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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00727

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Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons

Giovanna Lalli1,*, Stephen Gschmeissner2 and Giampietro Schiavo1,{ddagger}

1 Molecular NeuroPathoBiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
2 Electron Microscopy Unit, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK



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  		Fig. 1. TeNT HC labelled with different fluorophores is specifically transported in MNs. (A) TeNT and the recombinant TeNT HC used in this study. TeNT is composed of two chains (H and L). The HC fragment (dark grey) is responsible for neurospecific binding and retrograde transport. TeNT HC 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 {alpha}-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 HC in living rat MNs imaged by low-light microscopy. TeNT HC-FLASH (B), TeNT HC-Alexa488 (C), TeNT HC-Alexa 546 (D) and TeNT HC-Alexa594 (E) (all 40 nM) are internalized and transported in vesicular carriers (arrowheads). (F) Binding and transport of TeNT HC-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.

 


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  		Fig. 2. Structures associated with TeNT HC binding and internalization. (A-D) Distribution of TeNT HC observed by EM in MNs after incubation with TeNT HC at 4°C followed by 10 nm gold-conjugated antibody. Gold (arrows) is found along neurite surfaces (a) and in forming coated pits (B). TeNT HC is also observed at synaptic sites (C) and neurite contacts (D). (E-H) MNs were treated as in AD, washed and then warmed to 37°C for 5 (E and F) or 20 minutes (G and H). TeNT HC is found in coated pits (E) and coated vesicles (F), whereas at later internalization timepoints gold particles are observed in tubular (G) and round organelles (H). Control samples where TeNT HC was omitted showed negligible gold labelling. Bars, 0.2 µm.

 


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  		Fig. 3. Retrograde transport of TeNT HC depends on F-actin. A scheme of the experiment is shown at the top. MNs were treated with DMSO (Ctrl) (A) or with 0.5 µM Lat B (B). Intervals between frames are 5 seconds. Cell bodies are located at the bottom. (A) Control neurons display retrograde transport (arrowhead, arrow, asterisk). See video 1. (B) Lat B causes the majority of carriers to stop or oscillate (arrowhead). See video 2. (C) Quantitative analysis of TeNT HC transport after F-actin disruption. Carriers were classified as stationary/oscillatory when the extent of the movement did not lead to any significant progression (<0.2 µm). Results are expressed as a percentage of the total carriers observed (control = 437, Lat B = 315). Bars represent the s.d. of four independent experiments (P=0.01). (D) Treatment with Lat B does not affect MTs visualized by an anti-ß-tubulin antibody. F-actin staining in MNs (E) disappears after Lat B treatment (F). Bars, 5 µm (A,B and E,F) and 10 µm (D).

 


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  		Fig. 4. Retrograde transport of TeNT HC depends on intact MTs. A scheme of the experiment is shown at the top. (A) MNs were incubated with TeNT HC-Alexa488 and treated with 5 µM Vin after the appearance of fluorescent carriers. The majority of carriers stops or oscillates (arrowhead, arrow). Intervals between frames are 20 seconds. The cell body is located at the bottom. See video 3. (B) Quantitative analysis of TeNT HC transport after MT disruption. Results are expressed as a percentage of the total carriers observed (control = 364, Vin = 361; n=2). (C) EM analysis of neurite cross-sections shows that MTs are absent in Vin-treated cells, whereas their density is unchanged in Lat B-treated cells. Data are expressed as a percentage of MT density observed in untreated MNs (control = 58; Vin = 36; Lat B = 24 neurites). The same cells used in A were immunostained for ß-tubulin. Treatment with Vin causes the accumulation of tubulin in paracrystals (D). Vin treatment is specific and does not affect F-actin visualized with fluorescent phalloidin (E). Bars, 5 µm (A) and 10 µm (D,E).

 


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  		Fig. 5. Efficient retrograde transport of TeNT HC depends on multiple MT-based motors. (A-C) Distribution of speed values observed between two consecutive frames for TeNT HC-Alexa488 carriers (interval = 5 seconds). Retrograde movement is conventionally shown as positive. (A) Effect of dynein inhibition on TeNT HC transport. MNs were incubated with control medium (718 carriers, n=7 independent experiments; black bars) or with 1 mM EHNA (142 carriers; n=4; white bars). Note the drastic decrease in the frequency of fast retrograde speed movements and the increase in pauses and slow anterograde movements. (B) Effect of kinesin inhibition on TeNT HC transport. Incubation of MNs with 10 µM AA (308 carriers; n=3; white bars) increases the frequency of pauses and of slow anterograde movements, and induces a decrease of high speed values which is less marked than after EHNA treatment. (C) Simultaneous treatment of MNs with EHNA and AA (57 carriers; n=2; white bars) has similar effects on TeNT HC transport to EHNA alone. (D) The speed distribution profile of TeNT HC carriers in rat MNs is best described by the sum of three Gaussian components, centred at 0, 0.53 and 1.15 µm/second (718 carriers; n=7). Quantification of these speed components in the presence and absence of motor inhibitors allowed us to analyse the contribution of different molecular motors to retrograde transport (E). EHNA (white bars) abolished the fast component and caused a correspondent increase of pauses, whereas the intermediate component remained unaltered compared with control cells (black bars). AA alone (hatched bars) increased the frequency of stationary periods while reducing the contribution of the fast component. Simultaneous treatment of cells with EHNA and AA (grey bars) led to the same effects observed with EHNA alone. Error bars represent mean values ± s.e.m.

 


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  		Fig. 6. Myosin Va is required for efficient retrograde transport in living MNs. (A) In rat MNs myosin Va shows an abundant punctate distribution and co-localises with TeNT HC-Alexa 488 carriers (B, arrowheads). (C) Merged image of A and B. (D,E) Time-lapse imaging of TeNT HC-Alexa 488 retrograde carriers (arrowheads, asterisks) in an axon of a wild-type (D) or a dilute lethal (E) mouse MN. Intervals between frames are 5 seconds. The cell body is located at the bottom in both D and E. Bars, 5 µm. See video 4. (F) Relative frequencies of speed values measured between two consecutive frames (interval = 5 seconds) for TeNT HC carriers in wild-type MNs (193 carriers, filled circles; three embryos) and dilute lethal MNs (91 carriers, empty circles; two embryos). Wild-type and dilute lethal embryos are derived from the same litter. Retrograde movement is conventionally shown as positive. (G) In mouse MNs, the speed distribution profile of TeNT HC is best described by the sum of three Gaussian curves centred at 0, 0.53 and 0.95 µm/second. A decrease of the fast component and an increase of pauses are observed in dilute lethal MNs (white bars), whereas the intermediate component remains unaltered relative to wild-type cells (black bars). Data are derived from the analysis of 357 carriers from five wild-type embryos and 141 carriers from three dilute lethal embryos. Error bars indicate mean ± s.e.m.

 

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© The Company of Biologists Ltd 2003