Toward quantitative super-resolution methods for cryo-CLEM.

Cryogenic ultrastructural imaging techniques such as cryo-electron tomography have produced a revolution in how the structure of biological systems is investigated by enabling the determination of structures of protein complexes immersed in a complex biological matrix within vitrified cell and model organisms. However, so far, the portfolio of successes has been mostly limited to highly abundant complexes or to structures that are relatively unambiguous and easy to identify through electron microscopy. In order to realize the full potential of this revolution, researchers would have to be able to pinpoint lower abundance species and obtain functional annotations on the state of objects of interest which would then be correlated to ultrastructural information to build a complete picture of the structure-function relationships underpinning biological processes.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation.

Structure and activity of particulate methane monooxygenase arrays in methanotrophs.

Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane.

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells.

Cooperation and Interplay between EGFR Signalling and Extracellular Vesicle Biogenesis in Cancer.

Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs.

Mechanisms of Action of EGFR Tyrosine Kinase Receptor Incorporated in Extracellular Vesicles.

EGFR and some of the cognate ligands extensively traffic in extracellular vesicles (EVs) from different biogenesis pathways. EGFR belongs to a family of four homologous tyrosine kinase receptors (TKRs). This family are one of the major drivers of cancer and is involved in several of the most frequent malignancies such as non-small cell lung cancer, breast cancer, colorectal cancer and ovarian cancer.

SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection.

Serial cryoFIB/SEM Reveals Cytoarchitectural Disruptions in Leigh Syndrome Patient Cells.

The advancement of serial cryoFIB/SEM offers an opportunity to study large volumes of near-native, fully hydrated frozen cells and tissues at voxel sizes of 10 nm and below. We explored this capability for pathologic characterization of vitrified human patient cells by developing and optimizing a serial cryoFIB/SEM volume imaging workflow. We demonstrate profound disruption of subcellular architecture in primary fibroblasts from a Leigh syndrome patient harboring a disease-causing mutation in USMG5 protein responsible for impaired mitochondrial energy production.

Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses.

Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work.

A global sampler of single particle tracking solutions for single molecule microscopy.

The dependence on model-fitting to evaluate particle trajectories makes it difficult for single particle tracking (SPT) to resolve the heterogeneous molecular motions typical of cells. We present here a global spatiotemporal sampler for SPT solutions using a Metropolis-Hastings algorithm. The sampler does not find just the most likely solution but also assesses its likelihood and presents alternative solutions.

Structure and Dynamics of the EGF Receptor as Revealed by Experiments and Simulations and Its Relevance to Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is historically the prototypical receptor tyrosine kinase, being the first cloned and the first where the importance of ligand-induced dimer activation was ascertained. However, many years of structure determination has shown that EGFR is not completely understood. One challenge is that the many structure fragments stored at the PDB only provide a partial view because full-length proteins are flexible entities and dynamics play a key role in their functionality.

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution.

Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions.

Cluster Analysis of Endogenous HER2 and HER3 Receptors in SKBR3 Cells.

The Human Epidermal Growth Factor Receptor (HER) family of receptor tyrosine kinases consists of four, single pass, transmembrane receptor homologs (HER1-4) that act to regulate many critical processes in normal and tumor cells. HER2 is overexpressed in many tumors, and the deregulated proliferation of cancerous cells is driven by cooperation with its preferred receptor partner, HER3. The assessment of the organization of tagged HER2 and HER3 using super-resolution microscopy reveals quantitative Single Molecule Localization Microscopy (SMLM) as an ideal bioanalytical tool to characterize receptor clusters.

The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation.

Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface.

While targeted therapy against HER2 is an effective first-line treatment in HER2 breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms.

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution.

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5-50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.

Nanometric molecular separation measurements by single molecule photobleaching.

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes.

Measuring EGFR separations on cells with ~10 nm resolution via fluorophore localization imaging with photobleaching.

Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ~10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment.

A systematic investigation of differential effects of cell culture substrates on the extent of artifacts in single-molecule tracking.

Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties.