Dcas is Required for Importin-alpha3 Nuclear Export and Mechano-sensory Organ Cell Fate Specification in Drosophila

Developmental Biology. Apr, 2002  |  Pubmed ID: 11944946

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-alpha3 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.

Intracellular MRNA Localization: Motors Move Messages

Trends in Genetics : TIG. Dec, 2002  |  Pubmed ID: 12446149

Intracellular mRNA localization is a common mechanism of post-transcriptional regulation of gene expression. In a wide range of organisms, mRNA localization coupled with translational regulation target the proteins to their site of function. Here, we describe recent exciting evidence that some mRNAs are transported as particles along the cytoskeleton by the molecular motors dynein, kinesin or myosin. We discuss the key questions of how localized mRNAs might be linked to motors and what determines their cytoplasmic destinations.

Drosophila Gurken (TGFalpha) MRNA Localizes As Particles That Move Within the Oocyte in Two Dynein-dependent Steps

Developmental Cell. Mar, 2003  |  Pubmed ID: 12636913

In Drosophila oocytes, gurken mRNA localization orientates the TGF-alpha signal to establish the anteroposterior and dorsoventral axes. We have elucidated the path and mechanism of gurken mRNA localization by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. We present evidence suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations.

A Helicase That Gets Oskar's Message Across

Nature Cell Biology. Apr, 2004  |  Pubmed ID: 15057239

MRNA Localisation Gets More Complex

Current Opinion in Cell Biology. Jun, 2004  |  Pubmed ID: 15145355

Recent advances in techniques for visualising mRNA movement in living cells have led to rapid progress in understanding the mechanism of mRNA localisation in the cytoplasm. There is an emerging consensus that in many cases the mRNA signals that determine intracellular destination are more complex and difficult to define than was first anticipated. Furthermore, the transacting factors that interpret the mRNA signals are numerous and their combinations change during the life of an mRNA, perhaps allowing the selection of many sub-destinations in the cell. Lastly, an emerging theme over the past few years is that many proteins that determine the destinations of mRNAs are recruited on nascent transcripts in the nucleus. They often function in many different processes in the biogenesis of mRNA and probably act in concert to provide specificity.

Gurken and the I Factor Retrotransposon RNAs Share Common Localization Signals and Machinery

Developmental Cell. Jul, 2005  |  Pubmed ID: 15992540

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.

Dynein Anchors Its MRNA Cargo After Apical Transport in the Drosophila Blastoderm Embryo

Cell. Jul, 2005  |  Pubmed ID: 16009136

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.

Bruno: a Double Turn-off for Oskar

Developmental Cell. Mar, 2006  |  Pubmed ID: 16516832

In Drosophila, the posterior localization of oskar mRNA and its translational regulation are essential for axis specification and germline formation. Recently in Cell, demonstrated that Bruno inhibits cap-dependent translation of oskar mRNA and uncovered a novel Bruno-dependent assembly of oskar mRNA into multimeric RNP particles, which are inaccessible to the translational machinery. This work provides a novel link between mRNA localization, particle formation, and translational regulation.

A Dynein-dependent Shortcut Rapidly Delivers Axis Determination Transcripts into the Drosophila Oocyte

Development (Cambridge, England). May, 2007  |  Pubmed ID: 17442699

The primary axes of Drosophila are set up by the localization of transcripts within the oocyte. These mRNAs originate in the nurse cells, but how they move into the oocyte remains poorly understood. Here, we study the path and mechanism of movement of gurken RNA within the nurse cells and towards and through ring canals connecting them to the oocyte. gurken transcripts, but not control transcripts, recruit the cytoplasmic Dynein-associated co-factors Bicaudal D (BicD) and Egalitarian in the nurse cells. gurken RNA requires BicD and Dynein for its transport towards the ring canals, where it accumulates before moving into the oocyte. Our results suggest that bicoid and oskar transcripts are also delivered to the oocyte by the same mechanism, which is distinct from cytoplasmic flow. We propose that Dynein-mediated transport of specific RNAs along specialized networks of microtubules increases the efficiency of their delivery, over the flow of general cytoplasmic components, into the oocyte.

RNA Localization Signals: Deciphering the Message with Bioinformatics

Seminars in Cell & Developmental Biology. Apr, 2007  |  Pubmed ID: 17452113

mRNA localization is an important posttranscriptional method of targeting proteins to their site of function. The sorting of transcripts to their correct intracellular destination is achieved by a number of mechanisms, including selective degradation or transport by molecular motors along the cytoskeleton. In all cases, this involves mRNA localization signals, or so called zip codes, being recognized by trans-acting cellular factors. In a few cases, primary sequence motifs for RNA localization can be identified, but in general, localization signals operate at the level of secondary (2D) and tertiary (3D) structure. This inevitably means that searching for localization signal motifs is a complex task requiring specialist knowledge of bioinformatics. Furthermore, the publications describing these searching methods tend to be aimed at the bioinformatics community. In this review, we have surveyed the major tools for folding, comparing, and searching potential mRNA localization signals in transcripts or across genomes. Our aim is to provide an overview for biologists, who lack specialist computer and bioinformatics training, of the major RNA bioinformatics tools that are available. The examples provided are focused on mRNA localization signals and RNA stem-loop structures, however, these tools can be applied to the study of any RNA signals.

Imaging the Migrating Border Cell Cluster in Living Drosophila Egg Chambers

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Oct, 2007  |  Pubmed ID: 17849456

Cell migration is a key process in animal development and central to the spread of cancer. Border cell migration in Drosophila egg chambers is an excellent general model for cell migration, but lacks techniques for studying this process in living cells. Here, we describe a simple and effective method of preparing egg chambers in halocarbon oil. The movement and behavior of the migrating border cells can reproducibly be followed in up to 25 egg chambers simultaneously by time-lapse microscopy using a variety of green fluorescent protein markers on a widefield microscope over a period of 4 hr. Our studies reveal a remarkably linear migration route of the border cell cluster and highly dynamic activity within their cluster. Migrating cells rapidly alter their relative positions and generate transient protrusions. These activities are likely to play key roles in the mechanism of migration and cannot readily be analyzed using fixed samples.

Dynactin Suppresses the Retrograde Movement of Apically Localized MRNA in Drosophila Blastoderm Embryos

RNA (New York, N.Y.). Nov, 2007  |  Pubmed ID: 17901156

Motor dependent transport of mRNA is a key mechanism in axis specification during development. Apical transport and anchoring of wingless and pair-rule transcripts in the Drosophila syncytial blastoderm embryo is mediated by cytoplasmic Dynein, the major minus end directed microtubule dependent molecular motor. Here, we show that, despite apical transport of mRNA being highly directional, mRNA particles often pause and move backward toward the plus ends of microtubules. We suggest that this retrograde movement helps overcome cellular obstructions. We show that the plus end movement of apical mRNA is independent of the major plus end microtubule motors Kinesin-1 and Kinesin-2. In contrast, Dynactin, a Dynein processivity factor, is required to suppress retrograde mRNA movements, as well as for efficient minus end motility. We propose that Dynein itself, rather than the activity of a plus end motor, is responsible for the plus end movements of the mRNA and that Dynactin is involved in preventing short reverse movements of the Dynein motor, known to occur in vitro.

Drosophila Squid/hnRNP Helps Dynein Switch from a Gurken MRNA Transport Motor to an Ultrastructural Static Anchor in Sponge Bodies

Developmental Cell. Oct, 2007  |  Pubmed ID: 17925228

In Drosophila oocytes, dorso-anterior transport of gurken mRNA requires both the Dynein motor and the heterogeneous nuclear ribonucleoprotein (hnRNP) Squid. We show that gurken transcripts are transported directly on microtubules by Dynein in nonmembranous electron-dense transport particles that also contain Squid and the transport cofactors Egalitarian and Bicaudal-D. At its destination, gurken mRNA is statically anchored by Dynein within large electron-dense cytoplasmic structures known as sponge bodies. Egalitarian and Bicaudal-D contribute only to active transport, whereas Dynein and Squid are also required for gurken mRNA anchoring and the integrity of sponge bodies. Disrupting Dynein function disperses gurken mRNA homogeneously throughout the cytoplasm, whereas the loss of Squid function converts the sponge bodies into active transport particles. We propose that Dynein acts as a static structural component for the assembly of gurken mRNA transport and anchoring complexes, and that Squid is required for the dynamic conversion of transport particles to sponge bodies.

The Salvador-warts-hippo Pathway is Required for Epithelial Proliferation and Axis Specification in Drosophila

Current Biology : CB. Nov, 2007  |  Pubmed ID: 17964161

In Drosophila, the body axes are specified during oogenesis through interactions between the germline and the overlying somatic follicle cells [1-5]. A Gurken/TGF-alpha signal from the oocyte to the adjacent follicle cells assigns them a posterior identity [6, 7]. These posterior cells then signal back to the oocyte, thereby inducing the repolarization of the microtubule cytoskeleton, the migration of the oocyte nucleus, and the localization of the axis specifying mRNAs [8-10]. However, little is known about the signaling pathways within or from the follicle cells responsible for these patterning events. We show that the Salvador Warts Hippo (SWH) tumor-suppressor pathway is required in the follicle cells in order to induce their Gurken- and Notch-dependent differentiation and to limit their proliferation. The SWH pathway is also required in the follicle cells to induce axis specification in the oocyte, by inducing the migration of the oocyte nucleus, the reorganization of the cytoskeleton, and the localization of the mRNAs that specify the anterior-posterior and dorsal-ventral axes of the embryo. This work highlights a novel connection between cell proliferation, cell growth, and axis specification in egg chambers.

UAP56 RNA Helicase is Required for Axis Specification and Cytoplasmic MRNA Localization in Drosophila

Developmental Biology. Mar, 2008  |  Pubmed ID: 18237727

mRNA export from the nucleus requires the RNA helicase UAP56 and involves remodeling of ribonucleo-protein complexes in the nucleus. Here, we show that UAP56 is required for bulk mRNA export from the nurse cell nuclei that supply most of the material to the growing Drosophila oocyte and for the organization of chromatin in the oocyte nucleus. Loss of UAP56 function leads to patterning defects that identify uap56 as a spindle-class gene similar to the RNA helicase Vasa. UAP56 is required for the localization of gurken, bicoid and oskar mRNA as well as post-translational modification of Osk protein. By injecting grk RNA into the oocyte cytoplasm, we show that UAP56 plays a role in cytoplasmic mRNA localization. We propose that UAP56 has two independent functions in the remodeling of ribonucleo-protein complexes. The first is in the nucleus for mRNA export of most transcripts from the nucleus. The second is in the cytoplasm for remodeling the transacting factors that decorate mRNA and dictate its cytoplasmic destination.

Changes in Bicoid MRNA Anchoring Highlight Conserved Mechanisms During the Oocyte-to-embryo Transition

Current Biology : CB. Jul, 2008  |  Pubmed ID: 18639459

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.

In Vivo Imaging of Oskar MRNA Transport Reveals the Mechanism of Posterior Localization

Cell. Sep, 2008  |  Pubmed ID: 18775316

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.

A Bioinformatics Search Pipeline, RNA2DSearch, Identifies RNA Localization Elements in Drosophila Retrotransposons

RNA (New York, N.Y.). Feb, 2009  |  Pubmed ID: 19144907

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-alpha mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem-loop required for Dynein-dependent localization to the anterior and dorso-anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a "family" of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles.

Building a Scientific Data Grid with DiGS

Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences. Jun, 2009  |  Pubmed ID: 19451103

We provide an insight into the challenge of building and supporting a scientific data infrastructure with reference to our experience working with scientists from computational particle physics and molecular biology. We illustrate how, with modern high-performance computing resources, even small scientific groups can generate huge volumes (petabytes) of valuable scientific data and explain how grid technology can be used to manage, publish, share and curate these data. We describe the DiGS software application, which we have developed to meet the needs of smaller communities and we have highlighted the key elements of its functionality.

The 'super-resolution' Revolution

Biochemical Society Transactions. Oct, 2009  |  Pubmed ID: 19754448

We are currently in the midst of an exciting revolution in microscopy. In many ways, this has been happening for several decades, but it is the rate of development of new methods that has increased recently. The last few years have seen an impressive proliferation of new instruments for imaging at higher resolution, imaging single molecules and faster and more sensitive multidimensional live cell imaging. These include light sheet microscopy, stimulated emission depletion, structured illumination and live cell imaging on the OMX (optical microscopy experimental) platform. However, new probes and image analysis methods have also been crucial for the development of these revolutionary methods.

Transmitting the Message: Intracellular MRNA Localization

Current Opinion in Cell Biology. Feb, 2010  |  Pubmed ID: 20022233

The intracellular localization of mRNA is an important mechanism for targeting proteins to their sites of function. Localized transcripts play key roles ranging from body axis determination to synapse plasticity, and recent studies highlight how common localized transcripts are in a variety of model organisms. In this review, we consider the full range of known mechanisms for delivering transcripts and anchoring them at their site of function. Finally, we evaluate the progress that has been made in the nervous system in understanding the mechanism and function of mRNA localization and translation regulation.

Distinguishing Direct from Indirect Roles for Bicoid MRNA Localization Factors

Development (Cambridge, England). Jan, 2010  |  Pubmed ID: 20023172

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. Here we use live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. We show that Swallow and bicoid mRNA are transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, and we present evidence that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. Our results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead we propose a model for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport.

Cohesin Cleavage and Cdk Inhibition Trigger Formation of Daughter Nuclei

Nature Cell Biology. Feb, 2010  |  Pubmed ID: 20081838

The metaphase-anaphase transition is orchestrated through proteolysis of numerous proteins by a ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C). A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves cohesin, a ring-shaped complex that entraps sister DNAs. It is a matter of debate whether cohesin-independent forces also contribute to sister chromatid cohesion. Using 4D live-cell imaging of Drosophila melanogaster syncytial embryos blocked in metaphase (via APC/C inhibition), we show that artificial cohesin cleavage is sufficient to trigger chromosome disjunction. This is nevertheless insufficient for correct chromosome segregation. Kinetochore-microtubule attachments are rapidly destabilized by the loss of tension caused by cohesin cleavage in the presence of high Cdk1 (cyclin-dependent kinase 1) activity, as occurs when the APC/C cannot destroy mitotic cyclins. Metaphase chromosomes undergo a bona fide anaphase when cohesin cleavage is combined with Cdk1 inhibition. We conclude that only two key events, opening of cohesin rings and downregulation of Cdk1, are sufficient to drive proper segregation of chromosomes in anaphase.

Isolation of Drosophila Egg Chambers for Imaging

Cold Spring Harbor Protocols. Apr, 2010  |  Pubmed ID: 20360356

The fruit fly Drosophila melanogaster is an important model for basic research into the molecular mechanisms underlying cell function and development, as well as a major biomedical research tool. A significant advantage of Drosophila is the ability to apply live cell imaging to a variety of living tissues that can be dissected and imaged in vivo, ex vivo, or in vitro. Drosophila egg chambers, for example, have proven to be a useful model system for studying border cell migration, Golgi unit transport, the rapid movement of mRNA and protein particles, and the role of microtubules in meiosis and oocyte differentiation. A crucial first step before imaging is preparation of the experimental material to ensure physiological relevance and to achieve the best conditions for image quality. Early- to mid-stage egg chambers cannot be mounted in an aqueous-based medium, because this causes a change in microtubule organization and follicle cell morphology. Such egg chambers survive better in Halocarbon oil, which allows free diffusion of oxygen, has low viscosity, and thus prevents dehydration and hypoxia. With a refractive index similar to glycerol, Halocarbon oil also has good optical properties for imaging. It also provides a good environment for injection and is particularly useful for long-term imaging of embryos. However, unlike with aqueous solutions, changes in the medium are not possible. This protocol describes the isolation of Drosophila egg chambers.

Collection and Mounting of Drosophila Embryos for Imaging

Cold Spring Harbor Protocols. Apr, 2010  |  Pubmed ID: 20360357

The fruit fly Drosophila melanogaster is an important model for basic research into the molecular mechanisms underlying cell function and development, as well as a major biomedical research tool. A significant advantage of Drosophila is the ability to apply live cell imaging to a variety of living tissues that can be dissected and imaged in vivo, ex vivo, or in vitro. For example, such imaging can be used for visual genetic screens such as analysis of morphological characteristics or of the distribution of fluorescently tagged proteins in living embryos. Indeed, Drosophila embryos have proven to be a useful model system for studying a variety of cellular processes such as asymmetric division, migration, wound healing, apoptosis, and fasciculation, as well as for tracking lipid droplet motility, protein recycling, fast mRNA transport, and the movement of chromosomal loci within individual cells. A crucial first step before imaging is preparation of the experimental material to ensure physiological relevance and to achieve the best conditions for image quality. Because it contributes to autofluorescence and lack of transparency, it is useful to remove the chorion before imaging by treating the embryos with bleach. This protocol describes the collection and mounting of Drosophila embryos for live cell imaging.

Drosophila Macrophage Preparation and Screening

Cold Spring Harbor Protocols. Apr, 2010  |  Pubmed ID: 20360358

Drosophila plasmatocytes, also known as macrophages, are part of the Drosophila innate immune system and also have roles during development. In late-stage embryos, it is possible to image macrophage migration in situ during development and when they converge at sites of wounding. This protocol describes the isolation of macrophages from third instar Drosophila larvae. The macrophages can be cultured for several hours, and fluorescently labeled macrophages can be screened using a fluorescence-imaging system.

Drosophila Larval Fillet Preparation and Imaging of Neurons

Cold Spring Harbor Protocols. Apr, 2010  |  Pubmed ID: 20360359

Drosophila is an established system in which to study synaptic development, function, and plasticity. A particular advantage of the larval neuromuscular system is its consistent well-defined segmental arrangement of neurons and muscle targets. Indeed, the motor neurons of the Drosophila central nervous system are particularly well characterized in terms of origin, identity, morphology, and electrophysiology, and have been used for studies on axonal transport of organelles, vesicle trafficking, and recycling. To facilitate the identification of nerves and synapses in vivo, specific fluorescent protein markers can be used. For example, UASmCD8-green fluorescent protein (GFP) and UASmyr-red fluorescent protein (RFP) both preferentially label plasma membranes, whereas Discs large (DLG) reveals synapses. Gal4 drivers can be used to target all neurons (e.g., elavGal4) or specifically label motor neurons (e.g., d42Gal4). This protocol describes the dissection of Drosophila larvae to isolate neurons for live cell imaging.

Live Cell Imaging in Drosophila Melanogaster

Cold Spring Harbor Protocols. Apr, 2010  |  Pubmed ID: 20360379

Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila.

Making the Message Clear: Visualizing MRNA Localization

Trends in Cell Biology. Jul, 2010  |  Pubmed ID: 20444605

Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy. Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

ParticleStats: Open Source Software for the Analysis of Particle Motility and Cytoskeletal Polarity

Nucleic Acids Research. Jul, 2010  |  Pubmed ID: 20542911

The study of dynamic cellular processes in living cells is central to biology and is particularly powerful when the motility characteristics of individual objects within cells can be determined and analysed statistically. However, commercial programs only offer a limited range of inflexible analysis modules and there are currently no open source programs for extensive analysis of particle motility. Here, we describe ParticleStats (http://www.ParticleStats.com), a web server and open source programs, which input the X,Y coordinate positions of objects in time, and output novel analyses, graphical plots and statistics for motile objects. ParticleStats comprises three separate analysis programs. First, ParticleStats:Directionality for the global analysis of polarity, for example microtubule plus end growth in Drosophila oocytes. Second, ParticleStats:Compare for the analysis of saltatory movement in terms of runs and pauses. This can be applied to chromosome segregation and molecular motor-based movements. Thirdly ParticleStats:Kymographs for the analysis of kymograph images, for example as applied to separation of chromosomes in mitosis. These analyses have provided key insights into molecular mechanisms that are not possible from qualitative analysis alone and are widely applicable to many other cell biology problems.

An Adaptive Non-local Means Filter for Denoising Live-cell Images and Improving Particle Detection

Journal of Structural Biology. Dec, 2010  |  Pubmed ID: 20599512

Fluorescence imaging of dynamical processes in live cells often results in a low signal-to-noise ratio. We present a novel feature-preserving non-local means approach to denoise such images to improve feature recovery and particle detection. The commonly used non-local means filter is not optimal for noisy biological images containing small features of interest because image noise prevents accurate determination of the correct coefficients for averaging, leading to over-smoothing and other artifacts. Our adaptive method addresses this problem by constructing a particle feature probability image, which is based on Haar-like feature extraction. The particle probability image is then used to improve the estimation of the correct coefficients for averaging. We show that this filter achieves higher peak signal-to-noise ratio in denoised images and has a greater capability in identifying weak particles when applied to synthetic data. We have applied this approach to live-cell images resulting in enhanced detection of end-binding-protein 1 foci on dynamically extending microtubules in photo-sensitive Drosophila tissues. We show that our feature-preserving non-local means filter can reduce the threshold of imaging conditions required to obtain meaningful data.

How the Sea Squirt Nucleus Tells Mesoderm Not to Be Endoderm

Developmental Cell. Oct, 2010  |  Pubmed ID: 20951340

Sea squirts are simple invertebrate chordates. In this issue of Developmental Cell, Takatori et al. show nuclear migration within ascidian mesendodermal cells enables polarized localization of Not mRNA, which encodes a homeobox protein that distinguishes mesoderm from endoderm fates. The link between nuclear migration and mRNA localization suggests exciting parallels with protostomes.

Nanometric Depth Resolution from Multi-focal Images in Microscopy

Journal of the Royal Society, Interface / the Royal Society. Jul, 2011  |  Pubmed ID: 21247948

We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

Identifying and Searching for Conserved RNA Localisation Signals

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21431757

RNA localisation is an important mode of delivering proteins to their site of function. Cis-acting signals within the RNAs, which can be thought of as zip-codes, determine the site of localisation. There are few examples of fully characterised RNA signals, but the signals are thought to be defined through a combination of primary, secondary, and tertiary structures. In this chapter, we describe a selection of computational methods for predicting RNA secondary structure, identifying localisation signals, and searching for similar localisation signals on a genome-wide scale. The chapter is aimed at the biologist rather than presenting the details of each of the individual methods.

A PAR-1-dependent Orientation Gradient of Dynamic Microtubules Directs Posterior Cargo Transport in the Drosophila Oocyte

The Journal of Cell Biology. Jul, 2011  |  Pubmed ID: 21746854

Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. We found that all MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. Our findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior.

OMX: a New Platform for Multimodal, Multichannel Wide-field Imaging

Cold Spring Harbor Protocols. Aug, 2011  |  Pubmed ID: 21807861

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-resolution Microscopy

PLoS Biology. Sep, 2011  |  Pubmed ID: 21931537

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

A Cell Biologist's Guide to High Resolution Imaging

Methods in Enzymology. 2012  |  Pubmed ID: 22264528

Fluorescence microscopy is particularly well suited to the study of cell biology, due to its noninvasive nature, high sensitivity detection of specific molecules, and high spatial and temporal resolution. In recent years, there has been an important transition from imaging the static distributions of molecules as a snapshot in time in fixed material to live-cell imaging of the dynamics of molecules in cells: in essence visualizing biochemical processes in living cells. Furthermore, in the last 5years, there have been important advances in so-called "super-resolution" imaging methods that have overcome the resolution limits imposed by the diffraction of light in optical systems. Live-cell imaging is now beginning to deliver in unprecedented detail, bridging the resolution gap between electron microscopy and light microscopy. We discuss the various factors that limit the spatial and temporal resolution of microscopy and how to overcome them, how to best prepare specimens for high resolution imaging, and the choice of fluorochromes. We also summarize the pros and cons of the different super-resolution techniques and introduce some of the key data analysis tasks that a cell biologist employing high resolution microscopy is typically interested in.

RNA:RNA Interaction Can Enhance RNA Localization in Drosophila Oocytes

RNA (New York, N.Y.). Feb, 2012  |  Pubmed ID: 22345148

RNA localization is a key mechanism for targeting proteins to particular subcellular domains. Sequences necessary and sufficient for localization have been identified, but little is known about factors that affect its kinetics. Transcripts of gurken and the I factor, a non-LTR retrotransposon, colocalize at the nucleus in the dorso-antero corner of the Drosophila oocyte directed by localization signals, the GLS and ILS. I factor RNA localizes faster than gurken after injection into oocytes, due to a difference in the intrinsic localization ability of the GLS and ILS. The kinetics of localization of RNA containing the ILS are enhanced by the presence of a stem-loop, the A loop. This acts as an RNA:RNA interaction element in vivo and in vitro, and stimulates localization of RNA containing other localization signals. RNA:RNA interaction may be a general mechanism for modulating RNA localization and could allow an mRNA that lacks a localization signal to hitchhike on another RNA that has one.