Richard M. Parton

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

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

A PAR-1–dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

A PAR-1–mediated bias in microtubule organization in the Drosophila oocyte underlies posterior-directed mRNA transport.

Making the message clear: visualizing mRNA localization.

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.

Drosophila patterning is established by differential association of mRNAs with P bodies

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization. In Drosophila melanogaster, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA in the oocyte and embryo. Both transcripts are translationally silent while being localized within the oocyte along microtubules by cytoplasmic dynein. Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF- α signal to the overlying somatic cells. In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos when it forms an anteroposterior morphogenetic gradient. How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live-cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron-dense bodies that lack ribosomes and contain translational repressors. These properties are characteristic of processing bodies (P bodies), which are considered to be regions of cytoplasm where decisions are made on the translation and degradation of mRNA. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Oo18 RNA-binding protein (Orb, a homologue of CEPB) and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodelling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

The 5' cap of tobacco mosaic virus (TMV) is required for virion attachment to the actin/endoplasmic reticulum network during early infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)‐labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3‐virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co‐injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell‐to‐cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3‐vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin‐dependent RNA movement. The 5′ methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5′ cap failed to form granules and was degraded in the cytoplasm. Removal of the 3′ untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual‐labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER‐bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.

Distinguishing direct from indirect roles for bicoid mRNA localization factors.

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.

An adaptive non-local means filter for denoising live-cell images and improving particle detection

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.

OMX: A New Platform for Multimodal, Multichannel Wide-Field Imaging

This article discusses the design principles and applications of the OMX microscope, a new platform that provides unprecedented mechanical and thermal stabilities coupled with a photon budget that is dramatically improved over traditional microscope platforms. These characteristics make the OMX microscope outstanding for fast live cell imaging and super-resolution imaging. Moreover, its open flexible architecture makes it particularly amenable to adding other modes of microscopy to the platform.

Subcellular mRNA localisation at a glance

ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms.

A single and rapid calcium wave at egg activation in Drosophila

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.