DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages.

Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with Mycobacterium marinum in murine RAW264.7 macrophages. We found that, shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for the recruitment of LC3 and for the acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to defend against mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages.

Two Types of Liposomal Formulations Improve the Therapeutic Ratio of Prednisolone Phosphate in a Zebrafish Model for Inflammation.

Glucocorticoids (GCs) are effective anti-inflammatory drugs, but their clinical use is limited by their side effects. Using liposomes to target GCs to inflammatory sites is a promising approach to improve their therapeutic ratio. We used zebrafish embryos to visualize the biodistribution of liposomes and to determine the anti-inflammatory and adverse effects of the GC prednisolone phosphate (PLP) encapsulated in these liposomes. Our results showed that PEGylated liposomes remained in circulation for long periods of time, whereas a novel type of liposomes (which we named AmbiMACs) selectively targeted macrophages. Upon laser wounding of the tail, both types of liposomes were shown to accumulate near the wounding site. Encapsulation of PLP in the PEGylated liposomes and AmbiMACs increased its potency to inhibit the inflammatory response. However, encapsulation of PLP in either type of liposome reduced its inhibitory effect on tissue regeneration, and encapsulation in PEGylated liposomes attenuated the activation of glucocorticoid-responsive gene expression throughout the body. Thus, by exploiting the unique possibilities of the zebrafish animal model to study the biodistribution as well as the anti-inflammatory and adverse effects of liposomal formulations of PLP, we showed that PEGylated liposomes and AmbiMACs increase the therapeutic ratio of this GC drug.

Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue.

Developments in single-molecule microscopy (SMM) have enabled imaging individual proteins in biological systems, focusing on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied dynamics of the membrane protein H-Ras, its membrane-anchoring domain, C10H-Ras, and mutants, using total internal reflection fluorescence microscopy. Our results consistently confirm the presence of fast- and slow-diffusing subpopulations of molecules, which confine to microdomains within the plasma membrane. The active mutant H-RasV12 exhibits higher diffusion rates and is confined to larger domains than the wild-type H-Ras and its inactive mutant H-RasN17. Subsequently, we demonstrate that the structure and composition of the plasma membrane have an imperative role in modulating H-Ras mobility patterns. Ultimately, we establish that differences between cells within the same embryo largely contribute to the overall data variability. Our findings agree with a model in which the cell architecture and the protein activation state determine protein mobility, underlining the importance of SMM imaging for studying factors influencing protein dynamics in an intact living organism. This article has an associated First Person interview with the first author of the paper.

Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

Polystyrene nanoplastics disrupt glucose metabolism and cortisol levels with a possible link to behavioural changes in larval zebrafish.

Plastic nanoparticles originating from weathering plastic waste are emerging contaminants in aquatic environments, with unknown modes of action in aquatic organisms. Recent studies suggest that internalised nanoplastics may disrupt processes related to energy metabolism. Such disruption can be crucial for organisms during development and may ultimately lead to changes in behaviour. Here, we investigated the link between polystyrene nanoplastic (PSNP)-induced signalling events and behavioural changes. Larval zebrafish exhibited PSNP accumulation in the pancreas, which coincided with a decreased glucose level. By using hyperglycemic and glucocorticoid receptor (Gr) mutant larvae, we demonstrate that the PSNP-induced disruption in glucose homoeostasis coincided with increased cortisol secretion and hyperactivity in challenge phases. Our work sheds new light on a potential mechanism underlying nanoplastics toxicity in fish, suggesting that the adverse effect of PSNPs are at least in part mediated by Gr activation in response to disrupted glucose homeostasis, ultimately leading to aberrant locomotor activity.