Detection and Quantification of Fluorescent Cell Clusters in Cryo-Imaging

We developed and evaluated an algorithm for enumerating fluorescently labeled cells (e.g., stem and cancer cells) in mouse-sized, microscopic-resolution, cryo-image volumes. Fluorescent cell clusters were detected, segmented, and then fit with a model which incorporated a priori information about cell size, shape, and intensity. The robust algorithm performed well in phantom and tissue imaging tests, including accurate (<2% error) counting of cells in mouse. Preliminary experiments demonstrate that cryo-imaging and software can uniquely analyze delivery, homing to an organ and tissue distribution of stem cell therapeutics

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling.

As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation

Characterising the formation and functional role of vesicular clustering during Influenza A virus infection

Tese de mestrado em Microbiologia Aplicada, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017Os vírus Influenza, que pertencem à família Orthomyxoviridae, estão divididos em quatro tipos A, B, C e D (Hause et al. 2013; Hause et al. 2014). Entre estes, o influenza A (IAV) é o vírus que causa maior impacto na saúde pública. O seu reservatório natural são as aves marinhas, no entanto possui a capacidade de gerar infeções zoonóticas encontrando-se disseminado em muitas espécies de aves e mamíferos (Acheson 2011; Taubenberger & Kash 2010). Nos seres humanos, o IAV é o patógeno responsável pela gripe, uma doença aguda respiratória associada a alta morbidade e mortalidade (Lofgren et al. 2007). O IAV é composto por um invólucro viral derivado da membrana plasmática da célula. Este envelope é revestido por uma camada externa de proteínas virais, designadamente hemaglutinina e neuraminidase. O interior do virião contém o genoma viral, constituído por oito complexos ribonucleoproteicos (vRNPs). Cada vRNP é composto por um dos oito segmentos de ácido ribonucleico viral que codificam para diferentes proteínas virais. Os vRNPs funcionam como unidades independentes sendo necessário um conjunto completo de oito vRNPs diferentes para a montagem de uma partícula infeciosa (Fields et al. 2007). Os vRNPs são transportadas por piggyback em vesículas destinadas à periferia da célula através da interação entre a polimerase 2 viral e a GTPase Ras related in brain 11 (Rab11) (Amorim et al. 2011; Eisfeld et al. 2011; Momose et al. 2011). Em homeostasia, o Rab11 está envolvido na reciclagem de proteínas endocitadas. Regula a formação, movimento e fusão de vesículas originárias do compartimento de reciclagem de endossomas e encontra-se espalhado pelo citoplasma (Grant & Donaldson 2009). Em células infetadas, o Rab11 acumula no citoplasma formando agregados vesiculares. Sabe-se que estas vesículas são heterogéneas, compostas por membrana simples e dupla (SMV e DMV, respetivamente), o que indica que o IAV conduz não só ao agrupamento vesicular mas também ao rearranjo das suas membranas (Vale-Costa et al. 2016). Este trabalho permitiu fundamentar um modelo alternativo para a montagem genómica do IAV, em que o agrupamento vesicular pode funcionar como uma plataforma induzida pelo virus para a criação de hotspots de vRNPs. A alta co-localização entre o conjunto de todas as vRNPs permitiria a ocorrência das interações RNA-RNA necessárias à junção do genoma completo composto pelos oito vRNPs. O agrupamento vesicular surge assim como um passo essencial no ciclo replicativo do IAV. Pelo que, a definição da cronologia de eventos que levam à sua maturação e as alterações na composição vesicular tornam-se fundamentais para compreender os mecanismos de montagem genómica do IAV. Neste trabalho experimental foi aplicada uma multi-metodologia de crio-microscopia eletrónica (EM). A razão da escolha dos métodos de crio-EM baseia-se na capacidade que estas técnicas têm para preservar os detalhes ultra-estruturais sem introduzirem os artefactos morfológicos de volume, forma e desnaturação de epitopos associados aos métodos cuja abordagem é puramente química (Studer et al. 1989). No entanto, para a sua aplicação, estes métodos tiveram que sofrer otimizações. Os mesmos permitiram elucidar o modo como a agregação vesicular se forma em células A549 que expressam constitutivamente GFP-Rab11 durante a infeção e qual a função das vesículas agregadas no ciclo replicativo do IAV. A metodologia de crio-EM usada incorporou duas técnicas conhecidas na área: a técnica de congelamento de alta pressão e de fixação química por substituição a baixas temperaturas e a técnica de Tokuyasu. A primeira utiliza a baixa temperatura do azoto (-196ºC) e alta pressão (aproximadamente 2000 bar) para crio-imobilizar as amostras. A técnica permitiu a preservação celular para análise morfológica e a quantificação vesicular (sem introdução de artefactos de fixação química). A segunda técnica, Tokuyasu, também tira partido da baixa temperatura do azoto para crio-imobilizar as amostras e permite manter as estruturas hidratadas para uma imunomarcação ótima. Esta técnica foi usada para discernir a origem das estruturas vesiculares em estudo. As metodologias usadas neste trabalho foram alvo de diversas otimizações, com o intuito de preservar as características ultra-estruturais o mais próximo possível do seu estado nativo (McDonald 2007). Na otimização da técnica de congelamento de alta pressão dois parâmetros foram avaliados: a vitrificação e a preservação morfológica das células GFP-Rab11. A vitrificação refere-se ao processo de transformação de um liquido em sólido sem cristalização. A cristalização induz artefactos nas células, reconhecíveis sob a forma de redes interligadas de espaços vazios onde outrora expandiram os cristais de gelo. Ambos os parâmetros de vitrificação e preservação morfológica são afetados pelo tipo de suporte de amostra utilizado para congelar, bem como pelo tipo de enchimento escolhido para preencher o espaço livre entre a amostra e o suporte. Os tipos de suporte para congelamento de amostras testados foram os discos planos e os suportes côncavos. O primeiro tipo – discos planos - permite o congelamento de células em camada única e foi testado em combinação com três tipos de enchimentos (hexadeceno, dextano a 20% (m/v) e albumina sérica bovina a 20% (m/v)). O segundo tipo – suportes côncavos – permite o congelamento em pellet e foram testados em combinação com hexadeceno. Estabeleceu-se que o melhor protocolo para processamento de células GFP-Rab11 por congelamento de alta pressão tendo em conta o objeto de estudo (a ultra-estrutura vesicular) foi alcançada ao usar a combinação de discos planos com hexadeceno. Para a otimização da técnica de Tokuyasu, quatro marcadores: Golgi Marker (GM) 130, calnexina, nucleoproteína (NP) e GFP-Rab11 foram estudados em células infetadas com IAV para a capacidade de manter sua imunorreatividade em diferentes condições de fixação. Três fixadores foram usados durante o teste: formaldeído a 2% (v/v) com glutaraldeído a 0,2%, 0,1% e 0,05% (v/v). Como controlo foi adicionado o fixador de referência em imunocitoquímica: formaldeído a 4% (v/v). O formaldeído permite a manutenção da imunorreatividade celular, no entanto o glutaraldeído conduz a uma melhor preservação morfológica da ultra-estrutura. O fixador que permitiu a preservação da imunorreatividade de todos os marcadores foi o formaldeído a 4% (v/v) e, como tal, o fixador usado no estudo. A combinação entre as técnicas de crio-EM, congelação por alta pressão e Tokuyasu, permitiu quantificar o número de vesículas de membrana simples e dupla (SMV e DMV, respetivamente) durante a infeção por IAV. Foi possível determinar que o número de SMVs - utilizadas como veículos para o transporte de vRNPs - aumenta durante a infeção por IAV e que as membranas destas vesiculas de membrana simples são reorganizadas para produzir as DMVs. Desta forma, é possível afirmar que as SMV juntamente com as DMVs parecem constituir os agregados vesiculares formados durante a infeção e que permitem a convergência espacial dos vRNPs. Para além das técnicas de crio-EM descritas, também a técnica de correlação entre microscopia de luz e eletrónica (CLEM) foi desenvolvida e otimizada para pesquisa da função dos agregados vesiculares. A técnica de CLEM aplicada combinou as técnicas de hibridização in situ fluorescente (FISH), microscopia estocástica de reconstrução ótica (STORM) e EM e contribuiu para uma caracterização adicional do papel das SMVs e das DMVs dentro dos agregados. A técnica FISH em conjunto com a técnica de STORM permitiu a identificação específica de segmentos do genoma do IAV em agregados de Rab11, enquanto a EM forneceu o contexto ultra-estrutural da célula. No entanto, a precisão da correlação alcançada foi baixa, não tendo permitido a identificação do conteúdo genómico transportado por cada vesícula dentro dos agregados. Neste sentido, podemos afirmar que, apesar de poder ser combinada com outras técnicas, a técnica de CLEM integrando FISH, STORM e EM é uma técnica ainda em desenvolvimento e que requer melhorias adicionais. Em resumo, o trabalho aqui descrito conduziu à conclusão de que as vesículas de membrana simples e dupla, portadoras de vRNPs, são originárias do compartimento de reciclagem de endossomas. Ambos os tipos de vesículas foram encontrados nos agregados e identificadas com os mesmos marcadores imunes. A sua relevância funcional para a montagem dos vRNPs continua por inferir, sendo necessária a contínua otimização da combinação das técnicas de microscopia de luz e de microscopia eletrónica para determinar um método de CLEM funcional.Influenza viruses, from the Orthomyxoviridae family, are divided in four types A, B, C and D (Hause et al. 2013; Hause et al. 2014). Influenza A (IAV) as the capacity to generate zoonotic infections and is widespread in many mammalian and avian species. (Acheson 2011; Taubenberger & Kash 2010). In humans, IAV is the pathogen responsible for flu, a respiratory acute disease associated with high morbidity and mortality (Lofgren et al. 2007). IAV as a segmented genome divided in eight different viral ribonucleoprotein complexes (vRNP). Each vRNP complex is composed by one of the eight viral ribonucleic acid segments. The vRNPs function as independent units and a complete set of eight different vRNPs is required for assembly of an infectious IAV particle (Fields et al. 2007). It is known that vRNPs are transported by piggybacking in vesicles destined to the cell periphery via interaction between the viral polymerase basic protein 2 and the host GTPase Ras-related in brain 11 (Rab11) (Amorim et al. 2011; Eisfeld et al. 2011; Momose et al. 2011). Also, it was stated that during infection there is an aggregation of vesicles creating clusters. The clusters were composed by vesicles with single and double membranes (SMV and DMV), indicating that IAV leads not only to clustering but also to the rearrangement of vesicular membranes (Vale-Costa et al. 2016). This work substantiated an alternative model for IAV genomic assembly in which vesicular clustering can function as a viral induced platform promoting the creation of vRNPs hotspots. The high co-localization among the pool of all vRNPs would allow the establishment (or completion) of the RNA-RNA interactions necessary for assembly of the eight RNA segments before budding. Vesicular clustering thus emerges as an essential step in IAV lifecycle. In this work, a multi electron microscopy (EM) cryo-methodology was not just applied but also optimized and developed to elucidate how vesicular clustering is formed in A549 cells constitutively expressing GFP-Rab11 during infection and what the vesicles functional role in the lifecycle of IAV. The EM cryo-methodology incorporated the High Pressure Freezing – Freeze Substitution (HPF-FS) and Tokuyasu techniques. The first permitted the morphological analysis of cells and vesicular quantification (without introducing chemical fixation artifacts) and the second enabled the immunolabeling of structures to discern their origin. Also, to further investigate the functional role of clusters a correlative light electron microscopy (CLEM) method that combines Fluorescent in situ Hybridization (FISH), Stochastic Optical Reconstruction microscopy (STORM) and EM was developed. The optimization of HPF-FS technique established that the best vitrification and morphological preservation of GFP-Rab11 cells was achieved when using flat disks, as specimen carriers for the freezing process, filled with 1-hexadecene, as a cryoprotectant. For Tokuyasu, 4% (v/v) formaldehyde was the fixative to enable the best preservation of immunoreactivity. Together, both techniques, lead to conjecture that the number of SMVs - used as vehicles for vRNP trafficking - increase during IAV infection and are modified to rearrange membranes and produce DMVs. Both Rab11 SMVs and DMVs seem to constitute the clusters formed during infection that enable the spatial convergence of vRNPs. The CLEM technique incorporating FISH, STORM and EM contributed to a further characterization of SMVs and DMVs role within the clusters. The FISH technique allowed the identification of IAV genome segments on Rab11 clusters imaged by STORM, whereas EM provided the ultrastructural context of the cell. A low accuracy of the achieved correlation did not allow the identification of the segment content of each vesicle within the clusters. This combinatorial but powerful method thus requires further improvement. In summary, the work herein described led to the conclusion that vesicles of single and double membrane, carrying vRNPs, originate from the endosomal recycling compartment. Both types of vesicles were found in clusters. Their functional relevance for vRNP assembly remains to be determined, as the CLEM method developed for its study needs further optimization

Intraoperative Quantification of Bone Perfusion in Lower Extremity Injury Surgery

Orthopaedic surgery is one of the most common surgical categories. In particular, lower extremity injuries sustained from trauma can be complex and life-threatening injuries that are addressed through orthopaedic trauma surgery. Timely evaluation and surgical debridement following lower extremity injury is essential, because devitalized bones and tissues will result in high surgical site infection rates. However, the current clinical judgment of what constitutes “devitalized tissue” is subjective and dependent on surgeon experience, so it is necessary to develop imaging techniques for guiding surgical debridement, in order to control infection rates and to improve patient outcome. In this thesis work, computational models of fluorescence-guided debridement in lower extremity injury surgery will be developed, by quantifying bone perfusion intraoperatively using Dynamic contrast-enhanced fluorescence imaging (DCE-FI) system. Perfusion is an important factor of tissue viability, and therefore quantifying perfusion is essential for fluorescence-guided debridement. In Chapters 3-7 of this thesis, we explore the performance of DCE-FI in quantifying perfusion from benchtop to translation: We proposed a modified fluorescent microsphere quantification technique using cryomacrotome in animal model. This technique can measure bone perfusion in periosteal and endosteal separately, and therefore to validate bone perfusion measurements obtained by DCE-FI; We developed pre-clinical rodent contaminated fracture model to correlate DCE-FI with infection risk, and compare with multi-modality scanning; Furthermore in clinical studies, we investigated first-pass kinetic parameters of DCE-FI and arterial input functions for characterization of perfusion changes during lower limb amputation surgery; We conducted the first in-human use of dynamic contrast-enhanced texture analysis for orthopaedic trauma classification, suggesting that spatiotemporal features from DCE-FI can classify bone perfusion intraoperatively with high accuracy and sensitivity; We established clinical machine learning infection risk predictive model on open fracture surgery, where pixel-scaled prediction on infection risk will be accomplished. In conclusion, pharmacokinetic and spatiotemporal patterns of dynamic contrast-enhanced imaging show great potential for quantifying bone perfusion and prognosing bone infection. The thesis work will decrease surgical site infection risk and improve successful rates of lower extremity injury surgery

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Visualization of the HIV-1 Nuclear Preintegration Complex Structure by High Precision Correlative Light - and Electron Microscopy and - Tomography

Upon fusion of the viral envelope with the host cell membrane, the capsid of the human immunodeficiency virus 1 (HIV-1) is released into the host cell cytoplasm. To productively infect a cell, the viral RNA genome needs to be reverse transcribed into viral DNA. This in turn needs to become integrated into the host cell genome. Integration can, however, only happen, after the viral genome is released from its capsid-container, in a process called uncoating. This is a vital process and needs to be regulated and orchestrated in certain ways – which are still elusive and controversially discussed. Some studies suggest that uncoating takes place soon after -, or concomitant with viral entry. Other researchers came to the result that the capsid needs to retain its structure to shield the viral components from being sensed by the innate cellular immune system. Both hypotheses, early uncoating and prolonged structural retention, are solidly supported by experimental data. Therefore, the timing and kinetics of uncoating remain unresolved. Based on previous results from our group, we had reason to believe that the capsid might indeed be retained, possibly even within the nucleus. A method was developed, that allows the detection of viral DNA. The presence of viral DNA was used as a criterion to discriminate between productive and nonproductive subviral particles in infected cells. Surprisingly, productive subviral particles displayed an intense, stable signal for capsid protein in immunofluorescence experiments, throughout the cytoplasm and even within the nuclei of infected cells. A strong signal is can be understood as a high concentration of labeled protein, which in turn might indicate the presence of a retained structure. However, intense immunofluorescence signals can also mean more efficient binding of antibodies due to structural rearrangements (such as uncoating), and a high spatial concentration of proteins cannot be directly interpreted as structure retention. In this study, we present a unique way to address and solve this important question. We specifically focused on the small fraction of productive particles. Light Microscopy allows specific labeling but has low resolution. Electron Microscopy yields much higher resolution, but specific (immuno)labeling is difficult and often detrimental to ultrastructural retention. We overcame both limitations by correlative light – and electron microscopy: Regions of interest were identified by specific nuclear subviral particle surrogate markers in light microscopy. On these regions, tilt series electron tomography was performed, to visualize the subviral particles’ structure, as well as the subcellular environment, around the region of interest. Performing high resolution tilt series electron tomography, we could repeatedly and convincingly visualize a capsid-reminiscent structure that underlies HIV-1 nuclear preintegration complexes. This apparent structure is very similar in shape, but smaller in size compared to capsids of virus particles of mostly identical preparations. The discovery of a retained capsid structure in the nucleus of an infected cell will advance on our understanding of nuclear entry and provides whole new insights into the overall understanding of HIV-1 in early steps of infection

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function