A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal

The protein kinase D (PKD) family comprises multifunctional serine/threonine-specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral-targeted transport carrier fission from the trans -Golgi network (TGN). To visualize site-specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi-specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD-specific pharmacological inhibitors and siRNA-mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole-induced Golgi complex break-up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant-negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.     

Mutant p53 promotes RCP-dependent chemoresistance coinciding with increased delivery of P-glycoprotein to the plasma membrane

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.Subject terms: Tumour-suppressor proteins, Preclinical research     

Rapid induction of immune density-dependent prophylaxis in adult social insects

The innate immune system provides defence against parasites and pathogens. This defence comes at a cost, suggesting that immune function should exhibit plasticity in response to variation in environmental threats. Density-dependent prophylaxis (DDP) has been demonstrated mostly in phase-polyphenic insects, where larval group size determines levels of immune function in either adults or later larval instars. Social insects exhibit extreme sociality, but DDP has been suggested to be absent from these ecologically dominant taxa. Here we show that adult bumble-bee workers (Bombus terrestris) exhibit rapid plasticity in their immune function in response to social context. These results suggest that DDP does not depend upon larval conditions, and is likely to be a widespread and labile response to rapidly changing conditions in adult insect populations. This has obvious ramifications for experimental analysis of immune function in insects, and serious implications for our understanding of the epidemiology and impact of pathogens and parasites in spatially structured adult insect populations.     

Examination of the success rate of secondary symbiont manipulation by microinjection methods in the pea aphid system

Insects harbor a wide range of microbial symbionts, but their influence on host phenotypes is described in a limited number of biological models. One experimental approach to gain knowledge on the effects of symbionts to their hosts is to create insect lines with and without symbionts and examine their phenotypes. However, the success rate of symbiont elimination and introduction methods is dependent on several parameters that are scarcely tested or described. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), is a model insect of symbiosis studies. It harbors a primary symbiont that supplies the host with essential amino acids, and an array of secondary symbionts whose effects have been assessed by manipulating their presence/absence in the insect. Here, we describe the influence of key parameters on the success rate of symbiont manipulation using the pea aphid–secondary symbiont system. We compared two elimination methods differing in antibiotic treatment using several aphid–symbiont combinations. We also created new aphid host–symbiont combinations by secondary symbiont introduction and examined the effects of larval stage of recipient aphids on introduction success. Our study revealed that the aphid–symbiont combination has strong influence on both symbiont introduction and elimination success rates, and that the type of antibiotics and the larval stage of recipient aphids influence the elimination and introduction success rate, respectively.     

Tissue‐type plasminogen activator induces plasmin‐dependent proteolysis of intracellular neuronal nitric oxide synthase

Background information. Despite its pro‐fibrinolytic activity, tPA (tissue plasminogen activator) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through NMDA (N‐methyl‐d ‐aspartate) receptors, LRP (low‐density lipoprotein receptor‐related protein) or annexin II. Results. We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase, thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of NMDA receptors, LRP signalling and calpain‐mediated proteolysis, it is dependent on the ability of tPA to promote the conversion of plasminogen into plasmin. Conclusion. Altogether, these results demonstrate a new function for tPA in the central nervous system, which most likely contributes to its pleiotropic functions.     

Populations,not clones,are the unit of vibrio pathogenesis in naturally infected oysters

Disease in oysters has been steadily rising over the past decade, threatening the long-term survival of commercial and natural stocks. Our understanding and management of such diseases are of critical importance as aquaculture is an important aspect of dealing with the approaching worldwide food shortage. Although some bacteria of the Vibrio genus isolated from diseased oysters have been demonstrated to be pathogenic by experimental infection, direct causality has not been established. Little is known about the dynamics of how the bacterial population hosted by oysters changes during disease progression. Combining experimental ecology, a high-throughput infection assay and genome sequencing, we show that the onset of disease in oysters is associated with progressive replacement of diverse benign colonizers by members of a phylogenetically coherent virulent population. Although the virulent population is genetically diverse, all members of that population can cause disease. Comparative genomics across virulent and nonvirulent populations identified candidate virulence factors that were clustered in population-specific genomic regions. Genetic analyses revealed that one gene for a candidate virulent factor, a putative outer membrane protein, is necessary for infection of oysters. Finally, analyses of oyster mortality following experimental infection suggest that disease onset can be facilitated by the presence of nonvirulent strains. This is a new form of polymicrobial disease, in which nonpathogenic strains contribute to increase mortality.     

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low ¹³C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.     

Storage of Carotenoids in Crustaceans as an Adaptation to Modulate Immunopathology and Optimize Immunological and Life‐History Strategies

Why do some invertebrates store so much carotenoids in their tissues? Storage of carotenoids may not simply be passive and dependent on their environmental availability, as storage variation exists at various taxonomic scales, including among individuals within species. While the strong antioxidant and sometimes immune‐stimulating properties of carotenoids may be beneficial enough to cause the evolution of features improving their assimilation and storage, they may also have fitness downsides explaining why massive carotenoid storage is not universal. Here, the functional and ecological implications of carotenoid storage for the evolution of invertebrate innate immune defenses are examined, especially in crustaceans, which massively store carotenoids for unclear reasons. Three testable hypotheses about the role of carotenoid storage in immunological (resistance and tolerance) and life‐history strategies (with a focus on aging) are proposed, which may ultimately explain the storage of large amounts of these pigments in a context of host–pathogen interactions.