Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite

Fig. 1. Polymerization kinetics of the divergent Leishmania major actin. a Phylogenetic tree of selected evolutionarily divergent actins based on amino acid sequences. Leishmania major parasite actin (LmActin) and α-skeletal muscle rabbit actin (Oryctolagus cuniculus, RbActin) used in this study are highlighted in bold. See the Source Data file for the sequence accession codes from Uniprot. b Pairwise comparison of the sequence identity between selected actins. c Methods applied to assemble and visualize LmActin filaments in microfluidics-coupled TIRF microscopy experiments. BE barbed end, PE pointed end. d Examples of observed actin filaments assembled using the methods described in panel c. e Typical “segmented” filaments assembled by alternating between a solution of Alexa-labeled RbActin (green) and a solution containing equimolar concentrations of unlabeled Lm-G-actin and LmProfilin. For each filament, the three LmActin segments were polymerized for 5 min with the same solution containing either 0.2, 0.3, or 0.4 µM LmActin-LmProfilin. With 0.2 µM LmActin-LmProfilin, the resulting segments were not long enough to measure the polymerization rate accurately. f Quantification of the…

Fig. 2. Leishmania actin filaments undergo rapid depolymerization from both ends. a Typical kymographs of RbActin and LmActin filaments (visualized using ATP-ATTO-488) depolymerizing from their barbed ends. Actin subunits bear an ADP-Pi nucleotide at time t = 0, which is released over time to result in ADP-actin filament, as observed by the acceleration of the depolymerization rate. Note the difference in time scales between the RbActin and LmActin kymographs. b–d Comparison of the barbed end depolymerization rates of Leishmania and rabbit muscle ADP-actins (panel b) and ADP-Pi-actins (panel c), and the Pi-release rates of LmActin and RbActin filaments (panel d). Green symbols represent an average depolymerization rate of “aged” actin filaments. Orange and blue symbols represent values obtained by fitting the depolymerization rate of Pi-loaded actin filament vs. time over different data points of one movie (see Methods “Pi-release fit”, see Supplementary Fig. 2b, c). Note that the RbActin labeling strategy, with either ATP-ATTO or Alexa568, does not affect the Pi-release rate. Each data point represents an average over all measured filaments (n) from independent experiments (N), and…

Fig. 3. Structures of Leishmania ADP- and ADP-Pi-actin filaments. a A cryo-EM map of Leishmania major ADP-actin filament. The overall model, ADP and waters modeled in the flattened density around the Mg2+ ion are shown. b A cryo-EM map of L. major ADP-Pi-actin filament. The overall model, ADP, Pi, and Mg2+ are shown. c Superimposition of L. major ADP-actin (magenta) to vertebrate ADP-actin (cyan, PDB ID: 6DJO) subunit. The pointed end tips of actin subdomain 4 (indicated with an arrow) follow slightly different paths in LmActin and vertebrate muscle actin. d Superimposition of L. major ADP-Pi-actin (green) to vertebrate ADP-Pi actin (cyan, PDB ID: 6DJN) subunits. The ADP-Pi conformations in L. major and vertebrate actins are nearly identical to each other. e Positions of residues that are not conserved between rabbit muscle and L. major actins are highlighted in the structure surface presentation. Gray color indicates identical, orange similar, and red different amino acids in the corresponding positions between the two actins (see Supplementary Fig. 3b). Neighboring subunits (n-1 and n+1) are shown in cyan and green cartoon representations. Note that the non-conserved residues are…

Fig. 4. The differences in filament inter- and intra-strand interfaces between Leishmania and vertebrate actins. a H-plug of LmActin (magenta) contains an insertion of an additional aspartate (Asp269, highlighted in green) in the core of the filament that is not present in vertebrate actin (cyan). Corresponding interaction partners at the neighboring D-loop also differ between the two actins. In LmActin, the electrostatic network, formed by Glu270, Lys39, and Asp286 to His173, is hindered by aspartate insertion. Ala272Pro and His275Pro substitutions, preceding the H-plug in LmActin, decrease the flexibility of the H-plug region in comparison with vertebrate actin. Lys263 (absent in the vertebrate actin) can form a salt bridge with Glu258 located in the pointed end helix of subdomain 4, and thus link the dynamics of the H-plug region to the pointed end of the monomer. b The pointed end face of LmActin subdomain 4 contains a single amino acid deletion leading to a shorter loop between α-helix 222–233 and β-strand 238–242. In vertebrate actin, this loop can adopt an α-helical conformation that stabilizes the upstream pointed end tip. In vertebrate actin, Leu236 is inserted into the…

Fig. 5. Rapid severing of Leishmania actin filaments by cofilin. a Typical kymographs showing the fragmentation of ATP-ATTO-labeled ADP-actin filaments by 500 nM cofilin, comparison between Leishmania and vertebrate proteins. White arrowheads indicate severing events. Note the difference in time scales, and that severed fragments disappear from the kymographs due to flow in the microchamber. b Comparison of the global fragmentation rate of LmActin by LmCofilin (green) and RbActin by vertebrate cofilin-1 (gray). Each data point was calculated by fitting the survival fraction over different F-actin segments (n) with a single exponential and normalized by the mean segment length (units: /s/µm, see “Methods”). Error bars represent 95% confidence intervals. For unlabeled LmActin: 0.25 µM N = 1, n = 90; 0.5 µM N = 1, n = 90; 1 µM N = 2, n = 90,90. For fluorescent LmActin: 0.25 µM N = 1, n = 20; 0.5 µM N = 1, n = 20; 1 µM N = 1, n = 20. For unlabeled RbActin with unlabeled cofilin-1: 0.5 µM N = 1, n = 50. For fluorescent RbActin with unlabeled cofilin-1: 0.25 µM N = 2, n = 30; 0.5 µM N = 2, n = 30; 1 µM N = 1, n = 30. For unlabeled RbActin with mCherry-cofilin-1: 0.5 µM N = 2, n = 22, 40. For…

Fig. 6. Structural basis of Leishmania actin filament severing. a Structure of the LmActin filament decorated with Leishmania cofilin. In comparison to vertebrate cofilin (in red), the Leishmania cofilin (in cyan) contains an extended C-terminal α-helix. b A close view of the C-terminal region of cofilin. In vertebrate cofilin, C-terminus forms a turn followed by a two-strand β-sheet, which stabilizes loop 91–96 to closer contact with the actin filament. This β-sheet is absent from Leishmania cofilin, which contains an extended C-terminal α-helix. c Amino acid contacts between the C-terminus of Leishmania cofilin and LmActin. The acidic C-terminus can form an ion pair with Arg210 in actin, and local hydrophobic contacts through Val139 to Met16 and Val30. His136 and Arg137 can form ion bonds to Asp288 and Asp56, respectively, in actin. d The N-terminus of Leishmania cofilin interacts with the barbed end of actin. In the cofilin-bound actin, the D-loop is unstructured (not shown in the figure), whereas in bare actin filaments the D-loop (shown in yellow) associates with the hydrophobic pocket (residues named in white) of the adjacent actin subunit. Hydrophobic Ile2 from Leishmania cofilin…

Fig. 7. Principles of rapid actin disassembly in Leishmania parasites. a, b Impact of Leishmania Twinfilin (LmTwf) on LmActin barbed end dynamics. (panel a) Barbed end polymerization rate in the presence of 1 μM LmActin, 1 μM Leishmania profilin, and indicated concentrations of LmTwf. Negative values indicate that there is net depolymerization. Each point represents the mean over different actin segments (n) from independent experiments (N). Error bars represent S.D. N = 4, n = 20, 20, 40, 40 for 0 µM; N = 1, n = 20 for 0.1 µM; N = 2, n = 40,40 for 0.2 µM; N = 1, n = 40 for 0.3 µM; N = 1, n = 40 for 1 µM; N = 1, n = 40 for 2 µM; N = 1, n = 40 for 4 µM. (panel b) Depolymerization rate of unlabeled ADP-LmActin exposed to F-buffer, with and without LmTwf. Large symbols represent averages over individual measures (n) (small symbols) from independent experiments (N). N = 9, n = 30, 32, 32, 19, 40, 20, 20, 20, 20 for 0 µM; N = 1, n = 20 for 0.1 µM; N = 2, n = 15, 20 for 1 µM. c Model for the rapid actin disassembly in vertebrate and Leishmania cells. In mammalian cells, Capping protein (CP) and twinfilin cooperate to control actin assembly at filament barbed ends. CP is not expressed in…