We are interested in molecules essential for transmission of forces. We have identified one candidate, the Dictyostelium talin (talB) that is preferentially expressed in the morphogenetic stages of development. Knockout mutants of talB are unable to develop beyond the tight mound stage. If mixed with wildtype cells, however, they can go further and differentiate into mature stalk and spore cells, but they are excluded from the tip region in the chimeric slug (see Fig.1 below). Observation of cell movements in pure as well as mixed populations suggest that the mutant cells are somewhat defective in motility within 3-dimensional tissues and also defective in the transmission of force; they appear to be a poor substratum for the surrounding cells to move on. Subcellular localizatioin of the protein revealed by immunofluorescence seems to support this notion. The other talin of Dictyostelium (talA) which was discovered earlier is expressed at a constant level during growth and development and plays important roles in cell adhesion during growth and early developmental stages, but is not required for later morphogenesis (Kreitmeier et al. 1995, J. Cell Biol. 129, 179-188. Niewöhner et al. 1997, J. Cell Biol. 138, 349-361). There seems to be division of labour between the two talins in Dictyostelium.
After aggregation, cells appear to change the way of movement --- cells very actively extend a small number of (often just one) large, cylindrical pseudopods with a smooth end. By contrast, during growth and earlier stages of development, cells tend to make more than one pseudopods at a time, often flat ones (lamellipodia) when in contact with the substratum. Pseudopods of vegetative cells are usually decorated with many filopodia on solid substratum. To study the slug-type cell movement, it is necessary to study cell movement in 3-D tissues, but it would be ideal if one can examine the movement of such cells in isolation as well. However, cells isolated from migrating slugs seem unable to move about even though they extend pseudopods very vigorously. One way to circumvent this problem is to sandwich dissociated slug cells between agar sheets. In this way one can make slug cells move freely just like pre-aggregation cells. Another way is to look at earlier cells that move in a way similar to slug cells. Pre-aggregative cells in suspension sometimes show a funny way of movement for a limited period of time just before settling on the substratum. Such cells appear as though they were 'swimming', and their speed is astonishingly high. Their appearance is very similar to slug cells indeed. Although I believe every cell is capable of this type of cell movement, it is kind of rare to come across this kind of cell movement. K.Yoshida in our group found that quinine induces similar cell movement in vegetative cells. In many cases, cells only extend a cylindrical protrusion without actually moving, but some cells continue to extend the protrusion and actually translocate over a distance a little longer than the cell length. Protrusion formation induced by quinine is triggered by fusion of a contractile vacuole with the cell membrane. A great advantage of using this experimental system is one can be sure to be watching the same sort of protrusion; in 'normal' conditions, a cell can form different kinds of pseudopods (such as lamellipodia, filopodia, lobopodia, etc.) depending on the condition, or quite probably mixture of these at the same time, which would certailnly complicate the interpretation of any observations. We have been exploring this rather artificial quinine-induced protrusion in the hope of gaining insight into the mechanism of pseudopod extension typically seen in slug cells. What we have noted thus far include the importance of myosin II (conventional myosin) in the generation of propulsive force and of actin polymerization in the canalization of the cytoplasmic flow.
Migrating slugs of Dictyostelium respond to external stimuli and change their course of movement. The effective stimuli known so far include light (phototaxis), tempertarue gradient (thermotaxis), and chemicals such as ammonia and a small molecule called Slug Turning Factor (chemotaxis). Slugs also make turns without obvious external stimulus (spontaneous turning). This turning behaviour is interesting in itself, but it may be expected that it would also provide an interesting opportunity to study the mechanism of cell organization in multicellular development. We are analysing the movement of individual cells in slugs in the process of turning as well as in straight forward movement. (Earlier results have been presented in the 1997 Dictyostelium meeting at Snowbird.)
Based on our earlier models of slug movement, we have developed a mathematical model for the formation of a tip from a mound and subsequent elongation and slug migration. We are currently studying the mechanism of slug turning using this model.
We have shown that an artificial drop of cytoplasmic pH by acid load triggers maturation of prestalk cells into stalk cells and that clamping of the cytoplasmic pH at neutral pH with a weak base prevents stalk cell maturation. It seems that stalk cell maturation is controlled by cellular acidification in normal fruiting body formation as well, because a steep drop of cytoplasmic pH is invariably observed at the site of stalk cell maturation in a fruiting body, both in Dictyostelium and in Polsphondylium. I am trying to clarify the causal relationship between the pH drop and the onset of stalk cell differentiation.