Super-resolution optical imaging reveals the accumulation of SARS-CoV-2 nucleocapsid protein at viral replication organelles.

Extrinsic cues trigger the local translation of specific mRNAs in growing axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors associate with distinct sets of mRNAs and RNA-binding proteins. Cue stimulation of growing Xenopus retinal ganglion cell axons induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by co-exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and suggest a generalizable model for cue-selective control of the local proteome.

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.

19During neuronal wiring, extrinsic cues trigger the local translation of specific mRNAs in axons 20 via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a 21 mechanism linking signals to local translation but it is not known how broadly this mechanism 22 operates, nor whether it can selectively regulate mRNA translation. We report that receptor-23 ribosome coupling is employed by multiple guidance cue receptors and this interaction is 24 mRNA-dependent. We find that different receptors bind to distinct sets of mRNAs and RNA-25 binding proteins. Cue stimulation induces rapid dissociation of ribosomes from receptors and 26 the selective translation of receptor-specific mRNAs in retinal axon growth cones. Further, 27we show that receptor-ribosome dissociation and cue-induced selective translation are 28 inhibited by simultaneous exposure to translation-repressive cues, suggesting a novel mode 29 of signal integration. Our findings reveal receptor-specific interactomes and provide a general 30 model for the rapid, localized and selective control of cue-induced translation. 31 Lin and Holt, 2008). Unbiased detection of newly synthesized proteins in the axon 47 compartment has revealed further complexity showing that different guidance cues stimulate 48 the regulation of distinct signature sets of >100 axonal nascent proteins within just 5 mins, 49 many of which are not cytoskeletal-related , Yao et al., 2006, Wu et al., 50 2005. However, the mechanisms underlying the 51 localization and selectivity of translation are unclear. Several mechanisms are known to 52 control different aspects of axonal translation, including microRNA regulation (Bellon et al., 53 2017), mRNA modification (Yu et al., 2018), modulation of the phosphorylation of eukaryotic 54 initiation factors (Cagnetta et al., 2019), RNA-binding protein (RBP) phosphorylation (Sasaki 55 et al., 2010, Lepelletier et al., 2017, Huttelmaier et al., 2005) and receptor-ribosome coupling 56 (Tcherkezian et al. , 2010). The latter is a particularly direct and attractive mechanism to link 57 cue-specific signalling to differential mRNA translation. However, this mechanism has been 58 shown only for the Netrin-1 receptor, deleted in colorectal cancer (DCC), in commissural 59 axon growth cones and HEK293 cells (Tcherkezian et al., 2010) and it is unknown whether 60 receptor-ribosome coupling is a widespread mechanism used by different receptors and in 61 different cell types, and whether it regulates selective local translation. 62 63 Here, we show in retinal ganglion cell (RGC) axon growth cones that receptor-ribosome 64 coupling is used by several different axon guidance receptors (DCC, Neuropilin-1 and 65 Robo2), indicative of a common mechanism. Upon stimulation by specific cues, ribosomes 66 dissociate from their receptors within 2 minutes. Interestingly, the receptor-ribosome 67 interaction is mRNA-dependent, and immunoprecipitation (IP) reveals that distinct receptors 68 associate with specific RNA-binding proteins (RBPs) an...

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.