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First published online 10 July 2007
doi: 10.1242/jcs.010876

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PTEN plays a role in the suppression of lateral pseudopod formation during Dictyostelium motility and chemotaxis

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

View larger version (58K): [in this window] [in a new window]   Fig. 1. pten– cells exhibit fundamental defects in basic motile behavior and pseudopod formation in the absence of chemoattractant. Cells were distributed on the glass wall of a perfusion chamber at low density and perfused with buffer at a rate that turned over chamber volume four times a minute. (A,B) Perimeter and centroid tracks of representative parental AX2 (A) and pten– (B) cells that had been pulsed for 6 hours with cAMP to achieve aggregation competence prior to motion analysis. (C,D) Perimeter and centroid tracks of representative AX2 (C) and pten– (D) cells developed on pads to achieve aggregation competence prior to motion analysis. (E,F) Representative pad-developed parental AX2 (E) and pten– (F) cell reconstructed in 3D with 3D-DIAS software over a 150 second or 125 second period, respectively, of cells migrating in buffer in a perfusion chamber. The arrows in A to D indicate net direction of cell migration. In E and F, pseudopods are colored red, the nucleus dark blue, tail fibers green and the cell body is shown in a transparent blue wireframe. Two views of the reconstructions are provided, at 30° and 90° angles. Note in F the abnormal extension of multiple lateral pseudopods by pten– cells from the posterior as well as anterior end of the cell. See Movie 1 in supplementary material for a 3D dynamic presentation of an AX2 and pten– cell migrating in buffer.

View larger version (40K): [in this window] [in a new window]   Fig. 2. pten– cells abnormally extend multiple pseudopods at the same time. Different symbols represent different pseudopods. (A,B) The orderly extension of pseudopods by two representative parental AX2 cells. Note that only one pseudopod expands at a time, and that a previous pseudopod usually retracts when a new pseudopod expands. (C,D) The overlapping expansion of multiple pseudopods by two representative pten– cells. Both anterior and lateral pseudopods were reconstructed and volumes estimated at each time point using 3D-DIAS software (Wessels et al., 1998; Heid et al., 2005).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Although pten– cells undergo positive chemotaxis up spatial gradients of cAMP, they exhibit the same fundamental behavioral defects manifested in the absence of attractant. (A,B) Perimeter and centroid tracks of representative parental AX2 (A) and pten– (B) cells pulsed for 6 hour with cAMP to achieve aggregation competence and then analyzed in a spatial gradient of attractant. (C,D) Perimeter and centroid tracks of representative parental AX2 (C) and pten– (D) cells developed on pads to achieve aggregation competence and then analyzed in a spatial gradient of attractant. (E,F) Pad-developed parental (E) and pten– (F) cells undergoing chemotaxis that were reconstructed with 3D-DIAS software over 150-second or 125-second periods, respectively. The thin arrows in A-D indicate the net direction of migration for each cell. The thick arrow in A-F indicates direction of increasing cAMP concentration. Plus (+) and minus (–) in A-D indicate a positive or negative chemotactic index, respectively, for each cell computed over the period of analysis. Color-coding of reconstructions in E and F are the same as in Fig. 1.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Histograms of chemotactic indices (CI) demonstrate that the majority of pten– cells undergo chemotaxis up a spatial gradient of cAMP, but do so with decreased efficiency. pten– cells have difficulty achieving high-end CIs.

View larger version (34K): [in this window] [in a new window]   Fig. 5. pten– cells respond to the increasing temporal gradient of cAMP in the last three of a series of four temporal waves generated in a perfusion chamber in the absence of established spatial gradients. These waves mimic the temporal dynamics of natural waves (Tomchik and Devreotes, 1981), but the response is lower than that of parental AX2 cells. (A) Time plots of the instantaneous velocity (Inst. Vel.) of two representative AX2 cells responding to four successive temporal waves of cAMP. AX2 cells exhibit an increase in velocity in the front of the last three of four successive temporal waves of attractant. They do not respond to the first wave, as previously reported (Varnum et al., 1985; Wessels et al., 1992). (B) The instantaneous velocity of three representative pten– cells responding to four successive temporal waves of cAMP. pten– cells exhibit, on average, an increase in velocity in the front of the last three waves, but the magnitude of the increase is far below that of AX2 cells. Behavior in the front of each wave is color-coded blue.

View larger version (31K): [in this window] [in a new window]   Fig. 6. pten– cells undergo natural chemotaxis, but less efficiently than AX2 cells. (A) Selected centroid tracks of one representative AX2 and three representative pten– cells undergoing chemotaxis in an aggregation territory made up of 90% AX2 and 10% pten– cells in a submerged culture. (B-D) Velocity plots of one representative majority AX2 cell and two representative minority pten– cells in a mixed aggregation territory made up of 90% AX2 cells and 10% pten– cells. (E) Selected centroid tracks of four representative pten– cells undergoing chemotaxis in an aggregation territory made up of 100% pten– cells in a submerged culture. (F-H) Velocity plots of three representative pten– cells in a homogeneous pten– aggregation territory in a submerged culture. Small arrows in A and E indicate net direction.

View larger version (44K): [in this window] [in a new window]   Fig. 7. The defects in the behavior of cells of the myosin heavy chain phosphorylation mutant 3XASP in the absence of attractant and in a spatial gradient of cAMP are remarkably similar to those of pten– cells. (A,B) Centroid and perimeter tracks of representative parental JH10 and representative 3XASP cells, respectively, migrating in buffer in a perfusion chamber in the absence of attractant. (Compare with data for corresponding pten– cells in Fig. 1B,D.) (C,D) Centroid tracks and perimeter tracks of representative JH10 and 3XASP cells, respectively, undergoing chemotaxis in a spatial gradient of cAMP. Arrows in A-D indicate net direction and + signs in C and D indicate a positive chemotactic index. (Compare with corresponding pten– data in Fig. 3B,D.) (E,F) 3D-DIAS reconstructions of a JH10 and 3XASP cell, respectively, translocating in buffer reveal that whereas the former extends a dominant anterior pseudopod with occasional lateral pseudopods, the latter extends multiple pseudopods from both anterior and posterior regions of the cell body. Color coding of reconstructions are the same as in Fig. 1. (Compare with corresponding data for AX2 and pten– in Fig. 1E and F, respectively.) (G) The defects in motility and chemotaxis reflected in measured parameters are remarkably similar for 3XASP and pten– cells. The ratio of the average mutant to parental parameter is presented for cells translocating in the absence of cAMP and for cells responding to a spatial gradient of cAMP. Instant. vel., instantaneous velocity; Dir. change, direction change; Pers., directional persistence; % Pos. chemotaxis, percentage positive chemotaxis. The 2D and 3D analyses of JH10 and 3XASP for A-F were performed anew for this study. The data for computing the parameter proportions for 3XASP and JH10 represent a combination from Heid et al. (Heid et al., 2004) and new experiments. The chemotactic index for 3XASP was recomputed from new data for which interval time was the same as that used for the pten– study.

View larger version (69K): [in this window] [in a new window]   Fig. 8. pten–, like 3XASP, is defective in myosin II and F-actin localization to the cortex in response to an increasing temporal gradient of cAMP in the front of a temporal wave. (A,B and C,D) Indirect immunofluorescent staining of myosin II heavy chain in parental AX2 and pten– cells, respectively, migrating in buffer in the absence of attractant. Note that in both cases, there is very little cortical localization of myosin II. (E,F and G,H) Indirect immunofluorescent staining of myosin II heavy chain and laser scanning confocal microscopic (LSCM) line scans of pixel intensity of AX2 cells and pten– cells, respectively, migrating in the front of the third temporal cAMP wave in a series. Note that whereas there is a substantial increase in cortical staining in AX2 cells, there is no detectable increase in cortical staining in pten– cells. (I,J and K,L) Phalloidin staining of F-actin in Ax2 and pten– cells, respectively, migrating in buffer. Note that whereas the pseudopodia stain brightly for F-actin, there is only a hint of cortical staining in both cell types in buffer. (M,N and O,P) Phalloidin staining of F-actin and LSMC line scans of pixel intensity of AX2 cells and pten– cells, respectively, in the third of a series of temporal waves of cAMP. Note that whereas there is a substantial increase in cortical staining in AX2 cells, there is no similar increase in pten– cells. a, anterior end; u, uropod; e, cell edge. Line across cells in A-H, and M-P, indicates position of scan.

View larger version (64K): [in this window] [in a new window]   Fig. 9. PTEN is constitutively localized in the cortex. AX2 cells expressing GFP-PTEN were analyzed in buffer, in a spatial gradient of attractant and in the front of the third in a series of temporal waves of cAMP. (A) Perfused with buffer in a perfusion chamber in the absence of attractant. (B) Oriented up a spatial gradient of cAMP. (C,D) In the front and at the peak, respectively, of the third in a series of temporal waves of cAMP. LSCM line scans of pixel intensity are presented below each LSCM image, and the position of each line scan is indicated by a white line across each image. Note the similarity of peaks at cell edges under all conditions.

View larger version (50K): [in this window] [in a new window]   Fig. 10. A model for the role of PTEN in the suppression of lateral pseudopod formation. In the absence and in the presence of attractant, PTEN localizes constitutively at the same level in the cortex of the main cell body. (A) In the absence of attractant, a low steady state level of MHC-dephosphorylation results in myosin II and F-actin localization in the cortex with PTEN. This steady state localization is responsible for the suppression of lateral pseudopod formation through the generation of cortical tension. Once every 2 minutes in the absence of attractant, a lateral pseudopod forms randomly, usually from the anterior half of the cell body at a site where the myosin II-F-actin cortex transiently weakens. (B) In response to the increasing temporal and positive spatial gradients of attractant in the front of wave, a receptor-mediated signal transduction pathway is activated that increases the rate of myosin II dephosphorylation, resulting in an increase in cortical myosin II and F-actin. This results in an increase in cortical tension, leading to the increased suppression of lateral pseudopod formation in the front of a wave. In this model, receptor-mediated dephosphorylation of MHC is responsible for increased cortical tension. The similar behavioral defects of the mutants myoA, myoB, myoF, clathrin and sphingosine-1-phosphate lyase suggest that they also play roles in steady state pseudopod suppression in the absence of attractant and increased suppression in the front of a wave. In the model, PTEN is essential for myosin II-F-actin localization and pseudopod suppression in the absence of attractant, and the increases in localization and suppression in the front of the wave. But because it remains at a constitutive level in the cortex, it has not been placed in the receptor-activated transduction pathway leading to increased pseudopod suppression. It should be emphasized that this model deals with a mechanism that affects the efficiency of the chemotactic response through lateral pseudopod suppression, not with a mechanism of gradient sensing or the maintenance of cellular polarity.