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Hamed, MM;Taniguchi, K;Duncan, MC.
Methods in molecular biology (Clifton, N.J.),
Membrane traffic at the Golgi and endosomes plays many critical roles in the polarization and the morphogenesis of epithelial tissues. Studies into the roles of traffic in morphogenesis in mammals are often complicated by early embryonic lethality of mutations in membrane traffic as well as the inherent difficulty in imaging developing embryos posed by their size and location. Increasingly, human pluripotent stem cell (hPSC)-derived embryo- and organ-like systems (e.g., embryoids, organoids) provide a useful platform to illuminate the requirements of traffic in human embryonic tissue morphogenesis because these in vitro models are highly amenable to fluorescence microscopy and provide the ability to examine the role of essential genes not possible with animal studies. Here, we present a method to generate hPSC-cysts, a 3-D hPSC-based model of human epiblast lumen formation. This system provides unique opportunities to examine the role of membrane traffic during epithelial morphogenesis. We also present methods to process hPSC-cysts for immunofluorescence and staining with commonly used fluorescence labels useful for detecting defects in polarization and morphogenesis caused by defects in membrane traffic.
Puri, D;Fang, X;Allison, KR.
Biofilms are surface-attached multicellular microbial communities. Their genetics have been extensively studied, but the cell-scale morphogenetic events of their formation are largely unknown. Here, we recorded the entirety of morphogenesis in Escherichia coli, and discovered a previously unknown multicellular self-assembly process. Unattached, single-cells formed 4-cell rosettes which grew into constant-width chains. After ∼10 cell generations, these multicellular chains attached to surfaces and stopped growing. Chains remained clonal throughout morphogenesis. We showed that this process generates biofilms, which we found are composed of attached clonal chains, aligned in parallel. We investigated genetics of chain morphogenesis: Ag43 facilitates rosette formation and clonality; type-1 fimbriae and curli promote stability and configuration; and extracellular polysaccharide production facilitates attachment. Our study establishes that E. coli, a unicellular organism, can follow a multistage, clonal, genetically-regulated, rosette-initiated multicellular life cycle. These findings have implications for synthetic biology, multicellular development, and the treatment and prevention of bacterial diseases.
Sgritta, M;Vignoli, B;Pimpinella, D;Griguoli, M;Santi, S;Bialowas, A;Wiera, G;Zacchi, P;Malerba, F;Marchetti, C;Canossa, M;Cherubini, E.
In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.
Baudon, A;Clauss-Creusot, E;Darbon, P;Patwell, R;Grinevich, V;Charlet, A.
Astrocytes are glial cells that exhibit calcium signaling-mediated activity. Here, we present a protocol to monitor and manipulate astrocyte calcium activity from mouse amygdala slices. In the first part of this protocol, we describe the procedure of astrocyte calcium imaging. In the second part, we detail how to disrupt astrocyte calcium activity by patch-clamp-mediated loading of BAPTA. These two approaches are presented separately but they can also be used simultaneously to monitor the effects of disruption on an astrocyte network. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021).
Swoger, M;Gupta, S;Charrier, EE;Bates, M;Hehnly, H;Patteson, AE.
ACS applied bio materials,
The ability of cells to take and change shape is a fundamental feature underlying development, wound repair, and tissue maintenance. Central to this process is physical and signaling interactions between the three cytoskeletal polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk among the cytoskeleton, but its role in how cells sense and respond to the surrounding extracellular matrix is largely unclear. To investigate vimentin’s role in substrate sensing, we designed polyacrylamide hydrogels that mimic the elastic and viscoelastic nature of in vivo tissues. Using wild-type and vimentin-null mouse embryonic fibroblasts, we show that vimentin enhances cell spreading on viscoelastic substrates, even though it has little effect in the limit of purely elastic substrates. Our results provide compelling evidence that vimentin modulates how cells sense and respond to their environment and thus plays a key role in cell mechanosensing.
Torrente, D;Su, E;Schielke, G;Warnock, M;Stevenson, T;Mann, K;Vivien, D;Lawrence, D.
SSRN Electronic Journal,
The protease tissue plasminogen activator (tPA) is linked to diverse functions in the central nervous system. Here we characterize the expression and localization of tPA in the substantia nigra (SN) and explore the role of tPA in dopaminergic neuron degeneration in a human α-synuclein (hα-SYN) mouse model of Parkinson’s disease. We found that striatal GABAergic neurons send tPA + axons that innervate the SN proximal to dopaminergic neuronal cell bodies and axons. tPA deficiency protected dopaminergic neurons from degeneration and reversed behavioral deficits induced by hα-SYN-induced neurotoxicity. tPA’s action was independent of its proteolytic activity, and could be blocked by treatment with Glunomab, a neutralizing antibody that selectively inhibits tPA interaction with N-methyl-D-aspartate receptor-1. Both tPA deficiency and Glunomab treatment prevented neuronal degeneration, and reduced microglia activation and T-cell infiltration. Together, these data demonstrate a previously unrecognized pathway promoting dopaminergic neuronal degeneration and suggest a potential therapeutic intervention with Glunomab.
Shen, L;Lin, C;Lu, W;He, J;Wang, Q;Huang, Y;Zheng, X;Wang, Z.
Colorectal cancer (CRC) is a common malignant cancer worldwide. Although the molecular mechanism of CRC carcinogenesis has been studied extensively, the details remain unclear. Small nucleolar RNAs (snoRNAs) have recently been reported to have essential functions in carcinogenesis, although their roles in CRC pathogenesis are largely unknown. In this study, we found that the H/ACA snoRNA SNORA24 was up-regulated in various cancers, including CRC. SNORA24 expression was significantly associated with age and history of colon polyps in CRC patient cohorts, with high expression associated with a decreased 5-year overall survival. Our results indicated that the oncogenic function of SNORA24 is mediated by promoting G1/S phase transformation, cell proliferation, colony formation and growth of xenograft tumors. Furthermore, SNORA24 knockdown induced massive apoptosis. RNA-sequencing and Gene Ontology (GO) enrichment analyses were performed to explore its downstream targets. Finally, we confirmed that SNORA24 regulates p53 protein stability in a proteasomal degradation pathway. Our study clarifies the oncogenic role of SNORA24 in CRC and advance the current model of the role of the p53 pathway in this process.
Zaccaria, C;Mancinelli, M;Vignoli, B;Auslender, I;Bettotti, P;Malkoc, A;Canossa, M;Pavesi, L.
Optogenetics and Optical Manipulation 2022,
To study the brain and the related neuronal network activity, many attempts were made to design and develop platforms able to induce and record neuronal signals. However, many brain processes – like memory formation and storage – and diseases – like amnesia or epilepsy – need more basic studies. For these, a bottom-up approach is needed, starting from 2D in-vitro neuronal cultures. In this work, we will present two experimental systems able to optogenetically interact with 2D neuronal networks with patternized light. One system consists in a Digital Light Projector (DLP) integrated in a microscope setup, which can illuminate neurons from the top; the other, is a compact and transportable photonic chip, properly designed to illuminate neurons plated on its surface.
Erbacher, C;Britz, S;Dinkel, P;Klein, T;Sauer, M;Stigloher, C;Üçeyler, N.
Traditionally, peripheral sensory neurons hold the monopole of transducing external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nociceptor interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of nociceptors and connexin 43 plaques at keratinocyte-nociceptor contact sites in healthy native skin. We further derived a fully human co-culture system, modeling ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and interaction sites marks a milestone in research at the neuro-cutaneous unit. These findings are mind-changers on the way to decipher the mechanisms of cutaneous nociception.
Rosito, M;Sanchini, C;Gosti, G;Moreno, M;Panfilis, S;Giubettini, M;Debellis, D;Catalano, F;Peruzzi, G;Marotta, R;Indrieri, A;Leonibus, E;Ragozzino, D;Ruocco, G;Angelantonio, S;Bartolini, F.
Microglia reactivity triggered by proinflammatory signaling entails a large-scale remodeling of cellular geometry, but the role of the microtubule cytoskeleton during these changes remains unexplored. Here we show that reactive proinflammatory microglia provide a heretofore unique example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, proinflammatory signaling induces recruitment of nucleating material nearby the centrosome and inhibition of de novo formed pericentrosomal microtubule-organizing centers enhances NLRP3 inflammasome activation and secretion of the interleukin IL-1β. Our results demonstrate that remodeling of the microtubule cytoskeleton is a hallmark of proinflammatory microglia reactivity and suggest that pericentrosomal microtubule nucleating material maturation may offer a valuable target to modulate cytokine-mediated inflammatory responses in chronic disease and tissue injury.
van Buren, L;Koenderink, G;Martinez-Torres, C.
Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery’s three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple pre-defined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of non-spherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in GUV research.
Metcalf, K;Hayward, M;Berens, E;Ironside, A;Stashko, C;Hwang, E;Weaver, V.
Matrix Biology Plus,
Tumors feature elevated sialoglycoprotein content. Sialoglycoproteins promote tumor progression and are linked to immune suppression via the sialic acid-Siglec axis. Understanding factors that increase sialoglycoprotein biosynthesis in tumors could identify approaches to improve patient response to immunotherapy. We quantified higher levels of sialoglycoproteins in the fibrotic regions within human breast tumor tissues. Human breast tumor subtypes, which are more fibrotic, similarly featured increased sialoglycoprotein content. Further analysis revealed the breast cancer cells as the primary cell type synthesizing and secreting the tumor tissue sialoglycoproteins and confirmed that the more aggressive, fibrotic breast cancer subtypes expressed the highest levels of sialoglycoprotein biosynthetic genes. The more aggressive breast cancer subtypes also featured greater infiltration of immunosuppressive SIGLEC7, SIGLEC9, and SIGLEC10-pos myeloid cells, indicating that triple-negative breast tumors had higher expression of both immunosuppressive Siglec receptors and their cognate ligands. The findings link sialoglycoprotein biosynthesis and secretion to tumor fibrosis and aggression in human breast tumors. The data suggest targeting of the sialic acid-Siglec axis may comprise an attractive therapeutic target particularly for the more aggressive HER2+ and triple-negative breast cancer subtypes.
Torrente, D;Su, E;Schielke, G;Warnock, M;Mann, K;Lawrence, D.
Background Noradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood. Methods Two mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD. Results Consistent with previous studies we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration whereas xamoterol a β-1AR agonist showed increased neuroinflammation and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity. Conclusion Our data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN driven neuropathology β2-AR specific agonists may have therapeutic benefit in PD.
Zhanghao, K;Li, M;Chen, X;Liu, W;Li, T;Wang, Y;Su, F;Wu, Z;Shan, C;Wu, J;Zhang, Y;Fu, J;Xi, P;Jin, D.
The number of colors in fluorescence microscopy is far less than the types of intracellular compartments, bringing gaps in studying live-cell anatomy and multiple organelles’ interactions. Here, we report that super-resolution imaging in association with deep convolutional neuronal networks can predict 15 subcellular structures at > 91.7% pixel accuracy using one laser excitation and two detection channels. It not only bypasses the limitations of multi-color imaging with single dye labeling but also accelerates the imaging speed by more than one order of magnitude. We find that the super-resolution ratiometric images well reflect the heterogeneity of organelles as the intrinsic “optical fingerprint” and the neuronal networks can be generalized with transfer learning to predict both 3D and 2D datasets from different microscopes, different cell types, and even complexed system of living tissues. It enables us to resolve the 3D anatomic structure of live cells at different mitotic phases and to track down the fast dynamic interactions among 9 intracellular compartments.
Chen, C;Humphrey, L;Jema, S;Ferrari, F;Joglekar, A.
The Spindle Assembly Checkpoint (SAC) is activated by unattached or laterally attached kinetochores in a dividing cell. It delays anaphase onset and thus prevents chromosome missegregation. To minimize chromosome missegregation, the last signaling kinetochore in the cell must produce the anaphase-inhibitory signal at a high rate to delay anaphase onset. Additionally, the large number of signaling kinetochores should be prevented from generating an unnecessarily large signal. How the cell balances these conflicting requirements remains unclear. Here, we show that the number of SAC proteins recruited by an unattached kinetochore is inversely correlated with the number of unattached kinetochores in HeLa cells. This correlation primarily results from the low amount of the protein Bub1, and this protein recruitment shortfall reduces the signaling activity of the kinetochore enabling chromosome missegregation. Conversely, Bub1 overexpression results in higher SAC protein recruitment and longer delays. Thus, the regulation of the signaling activity of unattached kinetochores by mass action kinetics promotes accurate chromosome segregation.
Gökçe, F;Kaestli, A;Lohasz, C;de Geus, M;Renggli, K;Bornhauser, B;Hierlemann, A;Modena, M.
Despite increasing survival rates of pediatric leukemia patients over the past decades, the outcome of some leukemia subtypes has remained dismal. Drug sensitivity and resistance testing on patient-derived leukemia samples provides important information to tailor treatments for high-risk patients. However, currently used well-based drug screening platforms are severely limited in predicting the effects of prodrugs, a class of therapeutics that require metabolic activation to become effective. To address this limitation, we developed a microphysiological drug-testing platform that enables co-culturing of patient-derived leukemia cells, human bone marrow mesenchymal stromal cells, and human liver microtissues within the same microfluidic platform that, at the same time, regulates the physical interaction between the diverse cell types. Our model recapitulates hepatic prodrug activation of ifosfamide, which cannot be assessed in traditional well-based assays. By testing the susceptibility of primary patient-derived leukemia samples to the prodrug ifosfamide, we identified sample-specific sensitivities to ifosfamide in primary leukemia samples. Our microfluidic platform enables the recapitulation of physiologically relevant conditions and the testing of prodrugs, particularly with short-lived and unstable metabolites, which provides a basis for clinical translation and precision chemotherapy selection.
Huayta Ramirez, J.
In developing countries, the number of elderly adults will continue to increase in the near future, causing a higher impact on society. Amongst the causes for these population tendencies is the improvement on health services experienced during the previous decades. Consequently, this has caused a rise in lifespan, defined as the maximum amount of time an organism in a species can live . Furthermore, elderly adults have a higher risk of exhibiting diseases such as Alzheimer’s disease, Parkinson’s disease, and several forms of dementia ; this underscores the relevance of performing studies on the effect of environmental stressors and their influence in the aging process. For that purpose, we have a powerful model organism for in C. elegans, a microscopic nematode that has been important in elucidating genetic and environmental factors affecting lifespan. This work would be the first of its kind to use an integrated approach using CRISPR/Cas9, deep learning, and machine learning tools to characterize lifespan and healthspan, while associating this metric to the spatiotemporal activity of longevity genes. Our systems biology approach will enable the advancement of our to understanding of the fundamental mechanisms by which genetic pathways regulate the lifespan and stress resistance. In Chapter 2 we modify and improve an existing CRISPR/Cas9 approach to allow us the generation of transgenic lines. These strains will enable tracking of endogenous levels of expression of transcription factors that regulate the stress response in C. elegans. We will also demonstrate that DAF-16 and PHA-4 have different responses to the same stimuli, giving insights on how a change in food source induces stress in these animals. In Chapter 3, we use our DAF-16 strain to explore further the response of C. elegans to dietary restriction and its role in dietary restriction. For this purpose, we describe the use of a custom/made MATLAB algorithm and deep learning tools to track endogenous DAF-16. To achieve this we expose the animals to various dietary restriction regimes and quantify the DAF-16 response and lifespan extension conveyed by its activity. We show that by combining these metrics, we are able to account for up to 78% of lifespan variation by using only DAF-16 activity as input. Furthermore, we describe that the main contributors to lifespan extension are neuron and intestinal cells. Finally, we describe a new interaction of DAF-16 in the cell nucleoli, where we observed DAF-16 migration, indicating a possible mechanism for lifespan extension in nucleolus previously undescribed. In Chapter 4 we describe the development of our custom-made MATLAB algorithm and other deep learning, and machine learning tools that we sued to extract the data in Chapter 3. This includes a new algorithm that is able to track the subtle translocation patterns of endogenous single-copy DAF-16, a Fine Tree Classifier that enables assessment of tissue contribution to DAF-16 activity, and a Mask R-CNN that can see potential use to track morphological changes of C. elegans pharynx. In Chapter 5 we develop a platform for the evaluation of combinatorial exposure to xenobiotics and environmental factors, by using a simplex centroid design. This experimental approach enables us to evaluate the oxidative response in C. elegans under conditions of food limitations and heat-shock. We determined that dietary restriction enhances the effect of ternary mixtures of naphthoquinones while heat-shock abrogates the oxidative response to these chemicals. This puts in perspective the importance of performing these combinatorial analyses using our approach. In Chapter 6, we explore methods and techniques for the assessment of healthspan and lifespan in short-lived C. elegans mutants. We describe that these metrics are robust to indicate that the strains evaluated not only have a diminished longevity but also have an early onset of aging phenotypes in the pharynx and their locomotion.
Giant unilamellar vesicles (GUVs) are a desired membrane‐mimetic system for the study of many membrane‐related phenomena, the function of MPs and the creation of synthetic cells. These micrometer sized vesicles are similar in size to bacteria and eukaryotic cells, and thus mimic these organisms more closely in terms of surface and volume. The size allows the integration of complex systems and entire metabolic processessuch astranscription and translation, as well asthe investigation of individual vesicles using light microscopy techniques, potentially cutting the costs of purified MPs needed to perform experiments by a factor of 100 compared to bulk methods. This makes them a very attractive system to investigate MPs, for example to test and develop novel drugs, and to create a bottom‐up synthetic cell. However, lipids do not spontaneously assemble into cell‐sized vesicles which has prompted the development ofseveral different techniquesfor GUV formation. For the same reason, these vesicles are more fragile towards the use of detergents, which complicates MP reconstitution. To harness the power of light microscopy measurements, GUVs have to be immobilized to enable real‐time observation over several minutes to hours. Lastly, the success of measuring MP function in a GUV also depends on the choice of detection system. In previous work in our lab, GUV electroformation on indium‐tin‐oxide (ITO) coated glass slides and reconstitution of MPs using charge‐mediated fusion of oppositely charged vesicles was established. GUVs were immobilized using a streptavidin‐biotin system to enable measurement of MP function. One of the disadvantages of GUV electroformation on ITO coated glass slides is the poor compatibility with high ionic solution, which could result in low protein activities due to formation at non‐physiological conditions. One of the aims of this project wasto establish GUV formation under physiologically relevant conditions to allow formation in buffer compositions optimal for MP function. We thus compared previously established electroformation on ITO coated glass slides with electroformation on platinum (Pt) wires and the more recently developed polymer assisted swelling using PVA. We observed that both Pt wire and PVA formation produced GUVs using various buffer compositions and that polymer assisted swelling produced a high yield of GUVs without much optimization, showing the potential and versatility of this method. Interestingly, we discovered that the immobilization was affected by the buffer composition, and that strong adhesion can lead to leakage and loss of encapsulated cargo, especially in PVA GUVs. This is an important finding as MPs are frequently followed using encapsulated fluorescent dyes, showing that immobilization conditions have to be tuned according to the buffer composition to provide sufficient immobilization while preventing too much cargo loss. Protons play an important role in many cellular processes, they are involved in many transmembrane transport reactions as well as in the production of ATP by the ATP synthase. Thus, GUVs should be able to maintain a proton gradient. Our measurements suggest that this is indeed the case also in immobilized vesicles that have not leaked vi encapsulated fluorophores. We further presented a simple strategy that could be used to estimate the protein concentration in GUVs after charge‐mediated reconstitution by fusion of GUVs with small vesicles containing labeled lipids and labeled MP. We could show that GUVs with more lipid‐coupled dye signal also showed more MP‐coupled dye signal, which could simplify the quantification of MPs in GUVs after fusion by following lipid‐coupled dye incorporation without the need for MP labeling. However, this strategy is not able to distinguish between simple adhesion or hemifusion of small vesicles and full fusion, which would be required for functional reconstitution of a MP. However, the same is true for simply following labeled MP signal, meaning that potentially other methods such as content mixing assays would be needed to get a better idea on the amount of functionally reconstituted MP. Finally, knowledge gained from the characterization of GUVs was applied for the reconstitution and measurement of cytochrome c oxidase from Rhodobacter sphaeroides using carboxyfluorescein and pyranine (HPTS). The former produced only weak and unclear signals and was prone to fast bleaching. Using ratiometric dyessuch as HPTS, pH calibration can be performed, where the observed ratiosshould be independent of dye concentration and bleaching. This proved to be challenging in GUVs, as the vesicles size seemed to have an effect on the observed HPTS ratio. Despite that, cytochrome c oxidase measurements using HPTS yielded better results and by characterization of vesicle shape and measurement of lipid‐coupled dye signal introduced via fusion, a correlation between the proton translocation and the relative amount of MP per vesicle could be observed, showing that thorough characterization of the GUVs can help to relate vesicle activities. Nonetheless, the different sizes of the vesicles were a considerable challenge for data analysis. We thus plan to use monodisperse GUVs produced by microfluidic techniques in collaboration with members of the deMello group form the ETH Zürich and we could show that these GUVs are fusogenic and can be potentially used to measure proton translocation. In a second project, we used bifunctional DNA duplexes to establish a new tool for the measurement of MP function. By linking pH‐sensitive dyes via DNA oligomers to cholesterol moieties, fluorescent probes can be anchored to the lipid membrane, allowing more efficient encapsulation compared to soluble dyes which could safe costs using precious probes. Addition of DNase I allows fast and simple removal of probes facing the liposome exterior, which are exposed to the buffered solution and thus do not contribute to the measurement of proton translocation in or out of the vesicle, a common problem with lipid‐coupled probes. Incorporation of bifunctional DNA into GUVs was slightly more challenging. Depending on the GUV formation method, as well as lipid and buffer composition, differences in the degree of incorporation into the membrane were observed, ranging from no membrane localization to complete incorporation. Formation furtherseemed to be negatively affected by the probe. Thus, further optimization of the probe might be needed to enable measurement of MP function in GUVs.
La Torre, M;Merigliano, C;Maccaroni, K;Chojnowski, A;Goh, WI;Giubettini, M;Vernì, F;Capanni, C;Rhodes, D;Wright, G;Burke, B;Soddu, S;Burla, R;Saggio, I.
Journal of experimental & clinical cancer research : CR,
Lamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53-/- mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins.We performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology.In HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized.Our data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.
Mori, M;Stelitano, G;Griego, A;Chiarelli, LR;Cazzaniga, G;Gelain, A;Pini, E;Camera, M;Canzano, P;Fumagalli, A;Scarpa, E;Cordiglieri, C;Rizzello, L;Villa, S;Meneghetti, F.
Pharmaceuticals (Basel, Switzerland),
Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure-activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.