Fig. 1. TeNT H_C labelled with different fluorophores is specifically transported in MNs. (A) TeNT and the recombinant TeNT H_C used in this study. TeNT is composed of two chains (H and L). The H_C fragment (dark grey) is responsible for neurospecific binding and retrograde transport. TeNT H_C with a cysteine-rich tag at the N-terminus (hatched box) was expressed as a glutathione S-transferase (GST) fusion protein. The boxed segments are predicted to form -helices with cysteines (in black) favourably oriented to bind FLASH or Alexa maleimides. Scissors indicate the thrombin cleavage site. (B-G) Binding and internalization of TeNT H_C in living rat MNs imaged by low-light microscopy. TeNT H_C-FLASH (B), TeNT H_C-Alexa488 (C), TeNT H_C-Alexa 546 (D) and TeNT H_C-Alexa594 (E) (all 40 nM) are internalized and transported in vesicular carriers (arrowheads). (F) Binding and transport of TeNT H_C-Alexa488 are abolished by pre-incubation with a 100x molar excess of native TeNT. (G) Phase contrast picture of the corresponding image in F. Bar, 10 µm.