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First published online 26 April 2005
doi: 10.1242/jcs.02342

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Computer-assisted analysis of filopod formation and the role of myosin II heavy chain phosphorylation in Dictyostelium

W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA

View larger version (65K): [in a new window]   Fig. 1. The computer-assisted DIAS 4.0 software program provides 3D reconstructions of living cells that include color-coded representations of the general cell body, nucleus, pseudopodia and filopodia. (A) The in-focus edges of cell compartments and filopodia are outlined in each of 60 optical sections beginning at the substratum and ending 20 µm above the substratum. Cells are imaged by differential interference contrast (DIC) microscopy. The set of 60 optical sections is collected in a 2-second period and the procedure repeated at intervals of 4 seconds. The outline of the in-focus cell body containing particulate cytoplasm is color-coded green, outlines of the in-focus pseudopodial protrusions containing non-particulate cytoplasm are color-coded blue, the outline of the nucleus is color-coded fuchsia, filopodial segments emanating from the general cell body and pseudopodia are color-coded red and filopodia emanating from the uropod, referred to here as tail fibers, are color-coded yellow. Only the first 12 outlined optical sections, beginning at the substratum (0 µm) are presented here, of a cell turning towards an aggregation stream in response to a gradient of chemoattractant late in the Dictyostelium aggregation process. (B) Reconstruction of the outlined cell viewed at 10°, 30° and 55° from three rotational vantage points. The arrows in each rotation illustrated at the top of the panels reflect the posterior-anterior axis deduced from the prior history of cellular translocation. The capacity to view the cell from different angles provides a more complete picture of filopod location and interaction with the substratum. The cell body is a transparent mesh and color-coded green, the nucleus is color-coded fuchsia, the pseudopodial regions are color-coded gray, the filopodia are color-coded red and the tail fibers emanating from the uropod are color-coded yellow. Bar, 5 µm.

View larger version (48K): [in a new window]   Fig. 2. Filopod formation, retraction and relocation for a representative cell migrating in buffer. (A) Reconstruction of the full cell, including nucleus, pseudopodia and filopodia. (B) Reconstruction only of pseudopodia and filopodia. The cell is viewed from the top (90°). Time is presented in seconds (sec). The arrows at 0 and 108 seconds in panel A reflect changes in orientation of the posterior-anterior axis during the time of analysis. See legend to Fig. 1B for color code. A, anterior end of cell; U, uropod; a, b, c, d, e, f, g, pseudopods in order of appearance. Note that the long yellow tail fiber is much longer than shown, and is truncated by the area of view and that the uropod and long tail fiber are stable throughout the period of analysis. Note also that the starred filopodia on the cell body in panel A, 40 sec, originated on pseudopod a and that the starred filopod on the cell body in panel A, 56 sec, originated on pseudopod c.

View larger version (43K): [in a new window]   Fig. 3. Filopod formation, retraction and relocation for a representative cell turning towards an aggregation stream in response to a spatial gradient of chemoattractant. (A) Reconstruction of the full cell, including nucleus, pseudopodia and filopodia. (B) Reconstruction only of pseudopodia and filopodia. The cell is viewed from 55°. Time is presented in seconds (sec). The arrow in the lower right corner of the 0 second panel denotes the direction of the aggregation stream, which is out of sight beyond the lower right-hand corner. See legend to Fig. 1B for color code. A, anterior end of cell; U, uropod; a, b, c, pseudopodia in order of appearance.

View larger version (43K): [in a new window]   Fig. 4. Frontal view of the cell reconstructed in Fig. 3 reveals the interactions of pseudopod and filopodia with the substratum. Time is in seconds (sec). See legend to Fig. 1B for color code. Note the interaction between filopodia and substratum.

View larger version (27K): [in a new window]   Fig. 5. The density of filopodia changes during three discrete phases associated with pseudopod extension in response to a spatial gradient of chemoattractant emanating from an aggregation stream. In this case, filopodial number was monitored during extension of pseudopod c of the cell in Fig. 2. (A) Filopod number per pseudopod c. (B) Filopod number per µm of pseudopod c unit length. Pseudopodia of four additional cells turning towards a stream exhibited similar pseudopod and filopod dynamics. Note that filopodia are dense during phase two, but decrease in density in phase three.

View larger version (58K): [in a new window]   Fig. 6. Frontal view of a representative pseudopod that formed away from an aggregation stream and was retracted. Time is in seconds (sec). See legend to Fig. 1B for color code. This pseudopod, which formed between 0 and 16 seconds (phase one), maintained the same approximate volume between 16 and 28 seconds (phase two) and retracted between 36 and 60 seconds (phase three). Note the interactions between filopodia and substratum. a, b, c, pseudopodia.

View larger version (39K): [in a new window]   Fig. 7. The effects of the rapid addition of 10–6 M cAMP. 10–6 M cAMP was added to a representative control cell (A-C) and representative cells of the myosin II mutants 3XALA (D-F), 3XASP (G-I) and HS1 (J-L). Cells in perfusion chambers were first perfused with buffer alone for 5 minutes and then with buffer containing 10–6 M cAMP for 4 minutes. See legend to Fig. 2 for color code. Deep purple protrusions on the cell surface represent blebs.

View larger version (30K): [in a new window]   Fig. 8. Histograms of total number of filopodia. Number of filopodia, including tail fibers, per cell (A) and per pseudopod (B) for control and myosin mutant cells translocating in buffer. The mean±s.d. is presented above the histogram of each strain.

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