A computer-assisted system for reconstructing and interpreting the dynamic three-dimensional relationships of the outer surface, nucleus and pseudopods of crawling cells.

Newly developed software additions to the three-dimensional dynamic image analysis system, 3D-DIAS, are described for simultaneously reconstructing and motion analyzing in three dimensions the outer surface, nucleus and pseudopods of living, crawling cells. This new system is then used to describe for the first time a nuclear behavior cycle in translocating Dictyostelium discoideum amoebae and to investigate the role of pseudopod extension in this process. The nuclear behavior cycle is tuned to the two phases of the general cell behavior cycle [Wessels et al., 1994], and includes nuclear migration both in the z- and in the x,y-axes from the proximal border of the prior anterior pseudopod to the proximal border of a newly expanding anterior pseudopod. Nuclear migration is cued by pseudopod-substratum contact, achieves velocities in excess of 50 microm/min, and is accompanied by characteristic changes in nuclear shape. The rules and characteristics of nuclear behavior are demonstrated to be intact in two mutants affecting pseudopod formation, a myosin IB null mutant (myoB-) and a myosin II heavy chain phosphorylation mutant (3XALA). The rules and characteristics of nuclear migration, however, are disrupted upon dissolution of microtubules by colcemid. Together the above results demonstrate that the newly developed 3D-DIAS system can be used to gain new insights into the dynamic changes in the intracellular 3D architecture associated with cellular translocation.