Bac­ulovirus is of inter­est since it gen­er­ates actin comets small enough to allow res­o­lu­tion of all actin fil­a­ments mak­ing up the comet tail by elec­tron tomog­ra­phy. Cul­tured insect cells that host bac­ulovirus are not suited for elec­tron tomog­ra­phy since their cytoskele­ton is poorly pre­served by cur­rent meth­ods used to expose the actin cytoskele­ton. How­ever, bac­ulovirus read­ily infects ver­te­brate cells and is pro­pelled at the head of actin comet tails in the same fash­ion as in insect cells.

Movie of a B16 melanoma cell that was trans­fected with GFP VASP and mCherry actin and infected with bac­ulovirus. Video frames were acquired on a con­fo­cal (spin­ning disc) micro­scope at inter­vals of 3 sec. Bac­ulovirus moves at speeds of up to 50 microns per minute.

Movie of a fish fibrob­last that was trans­fected with RFP-actin and infected with bac­ulovirus.

Enlarged region of a fish fibrob­last that was trans­fected with actin-GFP and infected with bac­ulovirus tagged with mCherry.

In neg­a­tively stained cytoskele­tons, actin comet tails are read­ily observed in the con­ven­tional trans­mis­sion elec­tron micro­scope. How­ever, their orga­ni­za­tion can­not be resolved in 2D pro­jec­tions. 

Bac­ulovirus asso­ci­ated actin comet in a negatively-stained cytoskele­ton of an infected fish fibrob­last. The image was recorded in a con­ven­tional trans­mis­sion elec­tron micro­scope.

Elec­tron tomog­ra­phy was used to deter­mine the three-dimensional orga­ni­za­tion of actin comet tails pro­pelling bac­ulovirus. The 3D orga­ni­za­tion of comet tails was best pre­served by rapidly freez­ing cytoskele­tons of infected cells and imag­ing the comet tails in ice over holes in the sup­port­ing film. Track­ing of fil­a­ments through the tomo­grams revealed a fish-tail like array of fil­a­ments (Mueller et al., 2014) linked by branch junc­tions located in the core region of the tail. From the man­ual track­ing of fil­a­ments it also appeared that there was not a con­tin­u­ous con­nec­tion of fil­a­ments through branch junc­tions along the comet tail, but that they were joined into sep­a­rate sub-sets by branch junc­tions. The sub­sets are indi­cated in dif­fer­ent colours in the model.
 

Movie shows a scan in Z of a cryo-electron tomo­gram of a bac­ulovirus comet tail in a B16 melanoma cell and a model of the tracked fil­a­ments. Actin fil­a­ments belong­ing to the cell cytoskele­ton appear in grey as also a sin­gle micro­tubule.

Details of the cryo-tomogram show­ing branch junc­tions in the comet tail (A, insets). Fil­a­ments marked in yel­low in B occupy the core region swept by the bac­ulovirus par­ti­cle. Tran­sec­tions of this region indi­cated that an aver­age of four actin fil­a­ments are required for propul­sion (C).

After dry­ing in neg­a­tive stain bac­ulovirus actin comets suf­fered some col­lapse in Z, but the fil­a­ments were well resolved and could be tracked through the tomo­grams. The res­o­lu­tion of fil­a­ment sub­struc­ture was higher in neg­a­tive stain and the branch junc­tions showed the same mor­phol­ogy as those observed in lamel­lipo­dia. As for the cryo-tomograms, the fil­a­ments in the comet tail appeared to be divided into sub­sets (coloured) linked by branch junc­tions.

Movie shows a scan in Z of an elec­tron tomo­gram of a bac­ulovirus comet tail in a B16 melanoma cell embed­ded in neg­a­tive stain. The res­o­lu­tion of the actin fil­a­ments was of high enough qual­ity to allow deter­mi­na­tion of the fast poly­mer­iz­ing “barbed” ends by image analy­sis. As shown, the fast poly­mer­iz­ing ends of the fil­a­ments in the fish-bone like array are directed for­wards.

 

Fur­ther tomo­grams of comet tails embed­ded in neg­a­tive stain, together with the mod­els derived by fil­a­ment track­ing. The tomo­grams can be down­loaded (tomo­gram A, tomo­gram C) and inspected with soft­ware pack­age IMOD.

Related Pub­li­ca­tions

  • Mueller J, Pfanzel­ter J, Win­kler C, Narita A, Le Clainche C, Nemethova M, Car­lier MF, Maeda Y, Welch MD, Ohkawa T, Schmeiser C, Resch GP, Small JV. Elec­tron tomog­ra­phy and sim­u­la­tion of bac­ulovirus actin comet tails sup­port a teth­ered fil­a­ment model of pathogen propul­sion. PLoS Biol. 2014 Jan;12(1):e1001765. PDF