Y.S. Ng

Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity

The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

Modulation of the organelle specificity in Re(I) tetrazolato complexes leads to labeling of lipid droplets

The biological behaviour in terms of cellular incubation and organelle specificity for two complexes of the type fac-[Re(CO)3(phen)L], where phen is 1,10-phenanthroline and L is either 3-pyridyltetrazolate or 4-cyanophenyltetrazolate, are herein investigated. The emission signal detected from the live insect Drosophila and human cell lines, generated by exploiting two-photon excitation at 830 nm to reduce cellular damage and autofluorescence, suggests photophysical properties that are analogous to those measured from dilute solutions, meaning that the complexes remain intact within the cellular environment. Moreover, the rhenium complex linked to 4-cyanophenyltetrazolate shows high specificity for the lipid droplets, whereas the complex bound to 3-pyridyltetrazolate tends to localise within the lysosomes. This differential localisation implies that in these complexes, organelle specificity can be achieved and manipulated by simple functional group transformations thus avoiding more complex bioconjugation strategies. More importantly, these results highlight the first example of phosphorescent labeling of the lipid droplets, whose important cellular functions have been recently highlighted along with the fact that their role in the metabolism of healthy and diseased cells has not been fully elucidated.

The role of osteocyte apoptosis in cancer chemotherapy-induced bone loss

Intensive cancer chemotherapy leads to significant bone loss, the underlying mechanism of which remains unclear. The objective of this study was to elucidate mechanisms for effect of the commonly used anti‐metabolite methotrexate (MTX) on osteocytes and on general bone homeostasis. The current study in juvenile rats showed that MTX chemotherapy caused a 4.3‐fold increase in the number of apoptotic osteocytes in tibial metaphysis, which was accompanied by a 1.8‐fold increase in the number of tartrate‐resistant acid phosphatase‐positive bone resorbing osteoclasts, and a 35% loss of trabecular bone. This was associated with an increase in transcription of the osteoclastogenic cytokines IL‐6 (10‐fold) and IL‐11 (2‐fold). Moreover, the metaphyseal bone of MTX‐treated animals exhibited a 37.6% increase in the total number of osteocytes, along with 4.9‐fold higher expression of the DMP‐1 transcript. In cultured osteocyte‐like MLO‐Y4 cells, MTX treatment significantly increased caspase‐3‐mediated apoptosis, which was accompanied by the formation of plasma membrane‐born apoptotic bodies and an increase in IL‐6 (24‐fold) and IL‐11 (29‐fold) mRNA expression. Conditioned media derived from MTX‐treated MLO‐Y4 cells was twice as strong as untreated media in its capacity to induce osteoclast formation in primary bone marrow osteoclast precursors. Thus, our in vivo and in vitro data suggested that MTX‐induced apoptosis of osteocytes caused higher recruitment of DMP‐1 positive osteocytes and increased osteoclast formation, which could contribute towards the loss of bone homeostasis in vivo. J. Cell. Physiol. 227: 2889–2897, 2012.

Atg9 is required for intraluminal vesicles in amphisomes and autolysosomes

ABSTRACT Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and Lamp1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to amphisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/Lamp-1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within amphisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development. Summary: The disappearance of intraluminal vesicles in amphisomes/autolysosomes upon Atg9 depletion suggests that Atg9 has a specific role in intraluminal vesicle formation in autophagic compartments.

At the Intersection of the Pathways for Exocytosis and Autophagy

Recent studies have suggested that there are molecular links between the two critical biological processes of exocytosis and autophagy. Exocytosis involves the transport of intracellular vesicles to the plasma membrane of the cell, where vesicular fusion results in the delivery of membrane and protein to the cell surface, and secretion of the vesicular contents. Exocytosis is utilized in, for example, hormone or antimicrobial peptide secretion, the delivery of proteoglycans to the cell surface, cell-cell communication and neurotransmission (Brennwald & Rossi, 2007; He & Guo, 2009). Autophagy is a mechanism for the recycling and degradation of cytoplasmic content, which involves surrounding an area of cytoplasm with a double membrane structure, which then interacts with degradative endosome-lysosome compartments (He & Klionsky, 2009). Autophagy has important functions in a range of cell processes including the maintenance of cellular homeostasis, starvation adaption, energy balance, organelle clearance, immunity and cell death. In human diseases, such as cancers, neurodegenerative disorders (e.g. Huntington’s disease), and chronic inflammatory diseases (e.g. Crohn’s disease), there have been reports of functional disparity in both of these important membrane-related cellular pathways. There is now increasing evidence that exocytosis and autophagy share molecular machinery and there are a number of reasons why this would be beneficial in terms of cellular function.

Molecular Machinery Regulating Exocytosis

Exocytosis is the major intracellular route for the delivery of proteins and lipids to the plasma membrane and the means by which vesicular contents are released into the extracellular space. The anterograde trafficking of vesicles to the plasma membrane is vital for membrane expansion during cell division; cell growth and migration; the delivery of specialised molecules to establish cell polarity; cell-to-cell communication; neurotransmission and the secretion of response factors such as hormones, cytokines and antimicrobial peptides. There are two major trafficking routes in eukaryotic cells, which are referred to as constitutive and regulated (Ory & Gasman, 2011). Constitutive exocytosis involves the steady state delivery of secretory carrier vesicles from the endoplasmic reticulum via the Golgi apparatus to the plasma membrane (Lacy & Stow, 2011). Regulated or granule-mediated exocytosis involves a specific trigger, usually a burst of intracellular calcium following an extrinsic stimulus. This system is utilized for secretion in neuronal cells and other specialist secretory cells, such as neuroendocrine, endocrine and exocrine cells (Burgoyne & Morgan, 2003; Jolly & Sattentau, 2007; Lacy & Stow, 2011). Regulated exocytosis enables a rapid response from a subpopulation of vesicles already primed and competent for fusion (Manjithaya & Subramani, 2011; Nickel & Seedorf, 2008; Nickel, 2010). Regulated exocytosis is also used for polarised traffic of vesicular membrane and cargo to specific spatial landmarks and this is particularly important during times of dramatic change in cell morphology, such as cell division, cell motility, phagocytosis and axonal outgrowth.

Bacterial challenge initiates endosome-lysosome response in Drosophila immune tissues

An effective innate immune response is critical for the protection of an organism against pathogen and environmental challenge. There is emerging evidence that an effective immune response depends heavily on the traffic and function of endosomes and lysosomes. However, there is very little understanding of the dynamics of an innate immune response, especially in vivo. Toward this aim, we have used two-photon microscopy to visualize the response to bacterial infection of the endosome-lysosome system in immune response tissues using intact Drosophila larvae. First, we set up the conditions to image intact larva in vivo and more specifically GFP-labeled endosomes-lysosomes in the fat body, and compared their distribution and size with those in tissue explanted ex vivo. Notably, we observed significant expansion of both Rab5 and Rab7 endosomal compartments upon both tissue isolation and minor aseptic wounding, indicating significant differences between live and explanted tissue. We also observed changes in endosome-lysosome vesicles within internal immune response tissues following in vivo bacterial infection by the oral route (to avoid a wounding response). We conclude that there are significant changes to the architecture of endosomes and lysosomes during an innate immune response, setting the scene for mechanistic studies to identify the signaling pathways that orchestrate this process.

Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role for 14–3–3ε in Metabolic Pathways

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.