REPORT


imaging) should be used to verify observations obtained using flow cytometry.


AUTOPHAGY IN HOST-PATHOGEN INTERACTIONS


Salmonella, pictured here stained by a direct fluorescent antibody technique, are detected by NDP52 after they enter the cytosol of human cells.


above, the plasma membrane contributes membrane to autophagosome precursors. This may be mediated by ARF6-dependent generation of PIP2, which influences endocytic uptake of plasma membrane into autophagosome precursors.


LIVE CELL IMAGING


Recent advances in live cell imaging allow researchers to follow the contribution of individual ATG proteins to autophagosome formation in real time. Dr Eleftherios Karanasios has recently contributed to our understanding of the sequence of steps leading to autophagosome formation. During his presentation, entitled ‘Dynamic Association of the ULK1 Complex with Omegasomes During Autophagy Induction’, Dr Karanasios revealed that the ULK1 protein complex (which includes ULK1, ATG13, ATG101 and VPS34) associates with omegasomes (membranes connected to the ER that can serve as a platform for autophagosome biogenesis). Using live imaging, Dr Karanasios showed that upon starvation, ULK1 complex forms puncta associated with the ER. If PI3P (synthesised by the VPS34 kinase) is available, these puncta become omegasomes. Then the autophagosomes are formed following ULK1 complex exit from omegasomes. The autophagy protein ATG13 was also shown to co-localise to omegasomes following starvation and play a part in the overall process.


The model for autophagosome formation (via an omegasome intermediate) was suggested; active ULK1 complex locates to ER-associated omegasomes and contributes to VPS34-mediated PI3P synthesis for the nucleation of omegasomes. Continuous synthesis of PI3P drives the expansion of the omegasomes, followed by the lipidation of LC3. This induces the expansion of the


DECEMBER 2013


isolated membrane that eventually buds off the omegasome and eventually forms mature autophagosomes.


AUTOPHAGY MONITORING Although live cell imaging is a very powerful way of visualising the autophagy process, it is time-consuming and has some practical limitations. Therefore, researchers in the field have been tempted to develop methods that can monitor autophagy in a fast but robust way. Dr Gary Warnes presented encouraging data regarding the use of flow cytometry to measure organelle autophagy, a process whereby specific cellular organelles, such as mitochondria (mitophagy) or ER (ER-phagy), are degraded via autophagy. Employing a range of autophagy inducing/inhibiting agents (eg rapamycin, starvation and chloroquine), mitophagy and ER-phagy were analysed by flow cytometry using MitoTracker Green and ER Tracker Green as markers for mitochondria and ER, respectively. This could be correlated with monitoring of autophagosome numbers using LC3-specific antibodies, indicating that flow cytometry measurement of mitophagy and ER-phagy could provide a fast, quantitative method to investigate these processes. However, it was also emphasised that care should be taken in interpretation of data obtained by flow cytometry, and other methods (eg Western blotting and cell


‘Autophagy is induced following viral infection of mammalian cells and is a barrier to efficient delivery of gene therapy vectors’


Cells can use autophagy to protect their cytosol against invading pathogens. The understanding of the mechanism of how pathogens, such as bacteria, are recognised in the cytosol prior to autophagic degradation is emerging. As presented by Dr Agnes Foeglein, some bacteria, such as Salmonella, are detected by NDP52 after they enter the cytosol of human cells, where they become coated with poly-ubiquitinated proteins. NDP52 therefore acts as a receptor for the selective autophagy of cytosolic bacteria by binding to the bacterial ubiquitin coat, and then delivers the bacteria into autophagosomes. To mediate the delivery of the cargo to autophagosomes, NDP52 binds to LC3C (ATG8 orthologue) on autophagosomal membranes. This interaction is crucial for innate immunity because cells lacking either NDP52 or LC3C cannot protect their cytoplasm against Salmonella.


AUTOPHAGY AS A BARRIER TO GENE DELIVERY


As mentioned above, autophagy is known to have evolved as an efficient defence against pathogens and therefore plays a key role in removing intracellular bacteria and viruses by delivering them to lysosomes for degradation. However, while this is clearly beneficial to the health of humans, it may also become a major obstacle to the development of viral and non- viral gene therapy vectors, which are being produced in an attempt to compensate for a defect in genes associated with specific diseases. Professor Tom Wileman presented results illustrating that autophagy is induced following viral infection of mammalian cells, leading to autophagic degradation of proteins delivered into the cells, indicating that autophagy becomes a barrier to efficient delivery of gene therapy vectors. These results are also relevant to researchers in the field, who often rely on cellular transfection of exogenous proteins in their autophagy-related work, as non-viral gene delivery vectors can also induce the autophagy process, leading to decreased expression of the protein of interest.


A ROLE FOR RAB8 AND AUTOPHAGY Frontotemporal dementia (FTD) is a condition resulting from the progressive deterioration of the frontal lobe of the brain and is the most common form of dementia in humans under 60 years of age. In a screen for enhancers and suppressors of the FTD phenotype, members of Dr Sean Sweeney’s laboratory identified Rab8, which also plays a role in endosome and autophagosome regulation. Examination of Rab8 mutants revealed deregulation of JNK signalling, leading to generation of synapse overgrowth.


THE BIOMEDICAL SCIENTIST 723


CDC


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