ER-mitochondria interactions and neurodegeneration
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Date
29/06/2019Author
Harmon, Mark
Metadata
Abstract
Physical membrane contact sites between the ER and mitochondria play a critical
role in regulating a variety of processes including calcium signalling, lipid exchange,
controlling mitochondrial dynamics and cell death signalling. These contact sites are
formed at specialised regions of membrane, termed mitochondrial associated
membranes or MAMs, that are enriched for a group of proteins acting as tethers to
hold the ER and mitochondria at appropriate distances from one another. The
distance of these junctions is usually defined between 10-30 nm but can vary in
response to certain cellular conditions and it is believed that heterogeneity in the
distance of the contact sites may be reflective of different protein compositions or
activities at these sites. Abnormal alterations to ER-mitochondria contacts are
observed in numerous neurodegenerative disorders including Alzheimer’s,
Parkinson’s and amyotrophic lateral sclerosis (ALS) and therefore, it is believed that
a dysfunction to the MAMs may be a common pathogenic mechanism underlying
neuronal cell death.
Due to the associated dysfunction of organelle contact sites in neurodegenerative
disorders, the ability to detect these structures could provide critical information on
the pathogenic mechanisms of neuronal cell death. Existing techniques for detecting
MAMs have numerous limitations or are restricted to fixed samples. The aim of this
thesis was to develop a fluorescent-based method for the visualisation of contact
sites that can also be applied to living systems to study organelle contact dynamics.
Here, we have generated split fluorescent Venus fragments targeted to the ER and
outer mitochondrial membrane respectively, as the basis for our bi-fluorescence
complementation (BiFC) system for the detection and quantification of MAMs in
living cells. The principle of this technique relies on close spatial proximity of the
reporter probes for the restoration of the fluorescent protein and the emission of a
detectable signal. Validation of this method highlighted several advantages over
existing methods of detecting ER-mitochondria contacts and we were able to report
on changes in agreement with previously published, high-resolution electron
microscopy studies. Adaptations to the technique allow for the detection of other
organelle contact sites by varying the targeting sequence of the complementary
Venus fragments.
As these reporter proteins detect junctions of a maximum distance of around 6-10
nm, we could use the BiFC system to correlate the Venus signal with specific
functions of the MAMs to try and elucidate the functional significance of these
particularly tight contact sites. Our results suggest that some of these contact sites
may represent sites of actively dividing mitochondria. Furthermore, our results
indicated that these tight ER-mitochondria contacts are formed on a sub-population
of mitochondria of higher than average resting membrane potential and
mitochondrial calcium levels, which may indicate differences in the bioenergetic
state or the health of mitochondria with tight ER-mitochondria contact sites.
Finally, this technique was used to investigate the role of a MAM-enriched protein,
VAPB, of which a proline to serine missense mutation is associated with a dominantly
inherited form of ALS termed ALS8. The data shows that expression of the mutant
disease-linked VAPBP56S significantly increases the mean number of contact sites per
cell whereas altering the levels of wild-type VAPB has no significant effect. This
finding suggests that the expression of VAPBP56S induces abnormalities in ER-mitochondria
tethering but it is still unclear whether this is through direct binding
properties of mutant VAPB or through an indirect secondary mechanism. As the
MAMs regulate many of the pathways that are commonly perturbed in
neurodegenerative disorders, alterations in ER-mitochondria contact sites may
represent a key early pathogenic event in ALS.