The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29–41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602–R605).

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.

MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the ␣-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease. IntroductionThe autosomal dominant, giant-platelet disorders-May-Hegglin anomaly (MHA), Fechtner (FTNS), Sebastian (SBS), Epstein (EPS), and Alport-like syndromes with macrothrombocytopeniasresult from mutations in the human MYH9 gene, which encodes nonmuscle myosin-IIA heavy chain. [1][2][3][4][5] Virtually all patients exhibit platelet macrocytosis, thrombocytopenia, and leukocyte inclusions. Some individuals also exhibit high-tone sensorineural deafness, cataracts, and nephritis to varying degrees. Additional genetic or environmental factors may contribute to the variable penetrance of the nonhematologic clinical manifestations. 6,7 A single family with predominantly hearing loss has been reported. 8 Together, these illnesses represent a spectrum of disorders collectively termed MYH9-related disorders. 6,7,9 Of the 82 mutations identified in MYH9-related disorder families, only 21 amino acids, out of a possible 1961, are mutated. 3,7,10 Identical MYH9 mutations occur in unrelated families and can arise spontaneously, suggesting that these disorders result from highly specific disruption of the MYH9 structure and/or function. Mutations at other sites may occur and remain undetected owing to (1) a completely recessive alteration in myosin-II function, (2) a weakly penetrant dominant effect that escapes clinical detection, or (3) severe dysfunction that is incompatible with life.In humans, the 3 nonmuscle myosin-II heavy chains (IIA, IIB, and IIC) are encoded by distinct genes (MYH9, MYH10, and MYH14, respectively). 11 Only nonmuscle myosin-IIA is expressed in platelets, 12,13 and no mutations in nonmuscle myosin-IIB or myosin-IIC have been reported. ...