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Table 1 Influence of mutations in the WxxxE motif of BtpA and BtpB on subcellular localisation, microtubule-protection and filopodia induction

From: The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation

BtpA or variant Localisation of GFP-BtpA in filaments (% of transfected cells ± SD) Protection of microtubules after NC treatment(% of cells with BtpA arranged in filaments)a Induction of filopodia(% of cells with BtpA arranged in filaments)b
BtpA 90.5 ± 1.36 73,8 ± 3.45 75.9 ± 0.26
BtpA W213S 0 0 0
BtpA W213A 90.1 ± 3.86 75,6 ± 5,00 0
BtpA W213F 81.9 ± 1.81 87,1 ± 5,36 0
BtpA E217D 1.3 ± 0.78 83.9 ± 9.89 0
BtpA E217A 52.6 ± 6.67 0 0
BtpA I226S 14.4 ± 1.93 76,1 ± 3,66 69.7 ± 3.5
BtpA G183A 0 0 0
BtpB or variant Protection of microtubules after NC treatment (% of cells with) c Induction of filopodia
  Diffuse BtpB pattern Punctate BtpB pattern  
BtpB 24.5 ± 0.71 82,2 ± 1.59 0
BtpB W263S 0 0 0
BtpB E267A 0 0 0
  1. HeLa cells were transfected with plasmid DNA harbouring pCMV:GFP-BtpA, pCMV:GFP-BtpB or the respective Btp variants. After 16–20 hours cells were left untreated or treated with 1 μg/ml nocodazole for 30 min and fixed. Microtubules were visualised using β-tubulin antibodies and detected with Texas-Red conjugated secondary antibodies. For localisation studies, at least 200 transfected cells were analysed per variant per experiment. Data are given in average percentage of transfected cells ± SD (of three independent experiments per variant). For analysis of filopodia, cells were fixed 16-20h hours after transfection, and actin detected with Rhodamine Phalloidin. Each variant was analysed in at least 3 independent experiments.
  2. aFor wildtype BtpA and variants, protection of nocodazole-induced microtubule destabilisation was observed only in transfected cells showing BtpA in filaments. Indicated is the average percentage of cells with filaments that show protection, calculated from two individual experiments (±SD). Cells that showed partial protection were not taken into consideration. At least 100 cells per experiment were counted for BtpA wildtype, BtpA W213A, and BtpAI226S. For BtpA W213F and BtpA E217D, seen the low percentage of transfected cells with BtpA localising in filaments, about 12 cells with BtpA organised in filaments were counted in 2 independent experiments. BtpA W213S, BtpAE217A and BtpAG183A did not show any protection.
  3. bWildtype BtpA and the I226S variant showed induction of filopodia only in transfected cells showing BtpA in filaments. Counts are based on a total of 67 cells with BtpA in filaments in each of 2 independent experiments.
  4. cBtpB transfected cells showed a large variation in ectopic expression phenotypes: Low GFP expression, high GFP expression, and with or without punctae (varying from 1 to over 50). As for BtpA, cells showing partial protection were not taken into account. In two independent experiments 62 and 48 cells (of which 32.7% ± 10.8 were cells without punctae), respectively were analysed for protection of microtubules.