cytochalasin-d and 3-3--dioctadecylindocarbocyanine

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Topics: Animals; Carbocyanines; Cell Movement; Cytochalasin D; Diacetyl; Fibroblasts; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Kinetics; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Video; Toxoplasma

The tissue movements of epithelial spreading and mesenchymal contraction play key roles in many aspects of embryonic morphogenesis. One way of studying these movements in a controlled manner is to make an excisional skin wound to an embryo and watch the wound heal. In this paper we report our studies of healing of a simple excisional lesion made to the limb bud stage mouse embryo. The wounded, living embryo is cultured in a roller bottle; under such conditions the wound heals with a highly reproducible time course and is completely closed by 24 hr. During the healing period the environment bathing the wound can be simply manipulated by adding drugs or factors to the culture medium. We have used DiI to label mesenchymal cells exposed at the margin of the initial wound and, by following their fate and measuring the area of mesenchyme remaining exposed at various time points during the healing process, we have quantified both the extent of mesenchymal contraction and the extent of reepithelialisation by movement of epidermis over mesenchyme. We show that the two types of tissue movement contribute almost equally (50:50) to the total wound closure rate. We have gone on to investigate the cell machinery underlying these processes. In adult wounds the epidermis migrates by means of lamellipodial crawling, but we show that reepithelialisation in the embryo is achieved instead by purse-string contraction of a cable of filamentous actin which assembles in the basal layer of cells at the free edge of the epidermis. Addition of cytochalasin D to the culture medium blocks formation of this actin cable and leads to failure of reepithelialisation. Contraction of adult wound connective tissue appears to be driven by conversion of dermal fibroblasts into a specialist smooth muscle-like fibroblast, the myofibroblast. However, using an antibody recognising the alpha-isoform of smooth muscle actin and specific for smooth muscle cells and myofibroblasts, we show that a similar conversion into myofibroblasts does not occur at any stage during the embryonic wound healing process. These observations indicate that both of the tissue movements of embryonic wound healing utilise cell machinery fundamentally different from that driving the analogous tissue movements of adult healing.

Topics: Actins; Animals; Carbocyanines; Connective Tissue; Cytochalasin D; Embryo, Mammalian; Epidermis; Epithelium; Fibroblasts; Hindlimb; Mesoderm; Mice; Microscopy, Electron, Scanning; Morphogenesis; Organ Culture Techniques; Prenatal Injuries; Reproducibility of Results; Wound Healing