Article Figures & Data
Figures
- FIG. 1.
Effects of anti-Gli349 antibodies on glass binding and gliding speed of wild-type cells. The effects on binding and speed are presented in the upper and lower panels, respectively. The antibodies and Fab were added at time zero. The numbers of bound cells in an area 280 μm square and their average gliding speeds are presented relative to the initial values on a logarithmic scale. The gliding speeds were analyzed for three consecutive intervals of 0.333 s by using Image J version 1.33v with the plug-in software MultiTracker ver1 (http://rsb.info.nih.gov/ij/plugins/multitracker.html ) and are presented as the average for more than 40 cells on a logarithmic scale when sufficient numbers of cells were on glass. Antibody concentrations in mg/ml are as indicated in the central lower graph.
- FIG. 2.
Binding and gliding properties of each strain. (Left) The cell suspension was inserted into a tunnel slide at time zero, and the number of bound cells in an area 280 μm square was counted at various time points. (Right) Speeds were averaged for more than 40 cells and are presented with standard deviations. WT, wild type.
- FIG. 3.
Antibody binding sites and mutation points shown on a schematic of a Gli349 molecule. The gliding machinery is composed of the Gli123, Gli349, Gli521, and P42 proteins. The molecular shape of Gli349 was suggested from electron microscopy studies to be formed by three rods and one oval “foot,” which are tandemly connected by three hinges (1). A transmembrane segment is predicted for amino acids 9 to 31 (22). Repeat sequences of 100 amino acids with weak similarity are shown by ovals as follows: A, 118 to 222; E, 616 to 727; G, 830 to 938; H, 944 to 1047; I, 1048 to 1161; J, 1248 to 1343; K, 1344 to 1449; L, 1450 to 1546; M, 1553 to 1657; N, 1658 to 1762; O, 1765 to 1872; P, 1873 to 1972; Q, 1974 to 2080; R, 2084 to 2191; S, 2286 to 2391; T, 2396 to 2501; U, 2515 to 2608; and V, 2610 to 2720 (7). The binding sites of MAb7 and MAb33, comprised of regions 1193 to 1203 and 1477 to 1492, are shown by closed triangles. The mutation points of m26, the gli349(S1362W) mutant, and m23 at amino acids 1228, 1362, and 2770 are shown by open triangles.
Tables
- TABLE 1.
Antibodies and mutants
Antibody or mutant Binding activitya Gliding speedb Epitope or amino acid substitution in Gli349 or Gli521c Nucleotide changed Reference Isolation Mapping Antibodies MAb7 Reduced Reduced 1193-1203/Gli349 6 This study MAb3 Reduced No effect 1477-1492/Gli349 This study This study MAbR19 Reduced Reduced 3736-4020/Gli521 20 20 Mutants m9 None None E1670*/Gli521 G19148T 14 20 m12 None None Q523*/Gli123 C2627T 14 24 m13 None None Q1257*/Gli349 C8305T 14 22 m23 None None S2770L/Gli349 C12845T 14 This study m26 None None 1228RPTA1229/Gli349 8208-8219 tandem repeate 14 This study gli521(S859R) 54% 54% S859R/Gli521 T16717A This study This study gli521(P476R) 165% 75% P476R/Gli521 C15567G 23 23 gli349(S1362W) 59% 108% S1362W/Gli349 C8621G This study This study ↵ a Percentages refer to the cell density on glass compared to that of the wild-type strain.
↵ b Percentages refer to the gliding speed compared to that of the wild-type strain.
↵ c The protein fragments comprising amino acids 11 to 90, 109 to 223, 217 to 426, 421 to 821, 822 to 1021, 1024 to 1247, 1241 to 1608, 1581 to 1966, 1929 to 2380, 2374 to 2582, 2582 to 2703, 2685 to 2810, 2832 to 3066, 1159 to 1247, 1024 to 1182, 1024 to 1203, 1024 to 1212, 1024 to 1192, 1159 to 1342, 1242 to 1442, 1242 to 1522, 1242 to 1460, 1242 to 1476, 1242 to 1492, and 1242 to 1509 of the 3,183-amino-acid Gli349 protein were expressed in E. coli cells and analyzed by Western blotting. The total number of amino acids in translated Gli521 is 4,727, and processing gave 4,684. Asterisks indicate stop codons.
↵ d Numbering is as in the sequence under accession number AB084781 .
↵ e An additional C18522T mutation was found but is silent in the amino acid sequence.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Legends to Fig. S4 and S5.
Zipped MS Word document, 10K. - Supplemental file 2
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Fig. S4, behavior of M. mobile cells in the presence of MAb7; Fig. S5, centipede model.
Zipped MS PPT file, 17K.
- Supplemental file 1
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Legends to Fig. S4 and S5.
