Automatic Hotspots Detection for Intracellular Calcium Analysis in Fluorescence Microscopic Videos

In recent years, life-cell imaging techniques and their software applications have become powerful tools to investigate complex biological mechanisms such as calcium signalling. In this paper, we propose an automated framework to detect areas inside cells that show changes in their calcium concentration i.e. the regions of interests or hotspots, based on videos taken after loading living mouse cardiomyocytes with fluorescent calcium reporter dyes. The proposed system allows an objective and efficient analysis through the following four key stages: (1) Pre-processing to enhance video quality, (2) First level segmentation to detect candidate hotspots based on adaptive thresholding on the frame level, (3) Second-level segmentation to fuse and identify the best hotspots from the entire video by proposing the concept of calcium fluorescence hit-ratio, and (4) Extraction of the changes of calcium fluorescence over time per hotspot. From the extracted signals, different measurements are calculated such as maximum peak amplitude, area under the curve, peak frequency, and inter-spike interval of calcium changes. The system was tested using calcium imaging data collected from Heart muscle cells. The paper argues that the automated proposal offers biologists a tool to speed up the processing time and mitigate the consequences of inter-intra observer variability

Automated Analysis of Cellular Signalling Parameters based on Images and Videos of Fluorescence Microscopy

Rapid changes in computer vision technologies have enabled automatic perspectives for more disciplines that tend to need heavy intervention from human experts. Computational biology, which is the application field of this thesis, is one of those disciplines where computer technologies (software and hardware systems) are applied on cell biology research, drug discovery, and disease diagnosis. The research conducted in this thesis is primarily concerned with automating the analysis of calcium imaging data obtained by two-dimensional (2D) fluorescence microscopy (FM) over living cells. The thesis also presents a theoretical and empirical analysis of the state-of-the-art object detection techniques used in Region based Convolutional Neural Network (R-CNN) and proposes a new R-CNN scheme tailored for cellular object detection in FM data. The analysed images are either individual greyscale images or image stacks of cardiac myocytes stained with DNA markers or calcium indicators. The cells are special cardiac cells found in lung veins, called Pulmonary Vein sleeve Cells (PVC) and Neo-natal Rat Ventricular Myocytes (NRVM) extracted from heart muscle. PVC stains enable the analysis of Calcium signals effect on heart physiology, whereas NRVM images allow autophagy process measuring through accurate cell counting. In the thesis, we demonstrated that automated hotspot detection can be achieved with 79.75% of precision by a two-level segmentation procedure combining thresholding and statistical filtering of cellular regions. We also showed that photobleaching can be corrected by exponential curve fitting and baseline adjustment from normalised calcium traces with respectively a mean square error (MSE) varying between [0.09013, 6.41796] and an overall accuracy of 78.75% for a real-life dataset. Finally, based on the investigation carried over state-of-the-art object detection techniques developed in the past such as the Edge Boxes, the Selective Search, the Objectness Measure, and the Sliding Window paradigm, we demonstrated that a customised R-CNN framework based on a data-driven proposal box generation outperforms with less sampling rate (61 proposals/image) and more ground truth coverage ratio (GTCR of 99.40%)

Biophysical properties of blood-stage Plasmodium falciparum malaria: from single-cell host-pathogen interactions to human protective polymorphisms

Malaria is a mosquito-borne infectious disease responsible for half a million deaths every year and long-term economic stagnation in many countries where it is endemic. All symptoms and pathology of malaria are caused by Plasmodium falciparum parasite, and are initiated when parasites invade human red blood cells, then mature and multiply inside them in approximately 48 hours. The invasion process is completed in less than a minute and is one of the most crucial, yet least understood, phases of malaria infection. It also represents a brief window in which the parasites are extracellular and hence exposed to the host immune system, therefore representing a potential target for vaccines and treatments. The work described in this thesis firstly includes the optimisation of a real-time live microscopy platform for recording parasite egress-invasion sequences under controlled conditions, and to investigate their morphology and kinetics. This set-up was employed to address the role of calcium in mediating successful invasion by observing the invasion process simultaneously in bright-field and fluorescence. Elevated calcium signal was found to be absent during the early steps of the process, implying that calcium does not trigger invasion, and an alternative invasion mechanism was suggested. To investigate whether parasite and host cell physical parameters were actively involved in invasion, adhesion forces between parasites and red cells were measured with optical tweezers, while the biophysical properties of the red blood cells such as bending modulus, tension, radius, and viscosity were assessed by analysing their plasma membrane fluctuations. In particular, cells from the Dantu blood group, a rare blood variant found mainly in East Africa that provides up to 70% protection against malaria, and from Beta-thalassaemia individuals, were studied. A general correlation between red blood cell membrane tension, invasion efficiency and dynamics was established, determining a protective tension threshold above which cells are less likely to be invaded. Finally, mature parasites have the ability to bind to the endothelium of peripheral blood vessels, causing impair flow that can lead to a range of fatal conditions. To study malaria cytoadherence to endothelial cells, a microfluidic device was designed to produce an in vitro physiologically relevant model of human circulation. Increasing cytoadhesion was experimentally associated with endothelial glycocalyx disruption as initial factor for malaria pathogenesis. Live imaging methods and techniques adopted in this study highlight mechanisms crucial for malaria infection, and represent an innovative and complementary study of this disease with respect to purely biological approaches. These findings show how changes in red blood cell biophysics can be linked to human evolutionary response against malaria with tangible effects on the population

Astrocyte calcium activity mapping in behaving mice using anterograde axo-astrocytic AAV transfer

Astrocytes are considered active partners to neurons in information processing. Heterogeneous, bidirectional interaction between neurons and astrocytes alludes to circuit specific communication. However, the role of astrocytes in information processing, primarily established through ex vivo experiments, has been challenged by a series of controversies that highlighted the importance of studying astrocytes under fully physiological conditions in behaving mice. Astrocytes extend highly ramified processes that cradle synapses. They form functionally independent microdomains where they exhibit a rich repertoire of localized calcium signals. How astrocyte Ca²+ microdomain signals relate to neuronal activity and behaviour in vivo is still unclear. My objective was to investigate circuit specific, single-astrocyte Ca²+ microdomain activity in mice during behavioural states and sensory stimuli. I found that adeno-associated viruses (AAVs) can transfer anterogradely along thalamocortical projections to transduce cortical astrocytes and neurons. This axo-astrocytic AAV transfer enables the study of astrocytes and neurons embedded in specific neuronal circuits. Intersectional approaches, using anterograde axo-astrocytic AAV transfer in combination with membrane tagged genetically encoded calcium indicators (GECIs), enabled sparse, high contrast labelling of cortical astrocytes embedded in the somatosensory system of mice. Continuous imaging with two-photon microscopy of single astrocytes for about ≈1 hour combined with automatic, unbiased extraction of Ca²+ activity revealed a rich repertoire of subsecond, µm scale, localized Ca²+ signals. The number, size and duration of astrocytic Ca²+ signals were modulated with locomotion but not with intermittent whisker-touch stimulation. Locomotion and whisker stimuli evoked rapid [Ca²+]i elevation in thalamocortical axon boutons, whose activity was not correlated with nearby astrocyte Ca²+ microdomain signalling. Astrocyte fine processes exhibit heterogeneous, non-random, Ca²+ signalling patterns giving rise to hotspots of higher activity that are stable over time. Hotspot patterns allude to subcellular specialization. Our study: a) provides a new toolkit for studying neuron-astrocyte interactions within brain circuits, b) extends our understanding of astrocyte Ca²+ microdomain signalling and relationship to neuronal activity in behaving animals and c) suggests that there are astrocyte Ca²+ activity maps in the brain.Okinawa Institute of Science and Technology Graduate Universit

Zooming in on leukocyte extravasation:Discovering new pathways with innovative technologies

Under inflammatory conditions, leukocytes are directed to exit the vasculature and pass through the endothelial barrier. In this thesis, we focus on specific aspects of the multistep transendothelial migration (TEM) process. First, we give a literature overview of the changes in endothelial actin dynamics upon inflammation and during leukocyte TEM that facilitate this process. Next, we performed a proteomics analysis of the ICAM-1 adhesome that is recruited at the docking site of the leukocyte, mapping the entire complex for the first time. We follow up on this screen by demonstrating the importance of CD44 for efficient cytotoxic T-lymphocyte (CTL) extravasation. Another follow-up study demonstrated that CTL form a transmigration synapse that is essential extravasation via the transcellular route. In the second part of this thesis, we generated a new leukocyte TEM assay by adapting blood-vessel-on-a-chip (BVOAC) technology. Current models to study leukocyte extravasation are based on endothelium cultured on stiff surfaces, which means TEM can only be studied until the point where leukocytes cross the endothelium. We demonstrate that BVOAC can be used to study the complete leukocyte TEM process from the vascular lumen into the perivascular tissue matrix with real-time and 3D microscopy. We applied this model to characterize migration dynamics induced by the endothelium in CTL and neutrophils. Finally, we show with our BVOAC model that neutrophils from a patient with a deficiency in the ARPC1B gene are impaired in both traversing the endothelium and migration into the tissue matrix

Testing the role of extracellular vesicles in early left right patterning

Abstract Bilaterian animals, such as humans, are characterized by an external roughly mirror symmetry along the left – right axis that covers a pronounced internal asymmetric arrangement of the thoracic and abdominal organs. While external symmetry has been associated with health and beauty standards, the internal asymmetry may rely more on efficiency and functionality of the different physiological systems. The left – right asymmetry of visceral organs is established early on during embryonic development within a transient and specialized structure, commonly referred to as the left – right organizer (LRO). The LROs appear in many shapes and sizes, depending on the species, but a common feature in some vertebrates is the requirement of motile cilia. The movement of these tiny hair-like protrusions generate a directional fluid flow, that scales with the cube of cilia length, in order to become capable of triggering a differentiated response on the left side of the LRO. Such flow-dependent response involves Pkd2 channel activation and calcium signaling that subsequently drive the left sided expression of the Nodal signaling cascade. Nodal is a secreted protein that translates the asymmetries established at the LRO to the rest of the embryo, through the lateral plate mesoderm. As embryonic development evolves, at specific time points and locations along the anterior – posterior axis, Nodal induces the expression of genes involved in the formation of the heart, brain, gut and its derivatives, modulating the lateralization of these organs. With this work, we dedicated our efforts to understanding a few molecular and cellular steps missing in the establishment of the left – right axis within the LRO. In the Chapter 2, we explored how the fluid flow is sensed by the LRO cells. Between the two hypotheses in the field, one based on mechanosensing and other on chemosensing properties of the flow, we found that the number of extracellular vesicles is too low and variable to transport sufficient and efficiently a sidedness molecular signal towards the left sided LRO cells. Moreover, pharmacological impairment of distinct endocytic pathways did not impact on heart laterality arrangement. We also found out an upstream regulator of Notch signaling, syntenin-a, involved in the cell fate decision between motile and immotile cilia. We showed that syntenin-a loss-of-function severely affected the left – right axis development. By downregulating the levels of syntenin a, Notch signaling is activated increasing the expression of her12 and resulting in a higher number of immotile cilia, in concordance with our previous published data. We next described a potential molecular switch, downstream of Notch signaling, composed by the Rabconnectin complex. As this complex is known to promote V-ATPase assembly and consequently its activity, we inhibited the V-ATPase activity and we observed an increase in the number of motile cilia. Thus, suggesting that the link between Notch signaling and motile – immotile cilia ratio is through the modulation of pH. Lastly, in Chapter 3, we focused on the impact of ciliary dysfunction in the epithelial respiratory cells. We characterized the distribution pattern of several ciliary proteins in two siblings harboring a primary ciliary dyskinesia causing mutation on Zmynd10 gene. Recent studies showed that ZMYND10 is one of the cytoplasmatic factors responsible for stabilizing and driving axonemal dynein arm assembly. We showed here that outer and inner axonemal dyneins, that become mostly absent from the ciliary axoneme in Zmynd10 mutant respiratory ciliated cells, can sometimes enter the proximal part of the cilium. These results suggest that to a low extent the dynein arms can still assemble and be transported into the cilium in the absence of ZMYND10, thus opening an opportunity for small-molecule therapies that promote protein stability in primary ciliary dyskinesia disease management.Resumo Os animais bilaterais, como os humanos, são caracterizados por uma simetria externa ao longo do eixo esquerda – direita que cobre um arranjo interno pronunciadamente assimétrico dos órgãos torácicos e abdominais. Enquanto a simetria externa tem sido associada a padrões de saúde e beleza, a assimetria interna pode depender maioritariamente da eficiência e funcionalidade da montagem dos diferentes sistemas fisiológicos. Esta assimetria esquerda – direita dos órgãos viscerais é estabelecida durante o desenvolvimento embrionário dentro de uma estrutura transiente e especializada, normalmente conhecida por organizador esquerda – direita. Os organizadores esquerda – direita aparecem em várias formas e tamanhos, dependendo da espécie, mas uma característica comum em alguns dos vertebrados é a existência de cílios. Os cílios são organelos compostos por microtúbulos que são projetados da superfície da célula. E estes podem ser móveis ou imóveis dependendo da presença ou ausência de proteínas motoras, as dineínas do axonema, que geram energia suficiente para mover o cílio. No caso do organizador esquerda – direita, os dois tipos de cílios estão presentes e desempenham funções distintas: os cílios móveis promovem um fluxo direcional do fluido existente no lúmen dos organizadores, cuja velocidade é proporcional ao cubo do comprimento ciliar, e os cílios imóveis são potencialmente responsáveis por detetar esse mesmo fluxo. Por conseguinte, a deteção do fluxo desencadeia uma resposta assimétrica nas células do lado esquerdo do organizador esquerda – direita, que é dependente do canal de cálcio Pkd2 localizado nos cílios. Assim, os iões de cálcio entram pelo cílio e ativam a libertação de mais iões dos organelos internos, o que resulta numa onda de cálcio propagada pela célula que, por sua vez, é necessária para iniciar uma cascada molecular de sinalização composta por Nodal e os seus inibidores. Nodal é um factor secretado da família TGF-β inicialmente expresso em redor do organizador esquerda-direita de forma simétrica. Um dos seus antagonistas expresso no organizador, Dand5, impede a propagação precoce e simétrica de Nodal para a placa lateral da mesoderme. Contudo, a onda de cálcio que se forma nas células do organizador promove a degradação de dand5, tornando-se assim o primeiro gene assimetricamente expresso e libertando Nodal da sua repressão especificamente no lado esquerdo do organizador. Consequentemente, Nodal é capaz de ativar a sua própria expressão na placa lateral da mesoderme do lado esquerdo e a expressão de um segundo inibidor, lefty1, na linha mediana, de forma a impedir que Nodal ative a sua expressão no lado direito. À medida que o desenvolvimento embrionário evolui, Nodal propaga-se pela mesoderme ao longo do eixo anterior - posterior, que em estadios e regiões específicas, leva à expressão de genes envolvidos na formação do coração, cérebro, fígado, pâncreas, entre outros, modulando a lateralização destes órgãos. Este campo da biologia do desenvolvimento tem evoluído bastante ao longo dos últimos anos, contudo algumas questões continuam em aberto. A forma como o fluxo é detetado pelas células do organizador esquerda – direita é uma delas. Historicamente, o campo está dividido em torno de duas hipóteses principais – o modelo quimiossensor e o modelo mecanossensor. Por um lado, o modelo quimiossensor propõe que o fluxo serve para transportar vesículas e moléculas sinalizadoras para o lado esquerdo, onde serão internalizadas pelas células do organizador. Por outro lado, o modelo mecanossensor baseia-se na força hidrodinâmica que o fluxo exerce sobre os cílios imóveis. Com este projeto de doutoramento pretendemos fornecer novos dados do mecanismo biofísico impulsionado pelo fluxo usando o organizador esquerda – direita do peixe-zebra como modelo animal. Inicialmente, dedicámo-nos a inspecionar as características moleculares das células do organizador e o conteúdo de fluído para inferir sobre as possíveis contribuições do modelo quimiosensor na deteção do fluxo de fluidos pelo canal Pkd2. Para tal, gerámos uma linha transgénica para quantificar e permitir o rastreamento de vesículas extracelulares dentro do lúmen do organizador e usámos uma nova configuração de micromanipulação para modificar o conteúdo do fluido do organizador. Os nossos resultados mostram que o número de vesículas extracelulares detetadas é muito baixo e variável para transportar um sinal molecular de lateralidade de forma eficiente para as células do organizador do lado esquerdo. Adicionalmente, a inibição farmacológica de vias endocíticas distintas não teve impacto na lateralidade do coração. De seguida, analisámos a regulação do número de cílios móveis e imóveis no organizador esquerda – direita. No peixe zebra, todos os cílios têm a ultra estrutura necessária para se moverem, contudo, apenas alguns cílios se tornam móveis. O nosso grupo tinha anteriormente descoberto que a decisão entre móvel e imóvel é feita pela via de sinalização de Notch. Com este trabalho, nós identificámos novos moduladores a montante e efetores a jusante da sinalização de Notch envolvidos neste processo. Mostrámos que a perda de função da syntenin-a afeta severamente o desenvolvimento do eixo esquerda – direita, uma vez que ativa a sinalização de Notch e a expressão do seu gene alvo, her12, o que resulta num número maior de cílios imóveis e por conseguinte num fluxo do fluído menor. Também descrevemos um potencial botão molecular, a jusante da sinalização de Notch, composto pelo complexo Rabconnectin. Uma vez que este complexo é conhecido por promover a montagem da V-ATPase e consequentemente sua atividade, inibimos a atividade da V ATPase e observámos um aumento do número de cílios móveis. Assim, sugerimos que a ligação entre a sinalização de Notch e a proporção de cílios móveis – imóveis se dá através da modulação do pH. Por fim, no Capítulo 3, focámo-nos no impacto da disfunção ciliar nas células epiteliais respiratórias. Caracterizámos o padrão de distribuição de várias proteínas ciliares em dois irmãos portadores de discinésia ciliar primária causada por uma mutação no gene Zmynd10. Estudos recentes mostram que ZMYND10 é um dos fatores citoplasmáticos responsáveis por estabilizar e conduzir a montagem do braço de dineína que constituí o axonema do cílio móvel. Mostrámos aqui que as dineínas externas e internas do axonema, que se tornam principalmente ausentes do cílio em células respiratórias mutadas no gene Zmynd10, podem, no entanto, entrar na parte proximal do cílio. Estes resultados sugerem que uma pequena porção dos braços de dineína conseguem ser montados e transportados para o cílio na ausência de ZMYND10, abrindo assim uma oportunidade para terapias com pequenas moléculas que promovam a estabilidade de proteínas na gestão do tratamento da doença de discinésia ciliar primária

Characterization of cell uptake and intracellular trafficking of exosomes by quantitative live cell imaging: towards biomimetic delivery vehicles of therapeutic RNA

Exosomes are biological nanoparticles which play a role in long distance cell-to cell communication. These 40-100 nm sized vesicles are released by virtually all cells and derive from the multivesicular bodies within their parent cells. They modulate their target cell fate by induction of cell signaling as well as RNA and protein cargo transfer. Exosomes have also moved into the spotlight of clinical research, with potential use as biomarkers or next generation therapeutic delivery agents. Exosomes are thought to be highly efficient intercellular messengers but quantitative characterization is lacking. Also, their routes of cell uptake and subcellular fate within recipient cells remain elusive. This work introduces an in depth and quantitative characterization of exosome cargo, physicochemical properties, labeling, isolation and their recipient cell interaction at the single cell – single vesicle level. Basic protocols for exosome purification were revisited in order to allow for isolation of exosomes with sufficient yields and in as native state as possible to enable functional studies. Since exosome integrity and recovery yields after differential ultracentrifugation (UC), the most commonly used protocol for exosome isolation, turned out to be poor and unreproducible, we describe an alternative protocol based on ultrafiltration (UF) with subsequent gel filtration (GF) for recovering exosomes relatively selectively, with intact biophysical and functional properties and significantly higher yields. Next we establish methods for specific exosome labeling using fluorescent marker proteins transiently expressed in parent cells, which led to a focus on FP tagged CD63 constructs. CD63-emGFP labeled exosomes were extensively characterized and showed identical properties compared to unlabeled exosomes based on sucrose density gradient, CryoTEM microscopy and proteomics analysis. Furthermore, we successfully adapted fluctuation correlation spectroscopy for characterization of fluorescently labeled exosomes. In another part of this work we describe a high content screen for exosome uptake which we use to provide a first systematic and quantitative profiling of exosome uptake across a panel of exosome parent recipient cells, including HEK293, Huh7, B16F10 as parental cells and additional primary fibroblasts, primary keratinocytes, iPS derived motor neurons and HUVEC primary human endothelial cells as recipient cell lines. These quantitative profiling data reveals preferences in exosome internalization by different cell types and suggests that specific receptor ligand interactions may determine tissue specificity. Finally, we address one of the fundamental questions in the field of cellular communication: how exosomes released by one cell enter and interact with their recipient cell. Our data quantifies for the first time the cell uptake dynamics of exosomes at the single vesicle and single cell level and reveals a quantitative efficiency paralleling that of infective pathogens rather than artificial delivery vehicles. We demonstrate that exosome uptake is largely mediated by active recruitment and surfing on filopodia to reach endocytic hotspots for their internalization at the filopodia base. This provides a cell biological explanation for the remarkably high efficiency of exosomes in targeting recipient cells and discovers a new parallel to some viruses and other pathogens. We propose that the process of filopodia surfing may have evolved as a highway for exosomes into the cell, being hijacked by certain pathogens for host cell interaction. This data does not support the previously reported exosome uptake by vesicle fusion with the plasma membrane or cargo release by endosomal escape. Instead we observe intact exosome uptake to enter endocytic vesicles, which then scan along the endoplasmic reticulum (ER) and end up in lysosomes. Our data suggest a model of controlled cargo delivery to defined subcellular localizations like the ER, rather than vesicle fusion and free release into the cytoplasm

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin