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First published online 31 August 2004
doi: 10.1242/jcs.01358

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The role of myosin heavy chain phosphorylation in Dictyostelium motility, chemotaxis and F-actin localization

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

View larger version (45K): [in a new window]   Fig. 1. Perimeter tracks of representative control and mutant cells translocating in buffer, in the absence of chemoattractant. For each cell type, cells were selected that portrayed the range of behavior. The red cell image represents the last one in each track. Cells were reconstructed at 8-second intervals. Notice that while JH10 is the control for mhcA-, mhcA-/mhcA+ is the control for 3XALA and 3XASP.

View larger version (53K): [in a new window]   Fig. 2. Western blot analysis of MHC levels in JH10, mhcA-, mhcA-/mhcA+, 3XALA and 3XASP. Notice that the levels in mhcA-/mhcA+, 3XALA and 3XASP are equivalent, and at least twice that in JH10.

View larger version (30K): [in a new window]   Fig. 3. A comparison of translocation between a representative JH10 (A) and a representative mhcA- cell (B) in buffer. Top panels: centroid and perimeter plots of representative JH10 and fast mhcA- cells. The red image is the last in the track. Middle panels: vector plots of representative JH10 and mhcA- cells. Bottom panels: difference pictures of representative JH10 and mhcA- cell. Vectors were computed between centroids at 8-second intervals. Green and red zones represent expansion and contraction zones, respectively.

View larger version (64K): [in a new window]   Fig. 4. 3D-DIAS reconstruction of a representative JH10 cell (A), mhcA- cell (B), fast mhcA- cell (C), 3XALA cell (D) and 3XASP cell (E) in buffer. Cell bodies are color-coded pale blue and pseudopods yellow. Each cell was viewed at 0 degrees (0°) and 60 degrees (60°). Arrows in A, C, D and E indicate direction of translocation.

View larger version (36K): [in a new window]   Fig. 5. Perimeter tracks of representative control and mutant cells translocating in a spatial gradient of cAMP, the direction of which is shown by the arrow in each panel. The red cell image represents the last one in each track. Cells were reconstructed at 8-second intervals.

View larger version (60K): [in a new window]   Fig. 6. A comparison of translocation of control and mutant cells in spatial gradients of cAMP. For each representative mutant cell, a perimeter track, vector track and set of difference pictures are presented. In perimeter tracks, the red image is the last in the sequence. Vectors were computed between centroids at 8-second intervals. Green and red zones of difference pictures indicate expansion and contraction zones, respectively.

View larger version (46K): [in a new window]   Fig. 7. The behavior of a representative JH10 cell (A), mhcA- cell (B), 3XALA cell (C) and 3XASP cell (D) in a series of four simulated temporal waves of cAMP. Instantaneous velocity of each cell is plotted as a function of time for each cell. The estimated waves of cAMP are plotted at the top. Difference pictures are presented for each cell in the front, peak and back of the third wave. Velocity above an arbitrary threshold line drawn from 4 to 2 µm per minute over the 28-minute period of analysis was color-coded yellow to accentuate the velocity surges in response to the front of the second, third and fourth wave for JH10 and 3XALA cells, but not for mhcA- and 3XASP cells. In difference pictures, expansion zones are color-coded green and contraction zones red.

View larger version (39K): [in a new window]   Fig. 8. mhcA- and 3XASP cells are incapable, while 3XALA cells are capable, of chemotaxis in natural waves of cAMP relayed through an aggregation territory of control cells. mhcA-, 3XASP or 3XALA cells were mixed with control (JH10) cells at a ratio of 1:9 and examined early in the aggregation process, during the single cell chemotaxis stage. mhcA- cells in each case were stained with DiI before mixing, while control cells were unstained. Transmitted and fluorescent images were simultaneously collected at 20-second intervals. (A,C,E) Instantaneous velocity of a control and mutant cell in the same neighborhood (within 15 µm) as a function of time. (B,D,F) Centroid plots of neighboring control and mutant cells in the aggregation territory. The aggregation center is at the bottom. Curved lines represent the peaks of sequential waves interpreted from control centroid plots.

View larger version (192K): [in a new window]   Fig. 9. 3XALA cells exhibit aberrant streaming and also have problems entering streams of control cells. (A) Streaming during late aggregation of JH10 cells on agar. (B) Streaming during late aggregation of mhcA-/mhcA+ cells on agar. (C) Late aggregation of 3XALA cells on agar. (D) DiI-labeled 3XALA cells (red) mixed with unlabeled JH10 cells at a ratio of 1:9 failed to enter JH10 streams. Stained and unstained cells were co-imaged with simultaneous bright-field optics and laser scanning confocal microscopy.

View larger version (54K): [in a new window]   Fig. 10. F-actin localization analyzed by Oregon-Green-phalloidin staining in mhcA-/mhcA+, mhcA-, 3XALA and 3XASP cells in buffer, in response to the increasing temporal gradient of cAMP in the third in a series of four temporal and in response to the decreasing temporal gradient of cAMP in the back of the wave. For each representative cell, the first panel contains a projection image through all laser scanning confocal microscope (LSCM) sections, the second panel contains a single LSCM section at 1 µm above the substratum, and the third panel is a line plot of staining intensity in the single LSCM section across the cell behind the nucleus (indicated by the white line in the second panel).

View larger version (35K): [in a new window]   Fig. 11. Diagrams of F-actin localization in (A) control and (B) mhcA- or 3XASP and (C) 3XALA cells in buffer and in response to the increasing temporal wave of cAMP in the front of a wave. (D) Model of the regulation of F-actin localization in the cytoplasm and cortex in response to the increasing gradient of cAMP in the front of a wave.

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