Abstract. As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975. Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The aminoterminal two thirds of the protein comprises a head domain that is 29-33 % identical (60-65 % similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chainbinding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a ~100-amino acid region that is predicted to form an c~-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC).The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.
We present a molecular analysis of four dominant alleles of the pair-rule gene ftz. Three of these, the ftz"'^ alleles, cause anti-ftz segmentation defects and homeotic transformations of the first abdominal segment to the third. These alleles are shown to be missense changes affecting two nearby proline codons. Embryos homozygous for these mutations accumulate higher levels of ftz protein than wild type and show strong persistence of ftz protein, but not RNA. These effects appear to result from stabilization of the ftz protein, since ftz stripes decay much more slowly in mutant embryos than in wild type after injection of the protein synthesis inhibitor cycloheximide. We trace the origin of segmentation defects in ftz""^ embryos to repression of the pairrule gene even-skipped by excess ftz protein during stripe sharpening. Homeotic transformations are shown to be correlated with ectopic expression of the abd-A gene of the bithorax complex. A 12-amino-acid sequence containing the proline residues altered in the ftz^*^ mutants appears to be conserved in the proteins encoded by other segmentation genes and the vertebrate oncogene myc and may target these proteins for rapid degradation. The fourth allele examined, T(2}3)ftz^' {Rpl], also causes homeotic transformations and is a translocation broken within the /tz-coding region. Both ftz transcript and protein stripes are persistent in Rpl embryos, suggesting that the Rpl RNA is stabilized relative to wild type.
To examine the mechanisms of cell locomotion within a three-dimensional (3-D) cell mass, we have undertaken a systematic 3-D analysis of individual cell movements in the Dictyostelium mound, the first 3-D structure to form during development of the fruiting body. We used time-lapse deconvolution microscopy to examine two strains whose motion represents endpoints on the spectrum of motile behaviors that we have observed in mounds. In AX-2 mounds, cell motion is slow and trajectories are a combination of random and radial, compared to KAX-3, in which motion is fivefold faster and most trajectories are rotational. Although radial or rotational motion was correlated with the optical-density wave patterns present in each strain, we also found small but significant subpopulations of cells that moved differently from the majority, demonstrating that optical-density waves are at best insufficient to explain all motile behavior in mounds. In examining morphogenesis in these strains, we noted that AX-2 mounds tended to culminate directly to a fruiting body, whereas KAX-3 mounds first formed a migratory slug. By altering buffering conditions we could interchange these behaviors and then found that mound-cell motions also changed accordingly. This demonstrates a correlation between mound-cell motion and subsequent development, but it is not obligatory. Chimeric mounds composed of only 10% KAX-3 cells and 90% AX-2 cells exhibited rotational motion, suggesting that a diffusible molecule induces rotation, but many of these mounds still culminated directly, demonstrating that rotational motion does not always lead to slug migration. Our observations provide a detailed analysis of cell motion for two distinct modes of mound and slug formation in Dictyostelium.
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