Project title:

Analysis of three-dimensional cell movement during the migratory multicellular stage of  D. discoideum

In the cellular slime mold Dictyostelium discoideum, the multicellular migratory phase is termed “slug”. The movement of the cells directly powers the motility of slugs. However, details of how cell movement contributes to the slug migration and changes in slug shape is yet barely known. I study the three dimensional correlation between the cell movement and the phototactic turning of slugs. A novel analysis of three dimensional cell movement is also planned, which uses the computational detection of optical flow (vector-field method).

Background

Multicellular organism behaves as a single organism because cells within the organism move in a coordinated manner. Such a coordinated movement of cells is general in all multicellular organisms but complexity of the phenomena has been hindering the efforts to understand its mechanism. In cellular slime mould Dictyostelium discoideum, the mechanism of multicellular coordination has been relatively well addressed. It’s lifecycle has a unicellular stage and a multicellular stage. The multicellular stage involves chemotactic aggregation of the cells, multicellular migration and morphogenetic movement for constructing fruiting body. Molecular genetic approaches discovered many genes involved in these process. Multicellular movement is also reachable with current microscopic techniques. For these reasons, Dictyostelium discoideum is a powerful subject for studying the cell motility and the multicellular development, and is chosen as a model organism for biomedical research in NIH (USA).

 Amoebae of the Dictyostelium discoideum normally live as single cells in forest litter and feed on bacteria. Starvation induces the transition from the unicellular stage to the multicellular stage. Chemotactic aggregation of up to 10⁵ cells forms a multicellular mass which behaves as a single organism and ultimately transforms into a fruiting body consisting of a stalk and a spore head. Beginning 4 ~ 6 hours after removal of the food source individual amoebae start to aggregate in response to periodic cAMP (adenosine 3’,5’-monophosphate) signals initiated at the center of aggregation and transmitted outward from cell to cell as a travelling wave of cAMP. In turn, these cAMP waves direct the coordinated inward movement of the amoebae by chemotaxis. Aggregation results in the formation of a migrating slug. During the slug stage, up to 10⁵ cells coordinate their movement and migrate as a single organism. Slugs have a cylindrical shape with tip and tail; their morphological polarity corresponds to the polarity of migration. A large body of results suggest that cyclic AMP-mediated cell-cell signaling is the mechanism coordinating multicellular movement. Waves of cyclic AMP generated at the anterior tip propagate towards the tail and induce the chemotactic movement of cells toward the tip. Slugs exhibit highly sensitive environmental reactions: phototaxis, chemotaxis and thermotaxis. These abilities allow slugs to scan the environment to search for the best location to become fruiting body. 

 Although many studies have investigated how Dictyostelium slugs move toward a light source, the mechanism of phototaxis is still unclear. It has been known that slug turning is initiated from the anterior tip. In addition, previous research identified many mutations and drug treatments that interfere with phototaxis. Despite of these achievements, the methods for analyzing phototactic behavior both at the slug level and at the cellular level has been limited to low resolution both temporarily and spatially that the exact mechanism of how cells move in response to the light irradiation and turn the slug toward light source is still largely unknown. Taking these backgrounds into account, I analyzed the details of the slug phototactic behavior and found out following points:

 (1)     The light irradiation induces secretion of cAMP form the slug tip (Miura and Siegert, 2000).

(2)     Turning of the slug tip due to a bending of the anterior zone of the slug.

(3)     Two-dimensional analyses of the cell movement suggests that some of the cells move away from the light source, increase the volume of the slug side away from the light source and bend the anterior zone like a lever-arm action.

(4)     During phototaxis, slug increases speed, length and period of the up-down motion of the tip. Analyses of these data suggest that less up-down motion of the tip during phototaxis causes the increase of speed and the length.

Task

Among above findings the relationship between (1), (2) and (3) is currently the most interesting point, since the effect of light-induced cAMP secretion on the cellular behavior within the slug is the key for the slug phototactic turning. Within slug, intrinsic cAMP waves are propagating from front to back. How light-induced cAMP interferes with those intrinsic cAMP waves can be studied through analyzing the direction and the speed of cell movement within slugs. Previous analyses in (3) were done with two-dimensional time sequence due to the limitation in the current experimental set up but the actual slug and cell behavior is three dimensional. The task of this project is to study details of the three dimensional cell movement during slug turning thus to correlate those cell movement with the slug bending behavior during turning.  

In addition to the wildtype slugs, a mutant strain with defects in phototaxis will be analyzed. There are many phototaxis mutants with “bidirectional phototaxis” phenotype, in which their slug migration is directed obliquely towards the right or the left side of the light source (Fisher, 2000). It seems that the basic response to the light seems to be intact in these mutants, but a slight deficiency in three-dimensional cell movement within slug seems to be causing the falsely directed phototaxis. RasD-null mutant also exhibits bidirectional phototaxis (unpublished results) and I will analyze this mutant as a comparison to the wild type slugs. RasD protein is a small G protein expressed only during multicellular stage of Dictyostelium and the known phenotype of its knock-out mutant is only in the phototaxis deficiency (Wilkins et al., 2000).

The first step of the analysis will be to deconvolute the 4D sequence to extract precise 3D information. The second step is to analyze the cell movement quantitatively by using the vector-field method. This method calculates the average velocity vector for every pixel in a time series of digital images (Nomura et al., 1991). The cell movement in 2-dimension (projection) sequence was successfully analyzed by the vector-field algorithm (Siegert et al., 1994). The advantage of this method is that it is not necessary to identify single cells in order to measure cell movement. The program was originally written in the C language. I recently rewritten the program in the IgorPro macro language (Wavemetrics, Lake Oswego, OR ), which is more suited for such a calculation compared to the C language. In this project, the program will be extended to 3D. The method and conditions for the deconvolution of the original 4D sequence should be carefully chosen to reduce possible errors of the vector –field analysis.

References

Fisher, P. R. (2000). Genetic analysis of phototaxis in Dictyostelium. In “Review Series on Photobiology”, Vol. Volume 1 "Photomovement". Elsevier, Amsterdam.

Miura, K., and Siegert, F. (2000). Light affects cAMP signaling and cell movement activity in Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 97, 2111-2116.

Nomura, A., Miike, H., and Koga, K. (1991). Field theory approach for determining optical flow. Pattern Recog. Lett. 12, 183-190.

Siegert, F., Weijer, C. J., Nomura, A., and Miike, H. (1994). A gradient method for the quantitative analysis of cell movement and tissue flow and its application to the analysis of multicellular Dictyostelium development. J. Cell Sci. 107, 97-104.

Wilkins, A., Khosla, M., Fraser, D. J., Spiegelman, G. B., Fisher, P. R., Weeks, G., and Insall, R. H. (2000). Dictyostelium RasD is required for normal phototaxis, but not differentiation. Genes Devel. 14, 1407-1413.