DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin

Transition Metals Doped Nanocrystals: Synthesis, Characterization, and Applications

Doping is a technique that makes it possible to incorporate substitutional ions into the crystalline structure of materials, generating exciting properties. This book chapter will comment on the transition metals (TM) doped nanocrystals (NCs) and how doping and concentration influence applications and biocompatibility. In the NCs doped with TM, there is a strong interaction of sp-d exchange between the NCs’ charge carriers and the unpaired electrons of the MT, generating new and exciting properties. These doped NCs can be nanopowders or be embedded in glass matrices, depending on the application of interest. Therefore, we show the group results of synthesis, characterization, and applications of iron or copper-doped ZnO nanopowders and chromium-doped Bi2S3, nickel-doped ZnTe, and manganese-doped CdTe quantum dots in the glass matrices

A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions

Immunolocalization of HEX 70a in the testioles of pharate-adult drones (∼6 days after pupal ecdysis; developmental stage shown at the upper left corner).

<p>(<b>A, C</b>) Light microscopy of the testiole stained with methylene blue/basic fuchsin. Syncytial cluster of spermatids are evident (arrows). (<b>B, D</b>) Confocal microscopy images showing rhodamine/phaloidin labeled F-actin (green) and DAPI-labeled cell nuclei (blue). The association of actin with spermatid heads in the syncytial clusters is evidenced in B (arrows) and in the similar and amplified D image (arrows). (<b>E–G</b>) Confocal microscopy showing (<b>E</b>) DAPI-stained nuclei in the syncytial cluster of spermatids (asterisks) and in a cyst somatic cell nuclei (arrowhead); (<b>F</b>) HEX 70a foci detected with anti-HEX 70a/Cy3 (red) at the posterior end of the spermatid nuclei in the syncytial cluster (shown in higher magnification at the upper left side): arrows point to individualized spermatozoa and the arrowhead points to a cyst somatic cell nuclei. (<b>G</b>) The merged E–F images showing the amplified syncytial cluster of spermatids (insert), individualized spermatozoa (arrows) and a cyst somatic cell nuclei (arrowhead).</p

Colocalization of anti-HEX 70a/Cy3 and EdU/Alexa Fluor in the ovary of a pharate-adult worker (∼2 days after pupal ecdysis: seen at the upper left corner of the figure).

<p>(<b>A</b>) DAPI-stained ovarian cell nuclei (shown in blue). For clarity, the ovarioles are delineated by dashed lines above the basal portion of the ovary. Only the germarium region is seen in each ovariole. (<b>B</b>) HEX 70a foci detected with anti-HEX 70a/Cy3 (red). (<b>C</b>) EdU/Alexa Fluor foci in S-phase ovarian nuclei (yellow). (D) Merged B and C images. Circles in B–D emphasize groups of cystocytes (in the germarium region) showing double labeling (anti-HEX 70a/Cy3 and EdU/Alexa Fluor). The group of cystocytes encircled at the most right position in D is shown in higher magnification in the insert. Cell nuclei on the right margin of the ovary in B (arrowheads) show HEX 70a/Cy3 but not EdU/Alexa Fluor labels.</p

Detection of HEX 70a in ovarioles of workers at the beginning of the pharate-adult development (∼1 day after pupal ecdysis) (the developmental stage is illustrated at the upper left corner of the figure).

<p>(<b>A</b>) Light microscopy of ovarioles (covered by their respective peritoneal sheath) stained with methylene blue/basic fuchsin. Only the germarium is focused in this figure (the most anterior region of the ovariole, or terminal filament, is not shown). A rosette formed by germline cells (oocyte and nurse cell precursors) is distinguishable (circle) in the germarium. (<b>B, C</b>) Confocal microscopy image of rhodamine/phalloidin labeled F-actin (green) and DAPI-labeled cell nuclei (blue) showing aspects of the structure of the ovarioles (peritoneal sheath removed) at the time they were used for HEX 70A detection. The actin-rich polyfusomes (arrowheads in B) are seen in the center of the cystocyte rosettes in the upper region of the germarium. Ring canals derived from polyfusomes (arrows in B and C) are apparent in the lower region of the germarium shown in B and in higher magnification in C. (<b>D</b>) Confocal microscopy of an ovariole (upper portion of the germarium) stained with DAPI. (<b>E</b>) The same ovariole showing foci of HEX 70a detected with anti-HEX 70a/Cy3 (red). (<b>F</b>) The merged D and E images. The insert in F shows a “control” ovariole (upper portion of the germarium) incubated with the pre-immune serum and subsequently stained with Cy3/DAPI. Arrowheads in D-F show nuclei of germline cells. Arrows in D–F point to nuclei of follicle cell precursors. In all figures, the upper portion of the germarium is oriented upward.</p

Immunolocalization of HEX 70a in the testioles of drone pupae (1 day after pupal ecdysis; developmental stage shown at the upper left corner).

<p>(<b>A</b>) Light microscopy section of a testiole stained with methylene blue/basic fuchsin showing a region containing groups of cystocytes (spermatogonia) (arrows) involved by somatic cells (somatic cell nuclei pointed by arrowheads). (<b>B</b>) Confocal microscopy image showing rhodamine/phalloidin labeled F-actin (green) and DAPI-labeled cell nuclei (blue); somatic cell nuclei are pointed by arrowheads; insert shows a magnified image of a cyst containing cystocytes (cystocyte nuclei pointed by arrows) and ring canals (asterisks). (<b>C–E</b>) Confocal microscopy images of a testiole from a drone taken at the same developmental phase, showing (<b>C</b>) DAPI-stained cell nuclei, (<b>D</b>) foci of HEX 70a detected with anti-HEX 70a/Cy3 (red), and (<b>E</b>) the merged C and D images. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029006#pone-0029006-g001" target="_blank">Figure 1E</a> the thick arrows show germ cell nuclei, the thin arrows show HEX 70a foci in the cytoplasm and the arrowheads show somatic cell nuclei.</p

Effect of HEX 70a depletion on queen ovary growth and worker cuticle formation.

<p>(<b>A</b>) Width of the ovarioles of queens injected with anti-HEX 70a in 0.9% NaCl or saline vehicle only. Measurements were made in two regions of the germarium of 120 ovarioles, 60 of them dissected from 3 anti-HEX 70a injected queens (20 ovarioles per queen), and 60 from 3 control queens. Measurements obtained from bees injected with the antibody, or the antibody vehicle only, were compared using Two-Way ANOVA and the post-hoc Holm-Sidak multiple comparison test (Jandel SigmaStat 3.1 software, Jandel Corporation, San Rafael, CA, USA). (<b>B</b>) Western blot levels of HEX 70a in the hemolymph samples of workers at 4 and 72 h after injection with anti-HEX 70a or saline vehicle only (control). The levels of the ∼200 kDa lipophorin in the same samples were used as loading control. (<b>C</b>) Hind legs of workers injected with anti-HEX 70a in 0.9% NaCl, in comparison to workers injected with mouse IgG in 0,9% NaCl, or those of the 0.9% NaCl injected group.</p

Immunolocalization of HEX 70a in the queen ovariole.

<p>(<b>A</b>) Schematic representation of an ovariole of an egg laying queen (seen at the upper left corner): only the terminal filament, the germarium and early follicles initiating previtellogenic growth in the upper region of the vitellarium are shown in A. Confocal microscopy images: (<b>B</b>) Part of an ovariole showing the middle and lower regions of the vitellarium labeled with rhodamin/phalloidin (green) to highlight F-actin. The arrows and arrowheads show developing nurse cell- and oocyte- chambers, respectively. (<b>C–E</b>) the terminal filament (the lower region is oriented downward) shows HEX 70a foci in the nuclei (D, E) and in cytoplasm (arrows in D, E). (<b>F–H</b>) Nurse cell nuclei in the nurse cell chamber (lower region of the vitellarium as indicated by arrows in B). (<b>I–K</b>) Follicle cell nuclei covering an oocyte at the lower region of the vitellarium (as indicated by arrowheads in B). (<b>C, F, I</b>) DAPI-stained cell nuclei (blue); (<b>D, G, J</b>) anti-HEX 70a/Cy3-staining for HEX 70a detection (red) and (<b>E, H, K</b>) merged images.</p