QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 December 2007
doi: 10.1242/jcs.015602

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Veltman, D. M. Articles by van Haastert, P. J. M. Search for Related Content PubMed PubMed Citation Articles by Veltman, D. M. Articles by van Haastert, P. J. M. Social Bookmarking                         What's this?

The role of cGMP and the rear of the cell in Dictyostelium chemotaxis and cell streaming

Department of Biology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

View larger version (109K): [in this window] [in a new window]   Fig. 1. Development of gc-null cells. Wild-type AX3, gc-null and rescued gc-null cells were settled as a monolayer on non-nutrient agar plates and allowed to starve (t=0). Pictures of developing cells were taken during aggregation (t=5 hours) and after completion of formation of fruiting bodies (t=36 hours). To illustrate better the differential behaviour of the cell strains during aggregation, the area in the white box of the first panel is enlarged in the second panel and further enlarged in the third panel. Black bars indicate the scales used for each picture.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Speed and chemotaxis index during aggregation. (A) Wild-type AX3 and gc-null cells were allowed to aggregate on non-nutrient agar plates, and a movie of the aggregating cells was recorded (supplementary material Movies 1 and 2). The chemotaxis of the cells was analyzed from the time-point where cells first make a simultaneous movement surge in the direction of the aggregation centre (t=0 minutes) until the time-point where most cells are incorporated into streams (t=60 minutes). A representative cell in each frame is enlarged in the bottom frame. The arrow points in the direction of the aggregation centre, which is out of view. (B) The position of 25 cells was tracked throughout the duration of the movie. Cell tracks of three representative cells are shown. The tracks start at t=0 and are followed for at least 40 minutes. Unfilled dots were placed on the cell tracks representing the positions at intervals of 1 minute. Cells move in the direction of the aggregation centre, as indicated by the arrow. (C) The average chemotaxis index and speed of the cells was determined in each movie. The dotted lines indicate the progress of the minimum and maximum chemotaxis index, respectively, during the course of aggregation. The period where the minimum chemotaxis index increases with each subsequent wave is referred to as the `transition period'. (D) The waves of the chemotaxis index of aggregating cells during the transition period of two independent movies were averaged. Time on the horizontal scale is relative to the time-point of the peak of the wave. The CI that is indicated in the graph is the difference between the average of the lowest three points of the chemotaxis index both before and after the wave. (E) The number of protrusions in the front one-third and rear one-third of the cell was determined throughout aggregation. The graph shows the average number of pseudopodia during the first four waves and the last four waves of the movie. Error bars indicate the standard deviation.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Properties of cell contacts. (A) Wild-type AX3 and gc-null cells were starved in shaking suspension at 1x107 cells/ml. Cells were pulsed with 30 nM cAMP, starting at 3 hours after the onset of starvation, indicated by the dashed line in the figure. Each hour, a sample was drawn and incubated for 15 minutes in 10 mM EDTA. The number of cells that made no cell contact with other cells was determined using a haemocytometer. On the basis of these numbers, the percentage of cells in clumps was calculated. Each time-point was taken in duplicate and the experiment was repeated twice. Error bars indicate the standard deviation. (B) Either AX3 or gc-null cells were labelled with GFP. An equal amount of unlabeled starved cells from one strain was mixed with starved, labelled cells from the other strain, and the mixed cell suspension was settled on non-nutrient agar at 5x104 cells/cm2. After one hour of aggregation, thin streams of single-cell width were visible. Each cell in such a cell stream making polar head-to-tail contacts was identified as either a wild-type or gc-null cell. The graph shows the percentage of cells from each strain at the first position, middle (endo) positions and last position. The assay was repeated four times [two experiments with AX3(GFP)/gc-null and two experiments with AX3/gc-null (GFP) cells]. Error bars indicate the standard deviation. In total, 164 cell trains were analysed. (C) Starved wild-type and gc-null cells were settled on non-nutrient agar at a density of 5x104 cells/cm2 and a movie was recorded. The duration of all head-to-tail cell contacts that lasted for >30 seconds was noted and plotted as a percentage of the total number of cells analysed. The experiment was repeated two times and >90 individual head-to-tail cell contacts were analysed for each strain.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Development of cGMP-mutant cell strains. Mutant cells were starved as a monolayer on non-nutrient agar plates. Pictures of developing cells were taken at 5 hours after the start of starvation. gc-null cells lack cGMP synthesis, whereas gpbC-null cells lack the cGMP-target protein GbpC that mediates cell polarity. sGCN, expressed in gc-null cells, is catalytically active but cytosolic. sGCcat is catalytically inactive but localizes to the anterior of the cell, an event that previously was shown to stabilize the leading edge in a spatial gradient.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Proposed model of the effects of a stable rear of the cell on chemotaxis, cell-cell contact and cell streaming. (A) The strong spatiotemporal gradient at the rising flank of a cAMP wave induces a dominant pseudopod in both wild-type and gc-null cells. Wild-type cells suppress pseudopod formation in the posterior half of the cell. gc-null cells have an increased number of lateral pseudopodia, but their contribution to cell movement is relatively small during this phase. During the second half of the wave, wild-type cells maintain their polarized cell shape and extend pseudopodia only in the front. In the absence of a gradient-induced dominant pseudopod, the increased number of lateral pseudopodia in gc-null cells lead to a rapid decrease of directional cell migration during the second half of the cAMP wave. (B) The polarity of wild-type cells that is acquired during the first half of the wave results in a persistent orientation towards the aggregation centre in the second half of the wave. By contrast, gc-null cells, which do not properly polarize, lack the orientation towards the aggregation centre during the back of the wave. This decreased orientation leads to movement in apparently random directions and an increased tendency to break out of cell streams.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter