Chemotaxis

Chemotaxis
Page updated 13/11/02
Amoeba feed on other protist, algae and bacteria. They must be able to adapt to the temporary absence of suitable prey, for example by entering resting stages such as cysts, but it is obviously an advantage to be able to "smell out" prey items and craw toward the source. This ability is chemotaxis. It is likely that all amoebae have this ability since it would confer such a huge advantage to these organisms, indeed chemotaxis has been demonstrated in Amoeba proteus, Acanthamoeba, Naegleria and Entamoeba. The best studied chemotactic amoeboid organism however is Dictyostelium discoideum. Dictyostelium is chemotactic for bacteria, but has been extensively studied for its ability to climb gradients of cAMP, a signalling molecule involved in the development of the slug (figure 1). The term "Chemotaxis" was first coined by a W. Pfeffer in 1884 to describe the attraction of fern sperm to the ova, but since then the phenomenon has been described in bacteria and many eukaryotic cells in many different situations. Specialised cells within metazoans have retained the ability to crawl toward bacteria in order to eliminate them from the body and the machinery is very similar to that used by primitive eukaryotes to find bacteria for food. Much of what we know about chemotaxis has been learned from studying the slime mould Dictyostelium discoideum, and comparing this to our own neutrophils, the white blood cells that detect and consume invading bacteria in our bodies. Neutrophils are end differentiated and largely non-biosynthetic cells which means that we cannot use the usual molecular biological tools that we would other wise use, such as gene knockout, transient transfection, or expression of GFP-labelled proteins. Fortunately, Dictyostelium can be used for all such studies. An understanding of neutrophils chemotaxis is of obvious importance for the treatment of human disease and therapeutic intervention of these processes has resulted. Whereas chemotaxis is the sensing of a chemoattractant gradient and climbing it cells can also accumulate by chemokinesis. Chemokinesis is an activity that increases the overall speed of locomotion. In principle a cell be perceive gradients by a number of distinct mechanisms (Figure 1). These hypothesis have been offered in various guises by a number of authors.
	Figure 1. How cells may perceive chemo-attractant gradients (after Lackie, 1986). The temporal model assumes that the amoeba measures and "remembers" the chemo-attractant concentration at time zero and compares this to a second reading at some time point later as the cell crawls. In this way the amoeba gets clues like that particularly annoying party game "getting warmer" / " getting colder" (Arghhh!!). The spatial model assumes that the cell is able to read the concentration across the span of the cell so that it can compare the number of chemoattractant molecules. A related model is the pseudospatial model, where the cell tests concentrations by sending out pseudopods (P₁ and P₂) at various points to sense where the highest concentrations lie. The temporo-spatial model, detects waves of chemo-attractants coming toward the cell from a particular direction. Note that this last mechanism could not read a static gradient. A huge amount of work has been expended trying to work out which (if any) of these hypotheses is correct.
The distribution of cAMP receptors on Dictyostelium has been determined by replacing the gene with a gene consisting of the receptor fused to green fluorescent protein (GFP –see below). These studies (Xiao et al, 1997) indicate that there is no particular concentration of receptors within pseudopods as expected from the spatial, pseudospatial or temporo-spatial models. However we cannot discount these models on this basis since they could all function in principle without concentrated receptors (it would just work better). The temporo-spatial model cannot be valid for all cases as it is well established that cells can chemotax in stable gradients of attractions. The Boyden chamber. Two chambers are separated by a filter through which cells migrate (page 7, figure15). Chemotactic gradients can be set up by placing different concentrations of the putative chemo-attractant in the upper and lower chambers. The advantage of the Boyden chamber is that is can discriminate between chemo-kinetic and chemotactic influences. The use of this chamber requires that the cells under test have to move in three dimensions (most do) and to squeeze between the pore size of the particular filter. Filters are available in different average pore sizes, so this latter point is seldom a problem. The Boyden chamber is reproducible and the chemokinetic, chemotactic response easy to quantify.

Figure 2. The Boyden Chamber. The main advantage is that it can detect chemokinetic effects as well as chemotactic effects. The Checkerboard assay (right) is a way to organise the Boyden chamber experiment.
	Figure 3. The checkerboard assay can be used to differential chemokinesis from chemokinesis. If attractants are added to both chambers at various concentrations, the relationship of migration to attractant can be plotted. In this example, the attractant is both a chemokinetic and a chemoattractant.

Figure 4. The under agarose method (left) versus the over agarose method. The under agarose method (Laevsky & Knecht, 2001 ) is simple and can be used for a variety of cell types. Chemo-attractant gradients are set up as the attractant diffuses from the trough into the agarose. The presence of the agarose stabilizes the gradient that might otherwise be dispersed or changed by thermal motion or minor agitations. An advantage of the various under agarose methods is that it is possible to set up multiple gradients of various chemo-attractants to study their effect on cell behaviour simultaneously (Heit et al, 2002).
Figure 5. A stable gradient can be created by placing the putative attractant in one well and the cells are then placed in a second well.
Chemotaxis in Development Mammalian development begins with the meeting and fusion of the gametes, the female egg gives signals and the male sperm comes swimming (setting the pattern for life?). Chemotaxis helps the sperm find the egg in humans (Eisenbach & Tur-Kaspa, 1994), and in algae where a chemo-attractant increases the turning angle of the sperm cell so that it spirals in toward the egg like a captured moon. Development involves mass movements of cells.

Figure 6. Neutrophil migration into inflammatory regions. (1). Neutrophils in the microvascular vessels circulate passively in the blood. At sites close to inflammation (2), the endothelial cells alter their charge in response to interleukins such as IL-8, so that now neutrophils can stick. (3). Neutrophils now crawl along the "-dimensional surface of the vessel and then squeeze between the endothelial cells. (4) Neutrophils can now invade the 3-dimensional space and begin to detect the fMLP and/or C5a gradient at the same time as a negative IL-8, LTP4 gradient. It has been suggested (Heit et al, 2002), that such an arrangement is more stimulatory. (5) Neutrophils arrive at the site of infection drawn by the gradient and now consume the bacteria by phagocytosis. Eventually the neutrophils die full of now dead bacteria and accumulate as pus. The top line in the graph represents adhesive forces which peak at the endothelial surface, drop after that as the neutrophils crawl in the 3-dimension matrix. Adhesion then increases with fMLP/C5a gradient. The lower graph represents the expected gradient of the various classes of chemo-attractants (after Lackie).
Transduction of Chemo-attractant Signals Most of what is known about the signalling cascade has been gleaned from Dictyostelium, but recent contributions are available from knock out mouse models. From the top, chemotaxis requires the chemotactic receptor, heterotrimeric G-proteins, Whereas the distribution of chemotactic receptors is uniform across the cell (Xiao et al, 1997), other signalling molecules further down the cascade are found to have a polarized distribution. Mammalian PLCg become localised to the leading edge in a PI3-Kinase dependent manner (Piccola et al, 2002). PLCg contains a PH domain that binds PtdIns-3,4,5-P3, a product of PI3-Kinase, and so its probable that the PLCg localization is downstream of PI3-Kinase activity. The bg subunit of the activated hetero-trimeric G-proteins are weakly polarised fashion with concentrations at the leading edge (Jin et al, 2000). Another protein involved in chemotactic pathway AKT (protein kinase B), also contain PH domains and are also localized to the leading edge of cells (neutrophils)(Servant et al, 2000). Surprisingly, it has recently been reported that in mouse neutrophils, PLCs are not required for chemotaxis but are involved in priming the superoxide burst (Li et al, 2000). Dictyostelium too seems to chemotax in the absence of PLC (Drayer et al, 1994).
	Figure 7. G-protein linked chemo-attractant receptor dissociates trimeric G-protein and the b,g subunit activates both PLC and PI(3)-kinase. The products of PI(3)-kinase are short-lived messengers that bind a number of targets leading to their activation. The activation of PLC is important for activation of the superoxide burst after phagocytosis in neutrophils but not important in chemotaxis its self. Mice bred without PI(3)-kinase however have a reduced capacity to chemotax.
References:- Key, T. A., Foutz, T. D., Gurevich, V. V., Sklar, L. A. & Prossnitz, E. R. (2003) N-Formyl Peptide Receptor Phosphorylation Domains Differentially Regulate Arrestin and Agonist Affinity. J. Biol. Chem. 278, 4041-4047.