A study of SNARE-mediated autophagosome clearance using fluorescence lifetime microscopy

Cell survival requires the turnover of toxic cellular material and recycling of biomolecules in low nutrient conditions. An efficient degradation system is therefore essential for disease prevention and its dysfunction has been linked to both neurodegeneration and oncogenesis. Bulk degradation is accomplished through the collection of cytoplasmic material in a unique sequestration vesicle, which forms de novo and subsequently deposits cargo in the lysosome for degradation. This process, known as autophagy, therefore requires membrane fusion between the autophagosomal vesicle and the lysosome. SNARE proteins mediate membrane fusion events and therefore their careful regulation ensures the proper organisation of the membrane trafficking network. The SNARE proteins governing autophagosome clearance have been identified as syntaxin 17, SNAP29 and VAMP8 and SNARE assembly appears to be positively regulated by VPS33A. This well established model of SNARE-mediated autophagosome clearance has not, however, been demonstrated within the spatiotemporal framework of the cell and little is known about how VPS33A modulates SNARE function. The research presented in this thesis therefore aims to determine the applicability of the proposed SNARE model within the cellular environment and to investigate the regulatory mechanisms controlling syntaxin 17 function. To accomplish this, carefully validated fluorescence colocalisation and time-resolved fluorescence lifetime imaging techniques were primarily employed. The limitations of these techniques were also considered for data interpretation and a novel prototype SPAD array technology, designed for high-speed time-correlated single photon counting, was trialled for widefield FLIM-FRET. FLIM-FRET revealed that VAMP8 has been incorrectly assigned as the dominant autophagosomal R-SNARE and VPS33A studies evidence a multi-modal regulation of Stx17 that diverges from other studied syntaxin family modulation mechanisms. A new model of SNAREmediated autophagosome clearance is therefore proposed, where syntaxin 17 engages with SNAP29 and VAMP7 to drive membrane fusion with the endolysosome in a manner governed by VPS33A and dependent on the phosphorylation status of syntaxin 17

Estimation of fluorescence lifetimes via rotational invariance techniques

Estimation of signal parameters via rotational invariance techniques is a classical algorithm widely used in array signal processing for direction-of-arrival estimation of emitters. Inspired by this method, a new signal model and a new fluorescence lifetime estimation via rotational invariance techniques (FLERIT) were developed for multi-exponential fluorescence lifetime imaging (FLIM) experiments. The FLERIT only requires a few time bins of a histogram generated by a time-correlated single photon counting FLIM system, greatly reducing the data throughput from the imager to the signal processing units. As a non-iterative method, the FLERIT does not require initial conditions, prior information nor model selection that are usually required by widely used traditional fitting methods, including nonlinear least square methods or maximum likelihood methods. Moreover, its simplicity means it is suitable for implementations in embedded systems for real-time applications. FLERIT was tested on synthesized and experimental fluorescent cell data showing the potentials to be widely applied in FLIM data analysis

Translation Microscopy (TRAM) for super-resolution imaging

Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease

SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A’s AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability

Bit-plane Processing Techniques for Low-Light, High Speed Imaging with a SPAD-based QIS

Advances in SPAD sensor technology have recently yielded a >10kfps, 320x240 resolution imager [1] that can be operated as a Quanta Image Sensor (QIS) [2]. This device (labelled SPCImager) thus combines the high speed and sensitivity afforded by SPAD devices with the negligible readout noise and logarithmic compression of QIS. These attributes open the door to a number of potential applications. This work explores two applications: high speed imaging of fast moving objects and low light microscopy. Appropriate signal processing schemes are considered in both cases

Differences in Preoperative Health-Related Quality of Life between Women Receiving Mastectomy or Breast Conserving Surgery in a Prospectively Recruited Cohort of Breast Cancer Patients

As rates of total mastectomy rise, the relationships between surgery modality with domains of health-related quality of life is not well understood. This study reports differences in depression, anxiety, pain, and health status among a cohort of women scheduled to receive total mastectomy or breast-conserving surgery. Patient-reported outcomes measured preoperative differences between patients receiving total mastectomy or breast-conserving surgery in a cross-sectional design. Regression analyses was used to model health outcomes and adjust for patient demographics on patient measures. Participants scheduled for total mastectomy were more likely to report more severe symptoms of depression and anxiety. This association was non-significant after adjusting for demographic differences. Younger participants were more likely to be scheduled for total mastectomy. Age was negatively associated with symptoms of depression and anxiety. Screening patients for mental health symptoms may be particularly important among younger patients who were more likely to report depression and anxiety before their surgery and were more likely to receive total mastectomy