from the axon of neurons expressing Strep-KIFC1*-HA in the absence of biotin.

In (B) and (C), single-color images are represented in inverted grayscale. See also Figure S2.