310 research outputs found
LIVE-PAINT Supplementary Videos: Compressed versions
No full textCompressed versions of the files of Oi, Curran; Horrocks, Mathew; Regan, Lynne. (2020). LIVE-PAINT Supplementary Videos, [dataset]. University of Edinburgh. Quantitative Biology, Biochemistry and Biotechnology. https://doi.org/10.7488/ds/2801.
## File listing ##
* "SupplementaryMovie1_-_mKO_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mKO and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "SupplementaryMovie2_-_mOrange_blinking_compressed.mp4" LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mOrange and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "SupplementaryMovie3_-_cofilin_tracking_compressed.mp4" Video tracking cofilin in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 5 microns.
* "SupplementaryMovie4_-_cofilin_tracking_compressed.mp4" Tracking the diffusion of a single cofilin "spot" in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 1 micron
Super-resolution imaging of proteins in live cells using reversibly interacting peptide pairs
No full textSuper-resolution techniques have revolutionised our ability to observe cellular structures
with significantly higher resolution than traditional microscopy. Despite the
number of super-resolution microscopy techniques available, live cell super-resolution
imaging remains challenging. For example, while Photo-activated localisation microscopy
(PALM) can be used in vivo, it necessitates the direct fusion of a fluorophore
to the protein of interest. This approach can be problematic because a direct fusion
to a fluorescent protein can disrupt the normal function and localisation of the protein
being studied. Moreover, once the fluorescent protein is photobleached, no more data
can be collected from that molecule.
In this thesis, I describe the development and use of LIVE-PAINT, a novel live-cell
super-resolution microscopy technique. In LIVE-PAINT, a peptide-protein or peptidepeptide
pair, one fused to the protein of interest and the other to a fluorescent protein,
reversibly interact. When the peptide pair bind, a blink is observed, and the precise
location can be determined. In a few minutes, enough binding events occur to generate
an image of the protein of interest with a resolution of around 20 nanometres.
Initially, this work optimises and applies LIVE-PAINT for diffraction-limited and
super-resolution imaging of proteins within live budding yeast cells. I then demonstrate
that the small peptide tag used to label the protein of interest makes LIVE-PAINT a
valuable tool for imaging proteins that are sensitive to direct fusions to fluorescent
proteins. In addition, I validate that LIVE-PAINT enables replenishment of signal
throughout imaging. This is because the imaging peptide, the peptide-labelled fluorescent
protein, is expressed separately from the target protein, creating a pool of imaging
peptides within the cell that can replenish those that are photobleached during imaging.
I utilise this property of LIVE-PAINT to track moving proteins over long periods of
time.
Subsequently, I describe how I adapted the LIVE-PAINT system to apply this
technique to the more complex environment of live mammalian cells. I show that
LIVE-PAINT successfully yields diffraction-limited and super-resolution images of
proteins located in various organelles. This is the first time that interacting peptide pairs
have been used to facilitate point accumulation for imaging in nanoscale topography
(PAINT) based super-resolution imaging in live mammalian cells. These results are
obtained through both transient transfections of labelled proteins and stably integrated
versions. Through this work I generate several new cell lines which can be shared
with other researchers allowing them to use this technique to gain new insights into the
proteins they study.
Furthermore, this thesis explores improvements to the LIVE-PAINT method. I
demonstrate that peptides as small as 5 residues can be used for LIVE-PAINT imaging.
This will broaden the applicability of LIVE-PAINT to a wider range of proteins
that cannot tolerate modifications. To harness the increased brightness of synthetic
fluorescent dyes compared to fluorescent proteins, I developed mammalian cell lines
expressing a HaloTag fused to a LIVE-PAINT peptide. I show that the exogenous
addition of the binding partner to HaloTag, HaloLigand, labelled with a synthetic dye,
to these cells, enables LIVE-PAINT imaging with synthetic dyes. Lastly, I validate that
LIVE-PAINT can be multiplexed by using orthogonal peptide-protein pairs to image
two proteins concurrently in live cells.
In summary, this thesis presents the development and optimisation of LIVE-PAINT,
an innovative peptide-based super-resolution imaging technique tailored for live cell
imaging. While this work explores select applications of LIVE-PAINT, it is anticipated
that this novel technique will have a broad spectrum of applications
The design and synthesis of peptides for fluorescence imaging applications
No full textTesting new function - does this work?? Fluorescence microscopy is a tool
routinely utilised to address biological questions. Direct visualisation of features of interest, confirmation of protein identity or detecting the presence of a posttranslational modification enable researchers to study differences between healthy and pathological phenotypes. As the questions get more complex, more advanced imaging techniques are required to address the limit of resolution, probe specificity to target and fluorescent background. This thesis explores two advancements to the field of fluorescence microscopy: the development of a novel imaging platform
and the introduction of a new imaging modality.
The study covers the design and construction of the Full Spectrum Fluorescence Lifetime Imaging Microscope (FS-FLIM): a new imaging platform. The FS-FLIM maximises the information collected in a fluorescence imaging experiment. It is capable of 3-in-1 imaging, collecting fluorescence intensity and lifetime data at 512 wavelengths simultaneously. The instrument is compatible with a wide range of samples. The spectral region observed can be matched to the emitters present in the sample. Moreover, a wide range of lifetimes (from sub-nanosecond to tens of nanoseconds) can be recorded using the FS-FLIM. The performance of the
constructed instrument is validated through solution lifetime measurements of several fluorescent dyes, and its application in environment sensing is described using a model system.
Next, the thesis focuses on developing fluorescent peptides for imaging applications. Although peptides can be labelled with a range of fluorophores, this study mostly uses a selenium-derivatised nitrobenzoxadiazole dye - SeNBD, a recent exciting addition to the imaging toolbox. The fluorogenic properties of the new dye are characterised in this study, and the incorporation of the dye on-column using standard solid state peptide synthesis methods is described. The switch-on character, as well as the small size of the dye, are leveraged throughout the work, with short peptide sequences and internally-labelled peptide sequences developed as a new generation of peptide probes.
The specificity of peptides enables their application as therapeutic agents
for otherwise undruggable proteins. One such example is α-synuclein, a protein associated with Parkinson’s disease (PD). It is commonly thought that aggregation of α-synuclein and formation of cytotoxic inclusions is involved in PD progression. An α-helical bacteria-derived peptide, phenol soluble modulin α3 (PSMα), was previously shown to bind to small oligomeric α-synuclein species. Here, the peptide sequence was derivatised with several labels to explore its applications in imaging. Pilot experiments to detect α-synuclein species in vitro were conducted to confirm the binding to the protein-of-interest. A biotin-labelled peptide analogue was used to perform immunohistochemistry staining on patient tissue for in situ detection. Lastly, first attempts at fluorescence lifetime imaging of α-synuclein on the FS-FLIM instrument were made.
To further illustrate the capabilities of SeNBD dye specifically, and its potential
for super-resolution imaging, a range of sequences targeting the PDZ domain
(a common structural motif of anchoring and signalling proteins) were then
considered. The chosen sequences were only a few amino acids long, however they
demonstrated retained transient binding to the protein-of-interest upon labelling
with SeNBD. Moreover, an internally-labelled sequence was also synthesised and successfully used to target PDZ domains. The transient nature of the peptideprotein binding was utilised in a Point Accumulation in Nanoscale Topography (PAINT) experiment, where the on-off binding enabled precise localisation of the dye molecules and super-resolution image reconstruction. The PDZ contained in post-synaptic density proteins of a brain-derived sample was successfully imaged, and nanoclusters of the protein super-resolved, corroborating literature findings. Furthermore, an extension to the approach using a coiled-coil interacting peptide pair: 101A and 101B, was investigated as an alternative approach for proteiniv peptide interaction pairs for super-resolution microscopy. Whilst the delivery of
the synthesised targeting sequence (101B) proved challenging, expressing 101B attached to the target protein in cellulo, and subsequent staining with fluorescent 101A sequence produced promising results and enabled direct visualisation and super-resolution imaging of TOM-20 and LAMP-1 in fixed HEK cells.
Overall, the work presented herein showcased a range of fluorescent peptides that could be used across versatile fluorescence imaging platforms. The small novel dye, SeNBD, enabled super-resolution imaging using peptide sequences containing less than 10 amino acids. The small size of the peptides decreased the linkage error and ensured the fluorescent signal was localised at target of interest. The environment sensing properties of the dye were explored in preliminary experiments to observe α-synuclein on a purpose-built novel microscope, the FS-FLIM. It is hoped the techniques developed here could further progress research in the field
of neurodegeneration and beyond
Genetic diversity of Crocus antalyensis B. Mathew (Iridaceae) and a new subspecies from southern Anatolia
No full textCrocus antalyensis B. Mathew is a bulbous plant endemic to Turkey. It is morphologically variable within the western part of Anatolia. Amplified fragment length polymorphism (AFLP) marker system was used to detect genetic variation among the Crocus taxa. Twenty-two primer combinations were used to screen for polymorphism among the samples. Genetic variation ranged from 0.44 to 0.69. We demonstrated the efficiency of the AFLP marker system for discriminating between individual C. antalyensis specimens. A high level of genetic variation was present among C. antalyensis specimens collected from different locations in Turkey. We also observed that C. antalyensis subspp. are genetically distinct from their relative Crocus flavus Haw. subsp. dissectus Baytop & B. Mathew. A new subspecies of C. antalyensis B. Mathew from southern Turkey is described. It is characterized by striped outer perianth segments, waist-shaped flowers, and glabrous throat of the perianth. A composite image of the new subspecies is presented.Research Fund of Istanbul University, Istanbul, TurkeyIstanbul University [4155]; Turkish Research CouncilTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108G096]The first author appreciates Helmut Kerndorff and Eric Pasche for sharing of their knowledge of the genus Crocus. We are grateful to Prof. Dr. Neriman Ozhatay (ISTE) for her scientific advice. We also thank Mark Garland (Micanopy, Florida, USA) for Latin translation. This work was supported by the Research Fund of Istanbul University, Istanbul, Turkey (project number 4155). We also kindly thank the Turkish Research Council for supporting PhD student H. Betul Kaya's scholarship (project number 108G096)
LIVE-PAINT Supplementary Videos
No full textWe present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extended data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein. Please note, compressed versions of the mp4 files are available at https://doi.org/10.7488/ds/2839 .# FILE LISTING #
* "Supplementary Movie 1 - mKO blinking.avi"
LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mKO and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "Supplementary Movie 2 - mOrange blinking.avi"
LIVE-PAINT blinking behavior shown in S. cerevisiae using the fluorescent protein mOrange and the reversible interaction pair SYNZIP17-SYNZIP18. Scale bar is 1 micron.
* "Supplementary Movie 3 - cofilin tracking.avi"
Video tracking cofilin in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 5 microns.
* "Supplementary Movie 4 - cofilin tracking.avi"
Tracking the diffusion of a single cofilin "spot" in S. cerevisiae, where cofilin is C-terminally tagged with SYNZIP18 and labeled with separately expressed SYNZIP17-mNeonGreen. Scale bar is 1 micron.
MP4 versions of each file have also been included, for accessibility
PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations
No full textAim: to fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow.Methods: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes.Results: labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis.Conclusions: active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.</p
Probing TDP-43 condensation using an in silico designed aptamer
No full textAptamers are artificial oligonucleotides binding to specific molecular targets. They have a promising role in therapeutics and diagnostics but are often difficult to design. Here, we exploited the catRAPID algorithm to generate aptamers targeting TAR DNA-binding protein 43 (TDP-43), whose aggregation is associated with Amyotrophic Lateral Sclerosis. On the pathway to forming insoluble inclusions, TDP-43 adopts a heterogeneous population of assemblies, many smaller than the diffraction-limit of light. We demonstrated that our aptamers bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize TDP-43 condensates with super-resolution microscopy. At a resolution of 10 nanometers, we tracked TDP-43 oligomers undetectable by standard approaches. In cells, Apt-1 interacts with both diffuse and condensed forms of TDP-43, indicating that Apt-1 can be exploited to follow TDP-43 phase transition. The de novo generation of aptamers and their use for microscopy opens a new page to study protein condensation.</p
Single-molecule detection and characterisation of alpha-synuclein aggregates
No full textAberrant protein aggregation is a predominant feature of many neurodegenerative disorders.
It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in
Parkinson’s Disease (PD), and it is widely accepted that small α-syn oligomers are the key
cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise
using traditional biochemical ensemble methods due to their high level of heterogeneity
and low abundance. Single-molecule fluorescence microscopy techniques have emerged
as a suitable approach to circumventing this problem, enabling the detection of individual
aggregates amongst monomeric protein and thus facilitating the identification, quantification,
and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation
are poorly understood. Furthermore, there remains some limitations to the singlemolecule
techniques currently available. This thesis describes the work completed to address
some of these issues.
Chapter 1 provides the contextual background for the work presented in this thesis, detailing
the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining
the single-molecule techniques used to investigate aggregate species. Chapter 2 describes
the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made
using the single-molecule techniques which were utilised and developed in this work.
One primary approach for studying species in single-molecule experiments involves directly
labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation
have previously been identified using fluorescently tagged α-syn and single-molecule Förster
resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate
formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled
α-syn to detect and characterise aggregates formed both intracellularly and in vitro
via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and
iii
total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and
structures of the oligomers formed. This work reveals the presence of distinct oligomeric
species in vitro and in neurons resulting from structural conversion during early aggregate
formation.
The approach taken in Chapter 3 is highly suitable for investigating aggregate formation
resulting from the addition of exogenous α-syn to samples of interest. However, such an
approach is not ideal for the detection and characterisation of endogenous aggregates due to
issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used
as an alternative approach to labelled protein; however, such dyes are non-protein-specific
and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in
Chapter 4 describes a novel single-molecule method to specifically detect and characterise
α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with
orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule
confocal microscopy. This enables the quantification and size approximation of α-syn aggregates
at picomolar concentrations, both in vitro and in biological samples.
Although the kinetics of α-syn aggregation have been studied extensively, much of our current
knowledge stems from ensemble averaging techniques which are associated with high levels
of variability and are not conducive to detecting the earliest steps in aggregate formation.
In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational
modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects
of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D
mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4.
This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new
insights into the aggregation of α-syn variants.
Overall, the work presented here provides new insights into the aggregation of α-syn via the
use and development of single-molecule techniques. The advancements made have added
to the current understanding of the molecular mechanisms of α-syn aggregation, both in
vitro and in neurons, and have also been used to develop a novel single-molecule detection
method for α-syn aggregates. The work presented in this thesis has resulted in two published
papers, ’Pathological structural conversion of alpha-synuclein at the mitochondria induces
neuronal toxicity’ in Nature Neuroscience, and ’Single-molecule two-color coincidence detection
of unlabeled alpha-synuclein aggregates’ in Angewandte Chemie International Edition.
Furthermore, the novel detection method presented here holds promise for measuring α-syn
oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates.
This may therefore be used in future studies for identifying, detecting, and studying
potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected
that the work from this thesis will be used to aid researchers towards better understanding the
mechanisms of α-syn aggregation, both in vitro and in clinical samples
Fluoroaromatics as ¹⁹F NMR probes for chemical biology
No full textFluorine is almost entirely absent from nature but due to the high environmental sensitivity of the nucleus, coupled with its similar atomic radius to hydrogen, fluorine can be incorporated into biomolecules as a ¹⁹F NMR probe to study proteins with no background signal. Fluorine NMR can be used to study peptide and protein conformational changes, which play a role in most biological functions, including potentially malign processes including protein aggregation that can lead to neurodegenerative diseases such as Parkinson’s disease (PD), the cause of which remains mostly elusive. This thesis studied the use of ¹⁹F NMR for conformational analysis of peptide models as well as recombinant proteins. This work explored the two most convenient methods for incorporation of fluorine into peptides and proteins, firstly Chapter 2 explored the use of fluorinated amino acids such as 4-fluorophenylalanine, and then Chapters 3 and 4 investigated post-translational indirect fluorination using small covalently-reactive ‘fluoro-tags’.
In Chapter 2, a simple conformational switching event was studied by examining proline cis-trans isomerism – often the overall rate-determining step in protein folding – in over 60 simple X-Pro-Z pentapeptide models using distal ¹⁹F NMR reporters. Here, the ratio of cis- and trans-conformers was quantified in a variety of biologically relevant conditions (aqueous buffer, variable pH) that would not be possible using ¹H NMR due to the inherent background water and peptide proton signals. This work also confirmed that a) ¹⁹F NMR measurements of %cisPro in these simple models mirrored the reported populations in similar literature models, b) that the nature of the amino acid on the N-terminal side of proline had a larger impact upon %cisPro than the C-terminal residue, c) that aromatic amino acids afforded the greatest cisPro populations, and that the Pro conformational populations as dictated by X-Pro-Z sequences were largely found to be translatable between different peptide sequences.
In Chapter 3, the indirect fluorination of proteins was explored using small, fluorinated electrophilic compounds, mostly commercially available, that yielded the identification of two novel fluoro-tags for protein labelling. These compounds were initially used in a version of the above X-Pro-Z model peptide system to modify cysteine residues to probe a) the sensitivity of the tag and its fluorine environment to proline conformation, and b) the effect it has on %cisPro. The fluoro-tags proved to be non-biased reporters of proline cis-trans status as they measured the ratio of the conformers, from a distal position, in accordance with values expected from the literature and Chapter 2 of this work. The compounds were also successfully and chemoselectively used to conjugate to a mutant of alpha-synuclein (A100C), the protein linked to PD, with evidence of the fluoro-tags showing sensitivity to aggregation of the protein in a ¹⁹F NMR study.
Following on from this, in Chapter 4, a series novel aryl-DABCOnium reagents, quaternary ammonium salts based on 1,4-diazabicyclo[2.2.2]octane, were developed as water-soluble, highly fluorinated tags to give improved sensitivity in protein-observed ¹⁹F NMR protein conformational studies, but also with potential applications of the warhead beyond the scope of this work, such as for cell-penetrating covalent drugs. One of these reagents was used to selectively modify cysteine mutants of a) the steroid carrier protein type 2 like (SCP-2L) domain of human multifunctional enzyme 2 (MFE-2) to explore protein-observed ligand binding, and b) the A100C mutant of alpha-synuclein to observe oligomerization, both by ¹⁹F NMR. This revealed that the fluoro-tag was sensitive to the structural perturbations of SCP-2L during ligand binding by ¹⁹F NMR, suggesting a potential application as a sensor of protein-ligand interactions, and the tag was also sensitive to the oligomerization of alpha-synuclein, a process not well understood, potentially identifying different types of oligomers.
Finally, in Chapter 5, a fluorine tag was developed that could undergo a double-tagging to cross-link a pair of cysteine residues artificially introduced into the α-helix of a bacterially derived peptide PSMα-3, to conformationally constrain the peptide and introduce a fluorine reporter. This ‘fluoro-stapling’ method enabled the ¹⁹F NMR ligand-observed study of the binding of the peptide to fibrils of alpha-synuclein, with the potential application for a quantitative detection method for these malign protein structures. Overall, this work illustrated the power of fluorine tagging to study biomolecules and their conformational behaviour in absence of any background signals
Correction to: The role of resection in hepatocellular carcinoma BCLC stage B: A multi-institutional patient-level meta-analysis and systematic review (Langenbeck's Archives of Surgery, (2024), 409, 1, (277), 10.1007/s00423-024-03466-x)
No full textCorrection to: Langenbeck’s Archives of Surgery (2024) 409:277. https://doi.org/10.1007/s00423-024-03466-x. This article unfortunately contained a mistake. The author name ‘Mathew Vithayathil’ was incorrectly written as ‘Vithayathil Mathew K.’. The original article has been corrected
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