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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.01015

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Dynamics of novel feet of Dictyostelium cells during migration

Kazuhiko S. K. Uchida^* and Shigehiko Yumura

Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan

View larger version (100K): [in a new window]   Fig. 1. Distribution of actin and ConA receptors on the ventral membrane of cells. Cells were fixed and stained with TRITC-Con A, and immunostained with anti-actin antibodies. (A,C) Distribution of actin. Several actin foci were observed on the ventral membrane of the cell (A) and small dots were observed behind the cell (C). (B,D) Distribution of Con A receptors. Fluorescent dots (B) and CTs (D) were observed on the ventral cell membrane and behind the cell, respectively. Note that the fluorescent dots of ConA receptors were co-distributed with actin foci (see arrowheads in A and B). The dots in CTs and the actin dots behind the cell were also co-distributed (C,D). The cells in upper panels and lower panels are different. Scale bar: 5 µm.

View larger version (114K): [in a new window]   Fig. 2. Dynamics of actin foci in live cells as seem by interference reflection microscopy (IRM). (A-H) The ventral membrane of a quiescent Dictyostelium cell was analyzed at various time points, as indicated. (A'-H') The distance between the cell and the substratum is indicated as tone in IRM images. While most of the ventral membrane was gray, white rings were observed surrounding actin foci (1, 2 and 3). Scale bar: 5 µm. (I) Enlarged images of the actin foci numbered 1, 2 and 3 in A-H. (J) Intensity of the tone in the area enclosed by the bars across the white ring at the actin foci numbered 1, 2 and 3. The tone at the actin foci is darker than other areas, demonstrating that the actin foci were closer to the substratum than the rest of the ventral membrane. A movie is available (Movie 1, http://jcs.biologists.org/supplemental/).

View larger version (22K): [in a new window]   Fig. 3. Actin foci disappeared about 20 seconds after their appearance. (A) Time course of fluorescence intensity of the three actin foci shown in Fig. 2I. (B) Frequency distribution of the duration of actin foci in wild-type cells. The average±s.d. from 164 foci in 24 cells was 19.43±8.16 seconds. (C) Frequency distribution of the duration of actin foci in MHC-null cells. The average±s.d. from 192 foci in 14 cells was 20.26±7.39 seconds.

View larger version (94K): [in a new window]   Fig. 4. Actin foci were incorporated into retraction fibers and were eventually shed on the substratum during cell migration. (A-L) Some of the actin foci, especially in the posterior region, were incorporated into retraction fibers, ripped off from the cell and finally left on the substratum (arrowheads). The sites where actin foci had appeared and then disappeared were also incorporated into retraction fibers (circles). (M) Superimposed images of the posterior region of the cell shown in A-L. The black dots represent the positions where the actin foci had been located. Note that all these dots were located within retraction fibers. Scale bar: 5 µm.

View larger version (13K): [in a new window]   Fig. 5. The instantaneous velocity of cells was inversely proportional to the number of actin foci. The number of actin foci and instantaneous velocity were examined in sequential images of eight cells.

View larger version (43K): [in a new window]   Fig. 6. Traction force was transmitted to the substratum through actin foci. (A-C) Simultaneous observations of actin foci and bead movements beneath the cell. Time 0 indicates the beginning of the observation. Red areas represent the actin foci and green dots represent the position of the beads. Arrows represent the vectors of the bead movements during the previous 4.5 seconds. The length of the arrows is three times as long as actual displacement of the beads. Interestingly, extensive movement of the beads was observed only around the actin foci. A soft silicone substratum (about 4 nN/µm in stiffness) was used in order to exaggerate the movement of the beads, as described previously (Uchida et al., 2003). Scale bar: 5 µm. (D) Time-lapse images of the areas numbered 1, 2 and 3 in B and C. Around the actin foci, extensive bead movements were observed.

View larger version (40K): [in a new window]   Fig. 7. The number of actin foci decreased in the posterior region during the retraction phase. Panels A-L and A'-L' show sequential images of actin foci in two representative migrating cells. The actin foci were marked with white spots. M and M' show the time course of the area gained by the cells shown in A-L and A'-L'. The cells alternated between the extension and the retraction phases. Note that the changes in the distribution of actin foci were correlated with the cyclic phase changes during migration. From the late extension phase to the early retraction phase, the number of actin foci in the posterior region decreased (C-E, I-J, C'-D' and I'-K'). Scale bar: 5 µm.

View larger version (21K): [in a new window]   Fig. 8. Average number of actin foci in the anterior and the posterior regions during extension and retraction phases. The boundary between the anterior and the posterior regions of the cell was defined by the perpendicular line that crossed the half point of the cell migration axis between the extreme anterior and posterior edges (small protrusions were excluded). The cell migration axis was defined from the cell centroids in three successive images (Uchida and Yumura, 1999). Both extension and retraction phases were divided into early, middle and late extension (or retraction) phases respectively, and the number of the actin foci was counted. Vertical bars showed standard errors of 11 cases of six cells. In the anterior region, the number of the actin foci did not significantly fluctuate. In the posterior region, the number of the actin foci significantly decreased (t-test, P<0.01) in the late extension phase, and then gradually increased.

View larger version (46K): [in a new window]   Fig. 9. Dynamics of actin foci in MHC null cells. (A-M) Sequential images of actin foci in a representative MHC null cell. Actin foci marked with white spots were consistently observed in both the anterior and the posterior regions of MHC null cells, in contrast to wild-type cells. Scale bar: 5 µm. (N) Time course of the area gained by the MHC null cell shown in A-M. The MHC null cell also alternated between the extension and the retraction phases.

View larger version (31K): [in a new window]   Fig. 10. A schematic model illustrating how cells migrate across a substratum with coordinated changing steps. (Aa) Putative structure of cell-substratum adhesion sites. Actin filaments (pink) in an actin focus link the cortical actin layer (red) to the substratum through putative membranous cell-substratum adhesion proteins (vertical short bars). (b) Dynamic behavior of the adhesion structures. Following the appearance of actin foci, the putative membrane proteins are recruited to form adhesion structures. These proteins remain adhered to the substratum even after disappearance of the actin foci. (B) Coordinated changing steps in a wild-type cell. An open arrow represents the direction of cell migration. Green areas represent the putative region generating motive force (blue arrows) as suggested previously (Uchida et al., 2003). During the extension phase (a-c), the cell transmits traction force (black arrows) to the substratum through actin foci, which act as scaffolding for the anterior extension. (c) After anterior extension, the forefront of the newly extended region is anchored to the substratum by newly formed actin foci. (e-h) During the retraction phase, contraction and detachment occur in the posterior region. At that time, the actin foci disappear in the posterior region. Posterior contraction then generates a pushing force for anterior extension (blue arrows in f and g). The actin foci in the anterior region during this phase act as scaffolding for the anterior extension. (C) In the case of MHC null cells, generation of motive force is limited to the anterior region. These cells exhibit defects in contraction and detachment of the posterior region

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