Alexandra Sorvina

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.

Investigating Intracellular Localisation and Cytotoxicity Trends for Neutral and Cationic Iridium Tetrazolato Complexes in Live Cells

A family of five neutral cyclometalated iridium(III) tetrazolato complexes and their methylated cationic analogues have been synthesised and characterised. The complexes are distinguished by variations of the substituents or degree of π conjugation on either the phenylpyridine or tetrazolato ligands. The photophysical properties of these species have been evaluated in organic and aqueous media, revealing predominantly a solvatochromic emission originating from mixed metal-to-ligand and ligand-to-ligand charge transfer excited states of triplet multiplicity. These emissions are characterised by typically long excited-state lifetimes (∼hundreds of ns), and quantum yields around 5-10 % in aqueous media. Methylation of the complexes caused a systematic red-shift of the emission profiles. The behaviour and the effects of the different complexes were then examined in cells. The neutral species localised mostly in the endoplasmic reticulum and lipid droplets, whereas the majority of the cationic complexes localised in the mitochondria. The amount of complexes found within cells does not depend on lipophilicity, which potentially suggests diverse uptake mechanisms. Methylated analogues were found to be more cytotoxic compared to the neutral species, a behaviour that might to be linked to a combination of uptake and intracellular localisation.

Mitochondrial imaging in live or fixed tissues using a luminescent iridium complex

Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed samples. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir(ppy)2(MeTzPyPhCN)]+; where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand; and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue samples. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.

Imaging nuclear, endoplasmic reticulum and plasma membrane events in real time.

Live cell imaging can provide important information on cellular dynamics; however, the full utilisation of this technology has been hampered by the limitations of imaging reagents. Metal‐based complexes have the potential to overcome many of the issues common to many current imaging agents. The rhenium (I)‐based complex fac‐[Re(CO)3(1,10‐phenanthroline)(4‐pyridyltetrazolate)], herein referred to as ReZolve‐ER™, shows promise as a live cell imaging agent with rapid cell uptake, low cytotoxicity, resistance to photobleaching and compatibility with multicolour imaging. ReZolve‐ER™ localised to the nuclear membrane/endoplasmic reticulum (ER) and allowed the detection of exocytotic events at the plasma membrane. Thus, we present a new imaging agent for monitoring live cell events in real time, which is ideal for imaging either short‐ or long‐time courses.

A Drosophila model to image phagosome maturation.

Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3ε mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo.

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.

Drosophila Pkaap regulates Rab4/Rab11-dependent traffic and Rab11 exocytosis of innate immune cargo

ABSTRACT The secretion of immune-mediators is a critical step in the host innate immune response to pathogen invasion, and Rab GTPases have an important role in the regulation of this process. Rab4/Rab11 recycling endosomes are involved in the sorting of immune-mediators into specialist Rab11 vesicles that can traffic this cargo to the plasma membrane; however, how this sequential delivery process is regulated has yet to be fully defined. Here, we report that Drosophila Pkaap, an orthologue of the human dual-specific A-kinase-anchoring protein 2 or D-AKAP2 (also called AKAP10), appeared to have a nucleotide-dependent localisation to Rab4 and Rab11 endosomes. RNAi silencing of pkaap altered Rab4/Rab11 recycling endosome morphology, suggesting that Pkaap functions in cargo sorting and delivery in the secretory pathway. The depletion of pkaap also had a direct effect on Rab11 vesicle exocytosis and the secretion of the antimicrobial peptide Drosomycin at the plasma membrane. We propose that Pkaap has a dual role in antimicrobial peptide traffic and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens. Summary: Pkaap has a dual role in antimicrobial peptide trafficking and exocytosis, making it an essential component for the secretion of inflammatory mediators and the defence of the host against pathogens.

Label‐free imaging of healthy and infarcted fetal sheep hearts by two‐photon microscopy

Coronary heart disease is one of the largest causes of death worldwide, making this a significant health care issue. A critical problem for the adult human heart is that it does not undergo effective repair in response to damage, leaving patients with a poor prognosis. Unlike the adult, fetal hearts have the ability to repair after myocardial damage. Using two-photon microscopy, we have visualised the morphological and metabolic changes following myocardial infarction in sheep fetuses, to characterise response to cardiac injury in a mammalian model. Following myocardial infarction, fetal hearts showed no significant increase in collagen deposition in the region of the infarction, when compared to either the surrounding tissue or shams. In contrast, metabolic activity (i. e. NAD(P)H and FAD) was significantly reduced in the region of myocardial infarction, when compared to either the surrounding tissue or sham hearts. For comparison, we also imaged two hearts from preadolescent sheep (sham and myocardial infarction) and showed highly ordered collagen deposition with decreased metabolic activity within the infarcted area. Therefore, two-photon imaging had the capacity to image both morphological and metabolic changes in response to myocardial infarction and showed differences in the response with age. Picture: Two-photon imaging of myocardial infarction (b and d) enabled the visualisation of increased collagen (blue; Em=431 nm) and changes in other tissue autofluorescence (green; Em=489-606 nm) in fetal (a and b) and preadolescent (c and d) hearts, compared to shams (a and c). The excitation wavelength was 840 nm. Scale bars: 10 μm.

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.

A fibre optic fluorescence sensor to measure redox level in tissues

We report the design of a fibre optic-based redox detection system for investigating differences in metabolic activities of tissues. Our system shows qualitative agreement with the results collected from a commercial two- photon microscope system. Thus, demonstrating the feasibility of building an ex vivo and in vivo redox detection system that is low cost and portable.