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Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.11.029 Byline: Andrew M. Hudson (a), Lisa N. Petrella (a), Akemi J. Tanaka (a), Lynn Cooley (a)(b)(c) Keywords: Oogenesis; Visceral muscle; Fasciclin 3; Zasp; Filamin Abstract: Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. We have used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, we were able to use the Flp/FRT system to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle. Author Affiliation: (a) Department of Genetics, Yale University School of Medicine, 333 Cedar St. New Haven, CT 06520, USA (b) Department of Cell Biology, Yale University School of Medicine, 333 Cedar St. New Haven, CT 06520, USA (c) Department of Molecular, Cellular and Developmental Biology, Yale University, 260 Prospect St. New Haven, CT 06510, USA Article History: Received 5 October 2007; Revised 20 November 2007; Accepted 27 November 2007

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions, The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA, We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.028 Byline: Valerie Mermall (a), Nathalie Bonafe (a)(b), Lynn Jones (b), James R. Sellers (e), Lynn Cooley (a)(b)(c), Mark S. Mooseker (a)(c)(d) Keywords: Actin; Microtubules; Investment cones; Individualization complex; Gut; Oogenesis; CNS Abstract: Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex. Author Affiliation: (a) Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06510, USA (b) Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA (c) Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA (d) Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA (e) National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA Article History: Received 27 April 2005; Revised 21 July 2005; Accepted 22 July 2005

The Filamin family of actin binding proteins is required to maintain cell shape and promote cell locomotion. Using the Drosophila ovary, we provide a detailed description of Filamin-deficient cells during morphogenesis. Reduced expression of Filamin in follicle cells causes defects in the initial encapsulation of germline cysts and in the migration of border cells through the germline cyst. However, follicle cell morphogenesis is unaffected by point mutations that produce truncated Filamin proteins. In addition, mutant follicle cell movements can be partially rescued by a transgene encoding only the actin-binding domain and the first six filamin repeats. These data show that Filamin function in cell motility can be provided by a truncated Filamin protein that resembles Dictyostelium Actin Binding Protein-120. Keywords: Oogenesis; Filamin; follicle cell: morphogenesis; migration

We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin [beta]-like export receptor for importin [alpha]. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-[alpha]3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders. Key Words: Drosophila; dcas; CAS; Cse1p; importin alpha nuclear export; nucleocytoplasmic transport; mechano-sensory bristle development; peripheral nervous system.

During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG), midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.

Key Words ring canal, actin regulation, actin crosslinker, follicle cell, migration Abstract Much of our knowledge of the actin cytoskeleton has been derived from biochemical and cell biological approaches, through [...]

In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. Here we describe a new gene we named cheerio that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since hts and kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization.

Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo; [singed.sup.G409E] mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C-terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo; [singed.sup.S289N] mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of [singed.sup.S289N] sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.

Intercellular cytoplasm transport in Drosophila during oogenesis occurs in three stages, during germarium, early to mid oogenesis and late oogenesis, is selective and requires several cytoskeletal components and genes as the process mediators. Microtubules facilitate bcd mRNA transport in the early to mid oogenesis stage. Cytoplasm transport during late oogenesis requires actin filaments and armadillo, chickadee and quail genes in the cytoskeleton and results in a two-fold increase in oocyte volume.

The quail gene in Drosophila encodes a protein which is homologous to the vertebrate actin-binding protein villin. The villin-like quail protein controls the assembly of actin filament bundles in nurse cells and the gene expression is confined to the germ line in Drosophila adults. The quail protein regulates the polymerization and formation of actin filaments.

The role of intercellular bridges called ring canals during oocyte maturation in Drosophila was investigated. Oocyte maturation is supported by a cluster of 15 germline-derived nurse cells which contribute cytoplasmic material to the oocyte through ring canals. The study protocol involved the cloning of the kelch gene from a female sterile mutation which affects transport of cytoplasmic material through the ring canals. Two protein products from the kelch mRNA were produced. One of these proteins is conserved and is localized to the ring canal, suggesting that kelch is a ring canal component which regulates the flow of cytoplasm between cells.

Ring canals form between sister oocytes during the development of germ cells in Drosophila. An analysis of the formation of the ring canals in two protein kinase Drosophila mutants reveal the particular requirement of ring canal formation for the Src and the Tec kinases. Immunostaining reveals that Src localizes in the nurse cells while Tec localizes in the ring canal. It is postulated that Src is necessary for the localization of Tec in the ring canal.

During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitinproteasome system. [ABSTRACT FROM AUTHOR]