SummaryConfocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

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

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization 1 . In Drosophila, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA (mRNA) in the oocyte and embryo 1 . bcd and grk mRNA are both translationally silent while being localized within the oocyte along microtubules by cytoplasmic Dynein 1-4 . Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF-alpha signal to the overlying somatic cells 5 . In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos to form an anteroposterior morphogenetic gradient 6 . 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, characteristic of Processing bodies (P bodies), which are regions of cytoplasm where translational decisions are made. 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 Orb/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 remodeling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

Summary Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a wide range of vertebrates and invertebrates. In Drosophila, bicoid (bcd) mRNA is pre-localized at the anterior of the oocyte. After fertilization, this RNA is translated to produce a Bcd protein gradient that determines anterior cell fates [1, 2]. Recent analysis of bcd mRNA during late stages of oogenesis led to a model for steady-state localization of bcd by continual active transport [3]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4, 5] or retention of bcd at the anterior in mature oocytes, which can remain dormant, but developmentally competent, for weeks prior to fertilization [6]. Through temporal analysis of bcd RNA particle dynamics, we show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis, ensuring the developmental integrity of the oocyte during dormancy. Egg activation triggers release of bcd from the anterior cortex for proper deployment in the fertilized egg, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results highlight a conserved mechanism used by invertebrates and vertebrates to regulate mRNA anchoring and redeployment during the oocyte-to-embryo transition.