An actin-related protein in Drosophila colocalizes with heterochromatin protein 1 in pericentric heterochromatin

The actin-related proteins have been identified by virtue of their sequence similarity to actin. While their structures are thought to be closely homologous to actin, they exhibit a far greater range of functional diversity. We have localized the Drosophila actin-related protein, Arp4, to the nucleus. It is most abundant during embryogenesis but is expressed at all developmental stages. Within the nucleus Arp4 is primarily localized to the centric heterochromatin. Polytene chromosome spreads indicate it is also present at much lower levels in numerous euchromatic bands. The only other protein in Drosophila reported to be primarily localized to centric heterochromatin in polytene nuclei is heterochromatin protein 1 (HP1), which genetic evidence has linked to heterochromatin-mediated gene silencing and alterations in chromatin structure. The relationship between Arp4 and heterochromatin protein 1 (HP1) was investigated by labeling embryos and larval tissues with antibodies to Arp4 and HP1. Arp4 and HP1 exhibit almost superimposable heterochromatin localization patterns, remain associated with the heterochromatin throughout prepupal development, and exhibit similar changes in localization during the cell cycle. Polytene chromosome spreads indicate that the set of euchromatic bands labeled by each antibody overlap but are not identical. Arp4 and HP1 in parallel undergo several shifts in their nuclear localization patterns during embryogenesis, shifts that correlate with developmental changes in nuclear functions. The significance of their colocalization was further tested by examining nuclei that express mutant forms of HP1. In these nuclei the localization patterns of HP1 and Arp4 are altered in parallel fashion. The morphological, developmental and genetic data suggest that, like HP1, Arp4 may have a role in heterochromatin functions. Keywords: Chromatin, Actin-related protein, Drosophila, Heterochromatin-protein 1, Position effect variegatio

Roles for Drosophila melanogaster myosin IB in maintenance of enterocyte brush-border structure and resistance to the bacterial pathogen Pseudomonas entomophila

Author Posting. © American Society for Cell Biology, 2007. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 18 (2007): 4625-4636, doi:10.1091/mbc.E07-02-0191.Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both singed (fascin) and Dmoesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection.This work was supported by National Institutes of Health grants DK-25387 (to M.S.M.), DK-55389 (to Jon Morrow, Yale School of Medicine), and GM-52857 (to L.G.T.) and a research grant from the Crohns and Colitis Foundation of America (to M.S.M.)

Myosin-V stepping kinetics: A molecular model for processivity

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete ≈36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity