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Daniel P Kiehart

Publications and source records attributed to Daniel P Kiehart.

15 recordsLinked to original sources
PubMed2006

Dimerized Drosophila myosin VIIa: a processive motor.

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. We studied the molecular mechanism of Drosophila myosin VIIa, using transient kinetics and single-molecule motility assays. Myosin VIIa moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is approximately 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin VIIa is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate-limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin VIIa (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin VIIa to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter.

Animals↗
PubMed2006

Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster.

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners.

Amino Acid Sequence↗
PubMed2006

JNK signaling coordinates integrin and actin functions during Drosophila embryogenesis.

Epithelial movements are key morphogenetic events in animal development. They are driven by multiple mechanisms, including signal-dependent changes in cytoskeletal organization and in cell adhesion. Such processes must be controlled precisely and coordinated to accurately sculpt the three-dimensional form of the developing organism. By observing the Drosophila epidermis during embryonic development using confocal time-lapse microscopy, we have investigated how signaling through the Jun-N-terminal kinase (JNK) pathway governs the tissue sheet movements that result in dorsal closure (DC). We find that JNK controls the polymerization of actin into a cable at the epidermal leading edge as previously suggested, as well as the joining (zipping) of the contralateral epithelial cell sheets. Here, we show that zipping is mediated by regulation of the integrins myospheroid and scab. Our data demonstrate that JNK signaling regulates a set of target genes that cooperate to facilitate epithelial movement and closure.

Actins↗
PubMed2005

Nonmuscle myosin II generates forces that transmit tension and drive contraction in multiple tissues during dorsal closure.

BACKGROUND: The morphogenic movements that characterize embryonic development require the precise temporal and spatial control of cell-shape changes. Drosophila dorsal closure is a well-established model for epithelial sheet morphogenesis, and mutations in more than 60 genes cause defects in closure. Closure requires that four forces, derived from distinct tissues, be precisely balanced. The proteins responsible for generating each of the forces have not been determined. RESULTS: We document dorsal closure in living embryos to show that mutations in nonmuscle myosin II (encoded by zipper; zip/MyoII) disrupt the integrity of multiple tissues during closure. We demonstrate that MyoII localization is distinct from, but overlaps, F-actin in the supracellular purse string, whereas in the amnioserosa and lateral epidermis each has similar, cortical distributions. In zip/MyoII mutant embryos, we restore MyoII function either ubiquitously or specifically in the leading edge, amnioserosa, or lateral epidermis and find that zip/MyoII function in any one tissue can rescue closure. Using a novel, transgenic mosaic approach, we establish that contractility of the supracellular purse string in leading-edge cells requires zip/MyoII-generated forces; that zip/MyoII function is responsible for the apical contraction of amnioserosa cells; that zip/MyoII is important for zipping; and that defects in zip/MyoII contractility cause the misalignment of the lateral-epidermal sheets during seam formation. CONCLUSIONS: We establish that zip/MyoII is responsible for generating the forces that drive cell-shape changes in each of the force-generating tissues that contribute to closure. This highly conserved contractile protein likely drives cell-sheet movements throughout phylogeny.

Amino Acid Sequence↗
PubMed2005

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila.

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.

Animals↗
PubMed2004

Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly.

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 alpha-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.

Circular Dichroism↗
PubMed2004

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis.

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.

Animals↗
PubMed2004

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.

Amino Acid Sequence↗
PubMed2003

Forces for morphogenesis investigated with laser microsurgery and quantitative modeling.

We investigated the forces that connect the genetic program of development to morphogenesis in Drosophila. We focused on dorsal closure, a powerful model system for development and wound healing. We found that the bulk of progress toward closure is driven by contractility in supracellular "purse strings" and in the amnioserosa, whereas adhesion-mediated zipping coordinates the forces produced by the purse strings and is essential only for the end stages. We applied quantitative modeling to show that these forces, generated in distinct cells, are coordinated in space and synchronized in time. Modeling of wild-type and mutant phenotypes is predictive; although closure in myospheroid mutants ultimately fails when the cell sheets rip themselves apart, our analysis indicates that beta(PS) integrin has an earlier, important role in zipping.

Animals↗
PubMed2003

Myosins motor Miranda.

New evidence shows that myosin motors drive the spatial segregation of cell fate determinants during asymmetric cell division. How they do so remains a mystery.

Animals↗
PubMed2003

Genetic interactions between the RhoA and Stubble-stubbloid loci suggest a role for a type II transmembrane serine protease in intracellular signaling during Drosophila imaginal disc morphogenesis.

The Drosophila RhoA (Rho1) GTPase is essential for postembryonic morphogenesis of leg and wing imaginal discs. Mutations in RhoA enhance leg and wing defects associated with mutations in zipper, the gene encoding the heavy chain of nonmuscle myosin II. We demonstrate here that mutations affecting the RhoA signaling pathway also interact genetically with mutations in the Stubble-stubbloid (Sb-sbd) locus that encodes an unusual type II transmembrane serine protease required for normal leg and wing morphogenesis. In addition, a leg malformation phenotype associated with overexpression of Sb-sbd in prepupal leg discs is suppressed when RhoA gene dose is reduced, suggesting that RhoA and Sb-sbd act in a common pathway during leg morphogenesis. We also characterized six mutations identified as enhancers of zipper mutant leg defects. Three of these genes encode known members of the RhoA signaling pathway (RhoA, DRhoGEF2, and zipper). The remaining three enhancer of zipper mutations interact genetically with both RhoA and Sb-sbd mutations, suggesting that they encode additional components of the RhoA signaling pathway in imaginal discs. Our results provide evidence that the type II transmembrane serine proteases, a class of proteins linked to human developmental abnormalities and pathology, may be associated with intracellular signaling required for normal development.

Animals↗
PubMed2002

Actin dynamics: the arp2/3 complex branches out.

Several new findings point to novel functions for the Arp2/3 complex. The dendritic nucleation model that has been proposed to describe cell extension for locomotion may also be applicable to other actin-based processes.

Actin-Related Protein 2↗
PubMed2002

Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants.

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire length. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

3T3 Cells↗
PubMed2002

Drosophila RhoA regulates the cytoskeleton and cell-cell adhesion in the developing epidermis.

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. We have investigated its role in the developing Drosophila embryonic epidermis during the process of dorsal closure. By expressing the dominant negative DRhoA(N19) construct in stripes of epidermal cells, we confirm that Rho function is required for dorsal closure and demonstrate that it is necessary to maintain the integrity of the ventral epidermis. We show that defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA(N19) expression. Furthermore, we demonstrate that these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA(N19)-expressing cells.

Actomyosin↗