Asparagusic Golgi Trackers

Abstract

Thiol-mediated uptake (TMU) is thought to occur through dynamic covalent cascade exchange networks. Here we show that the cascade accounting for TMU of asparagusic acid derivatives (AspA) ends in the Golgi apparatus (G) and shifts from disulfide to thioester exchange with palmitoyl transferases as the final exchange partner. As a result, AspA combined with pH-sensitive fluoresceins, red-shifted silicon-rhodamines, or mechanosensitive flipper probes selectively labels the Golgi apparatus in fluorescence microscopy images in living and fixed cells. AspA Golgi trackers work without cellular engineering and excel with speed, simplicity, generality, and compatibility with G/ER and cis/trans discrimination, morphological changes, anterograde vesicular trafficking, and superresolution imaging by stimulated emission depletion microscopy. Golgi flippers in particular can image membrane order and tension in the Golgi and, if desired, at the plasma membrane during TMU.

Figure 1

Thiol-mediated uptake, Golgi tracking, and vesicle trafficking. (A) Golgi apparatus (G) and vesicle trafficking, with endoplasmic reticulum (ER), nucleus (N), cytosol (C), lysosome (LY), late (LE) and early endosome (EE). (b) Anterograde and (c) retrograde transport (gray arrows), (d) secretory pathway, (e) endocytosis. (B) TMU of AspA 1 is found to end in the Golgi. Disulfide exchange cascades I–III with membrane-associated exchange partners (P_m) to cross the plasma membrane (PM) through toroidal elastics (TE) are terminated by thioester exchange IV–VI with palmitoyl transferases (PATs) as intracellular P_i.

Figure 2

Structure and function of Golgi trackers. (A) Representative existing Golgi trackers. (B) SiR-AspA 1 with palmitoylation products 5 and 2 and HPLC traces of cell extracts (top) compared to pure 2 (middle) and 1 (bottom). (C) FITC-AspA 6 and controls (GFP = green fluorescent protein). (D) Golgi-Flipper 11, with compression of twisted 11t into planar 11p by physical forces σ.

Figure 3

Evaluation of Golgi trackers. (A–G) SDCM images of fixed HK cells with (A) 6, (B, C) 1, (C) 1 and GM130, (D) zoom of (C) with cell boundaries (white dashed), (E) 6 (green, cis) and 1 (red, all), (F) 6 (green, cis) and TGN46 (red, trans), (G) 6 (green, cis) and GM130 (red, cis-median) with nuclei boundaries (E–G, white dashed), (H, I) masks generated for (H) GM130 (red, cis-median) and TGN46 (green, trans), and (I) GM130 (green, cis-median) and 1 (red, all). (J) Colocalization ratios for (F–I). (K, L) Golgi/cytosol (G/C) ratios for (K) 1, 6–9 and for (L) 1 treated with tunicamycin (TM) and cerulenin (CL). (M–Q) SDCM images of live HK cells for sequential tracking (Q) with 6 (green, 20 μM) for (M) 1 h, then (N) 5 min after the addition of (O) 1 (red, 500 nM, (P) = (N) + (O) merged). (R, S) SDCM images of live HK cells with 1 with (R) and without (S) brefeldin A for 0, 10, 30, 60 min (from left). (T, U) Live-cell STED images of HeLa cells with 1 (green) expressing endogenously tagged (T) CLCa^EN-Halo and (U) βCOP^EN-Halo labeled with JF₅₇₁–CA (magenta). Blue: Hoechst 33342 (HOE), scale bars: 50 μm (A–C), 20 μm (D), 10 μm (M–S), 5 and 0.5 μm in the crops (T, U).

Figure 4

Golgi flippers. Comparison of HK cells with either 11 (A–E, λ_ex = 480 nm, detection 600–650 nm), 3 (F–H, λ_ex = 480 nm, detection 510–540 nm), or 1 (I, J, λ_ex = 600 nm, detection 630–800 nm) in (A, F) CLSM images, (B, G) FLIM images reconstructed from their (C, H) phasor hemispheres, (D, I) FLIM images under isoosmotic and (E, J) hyperosmotic conditions; scale bars: 20 μm. (K) Lifetimes and changes from isoosmotic to hyperosmotic HK cells (arrows, Δσ) for 11 in Golgi compared to HaloFlippers in Golgi and 11 in PM compared to Flipper-TR.