scholarly journals Optical reconstruction of murine colorectal mucosa at cellular resolution

2015 ◽  
Vol 308 (9) ◽  
pp. G721-G735 ◽  
Author(s):  
Cambrian Y. Liu ◽  
Philip E. Dubé ◽  
Nandini Girish ◽  
Ajay T. Reddy ◽  
D. Brent Polk
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Confocal Imaging ◽  
Host Cells ◽  
Cellular Mechanisms ◽  
Colorectal Mucosa ◽  
Cellular Resolution ◽  
Volume Imaging ◽  
Mucosal Layer ◽  
Optical Reconstruction

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.

scholarly journals Caveolin-1 Regulates Cellular Trafficking and Function of the Glucagon-Like Peptide 1 Receptor

2006 ◽  
Vol 20 (12) ◽  
pp. 3400-3411 ◽  
Author(s):  
Colin A. Syme ◽  
Lei Zhang ◽  
Alessandro Bisello
Keyword(s):  
Cell Membrane ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Cellular Trafficking ◽  
Cellular Mechanisms ◽  
Caveolin 1 ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Signaling Activity

Abstract The glucagon-like peptide 1 receptor (GLP-1R) mediates important effects on β-cell function and glucose homeostasis and is one of the most promising therapeutic targets for type 2, and possibly type 1, diabetes. Yet, little is known regarding the molecular and cellular mechanisms that regulate its function. Therefore, we examined the cellular trafficking of the GLP-1R and the relation between receptor localization and signaling activity. In resting human embryonic kidney 293 and insulinoma MIN6 cells, a fully functional green fluorescent protein-tagged GLP-1R was localized both at the cell membrane and in highly mobile intracellular compartments. Real-time confocal fluorescence microscopy allowed direct visualization of constitutive cycling of the receptor. Overexpression of K44A-dynamin increased the number of functional receptors at the cell membrane. Immunoprecipitation, sucrose sedimentation, and microscopy observations demonstrated that the GLP-1R localizes in lipid rafts and interacts with caveolin-1. This interaction is necessary for membrane localization of the GLP-1R, because overexpression of a dominant-negative form of caveolin-1 (P132L-cav1) or specific mutations within the putative GLP-1R’s caveolin-1 binding domain completely inhibited GLP-1 binding and activity. Upon agonist stimulation, the GLP-1R underwent rapid and extensive endocytosis independently from arrestins but in association with caveolin-1. Finally, GLP-1R-stimulated activation of ERK1/2, which involves transactivation of epidermal growth factor receptors, required lipid raft integrity. In summary, the interaction of the GLP-1R with caveolin-1 regulates subcellular localization, trafficking, and signaling activity. This study provides further evidence of the key role of accessory proteins in specifying the cellular behavior of G protein-coupled receptors.

scholarly journals Identification of Six Autographa californica Multicapsid Nucleopolyhedrovirus Early Genes That Mediate Nuclear Localization of G-Actin

2002 ◽  
Vol 76 (23) ◽  
pp. 12281-12289 ◽  
Author(s):  
Taro Ohkawa ◽  
Annette R. Rowe ◽  
Loy E. Volkman
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Autographa Californica ◽  
Host Cells ◽  
Filamentous Actin ◽  
Progeny Production ◽  
Cellular Mechanisms ◽  
Early Genes ◽  
Green Fluorescent

ABSTRACT Nuclear filamentous actin (F-actin) is required for nucleopolyhedrovirus (NPV) progeny production in NPV-infected, cultured lepidopteran cells. We have determined that monomeric G-actin is localized within the nuclei of host cells during the early stage of infection by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). With a library of cloned AcMNPV genomic fragments, along with a plasmid engineered to express enhanced green fluorescent protein-Bombyx mori G-actin in transient transfection experiments, we identified six AcMNPV early genes that mediate nuclear localization of G-actin in TN-368 cells: ie-1, pe38, he65, Ac004, Ac102, and Ac152. Within this subset, ie-1 and pe38 encode immediate-early transcriptional transactivators, he65 encodes a delayed-early product, and the products encoded by Ac004, Ac102, and Ac152 have not been characterized. We found that when driven by foreign promoters, ie-1, pe38, and Ac004 had to be expressed prior to Ac102 or he65 for nuclear G-actin to accumulate and that expression of Ac152 was no longer required. These results and others suggested that the product of Ac152 was a transactivator (directly or indirectly) of both Ac102 and he65 and that recruitment of G-actin to the nucleus was a temporally regulated process. Determining the functions of each of the six AcMNPV gene products with respect to our assay should provide valuable clues to basic cellular mechanisms of actin regulation and how AcMNPV infection affects them.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

scholarly journals Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 346
Author(s):  
Caitlin W. Lehman ◽  
Kylene Kehn-Hall ◽  
Megha Aggarwal ◽  
Nicole R. Bracci ◽  
Han-Chi Pan ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Dynamics Simulation ◽  
Luciferase Reporter ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

scholarly journals Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

2021 ◽  
Vol 14 (8) ◽  
pp. 757
Author(s):  
Iga Jakobowska ◽  
Frank Becker ◽  
Stefano Minguzzi ◽  
Kerrin Hansen ◽  
Björn Henke ◽  
...  
Keyword(s):  
Rate Constants ◽  
Cross Correlation ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Confocal Imaging ◽  
Binding Kinetics ◽  
Acceptor Site ◽  
Lactate Transport ◽  
Entry Site ◽  
Hydrogen Bond Acceptor

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay.

scholarly journals Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Takuya Tomita ◽  
Shoshiro Hirayama ◽  
Yasuyuki Sakurai ◽  
Yuki Ohte ◽  
Hidehito Yoshihara ◽  
...  
Keyword(s):  
Cell Function ◽  
Fluorescent Protein ◽  
Embryonic Fibroblasts ◽  
Α Subunit ◽  
Biochemical Alterations ◽  
Cellular Processes ◽  
Responsible Gene ◽  
Intracellular Protein Degradation ◽  
Green Fluorescent

ABSTRACT The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

scholarly journals Mycobacterium tuberculosis infection of host cells in space and time

2019 ◽  
Vol 43 (4) ◽  
pp. 341-361 ◽  
Author(s):  
Claudio Bussi ◽  
Maximiliano G Gutierrez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Cell Biology ◽  
Current Knowledge ◽  
Bacterial Pathogen ◽  
Infectious Agent ◽  
Host Cells ◽  
Cellular Mechanisms ◽  
Mycobacterium Tuberculosis Infection ◽  
Human Host

ABSTRACTTuberculosis (TB) caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb) remains one of the deadliest infectious diseases with over a billion deaths in the past 200 years (Paulson 2013). TB causes more deaths worldwide than any other single infectious agent, with 10.4 million new cases and close to 1.7 million deaths in 2017. The obstacles that make TB hard to treat and eradicate are intrinsically linked to the intracellular lifestyle of Mtb. Mtb needs to replicate within human cells to disseminate to other individuals and cause disease. However, we still do not completely understand how Mtb manages to survive within eukaryotic cells and why some cells are able to eradicate this lethal pathogen. Here, we summarise the current knowledge of the complex host cell-pathogen interactions in TB and review the cellular mechanisms operating at the interface between Mtb and the human host cell, highlighting the technical and methodological challenges to investigating the cell biology of human host cell-Mtb interactions.

scholarly journals Molecular and Clinical Insights into the Invasive Capacity of Glioblastoma Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Carlos Velásquez ◽  
Sheila Mansouri ◽  
Carla Mora ◽  
Farshad Nassiri ◽  
Suganth Suppiah ◽  
...  
Keyword(s):  
Treatment Resistance ◽  
Host Cells ◽  
Cellular Mechanisms ◽  
Poor Overall Survival ◽  
Cellular Processes ◽  
New Therapeutic Approaches ◽  
Invasive Capacity ◽  
Molecular Machinery ◽  
The Impact ◽  
Infiltrative Tumor

The invasive capacity of GBM is one of the key tumoral features associated with treatment resistance, recurrence, and poor overall survival. The molecular machinery underlying GBM invasiveness comprises an intricate network of signaling pathways and interactions with the extracellular matrix and host cells. Among them, PI3k/Akt, Wnt, Hedgehog, and NFkB play a crucial role in the cellular processes related to invasion. A better understanding of these pathways could potentially help in developing new therapeutic approaches with better outcomes. Nevertheless, despite significant advances made over the last decade on these molecular and cellular mechanisms, they have not been translated into the clinical practice. Moreover, targeting the infiltrative tumor and its significance regarding outcome is still a major clinical challenge. For instance, the pre- and intraoperative methods used to identify the infiltrative tumor are limited when trying to accurately define the tumor boundaries and the burden of tumor cells in the infiltrated parenchyma. Besides, the impact of treating the infiltrative tumor remains unclear. Here we aim to highlight the molecular and clinical hallmarks of invasion in GBM.

scholarly journals Critical Analysis of Non-Thermal Plasma-Driven Modulation of Immune Cells from Clinical Perspective

2020 ◽  
Vol 21 (17) ◽  
pp. 6226 ◽  
Author(s):  
Barbora Smolková ◽  
Adam Frtús ◽  
Mariia Uzhytchak ◽  
Mariia Lunova ◽  
Šárka Kubinová ◽  
...  
Keyword(s):  
Immune Cells ◽  
Thermal Plasma ◽  
Critical Analysis ◽  
Cell Function ◽  
Immune Cell ◽  
Real Life ◽  
Research Strategy ◽  
Cellular Mechanisms ◽  
Non Thermal Plasma

The emerged field of non-thermal plasma (NTP) shows great potential in the alteration of cell redox status, which can be utilized as a promising therapeutic implication. In recent years, the NTP field considerably progresses in the modulation of immune cell function leading to promising in vivo results. In fact, understanding the underlying cellular mechanisms triggered by NTP remains incomplete. In order to boost the field closer to real-life clinical applications, there is a need for a critical overview of the current state-of-the-art. In this review, we conduct a critical analysis of the NTP-triggered modulation of immune cells. Importantly, we analyze pitfalls in the field and identify persisting challenges. We show that the identification of misconceptions opens a door to the development of a research strategy to overcome these limitations. Finally, we propose the idea that solving problems highlighted in this review will accelerate the clinical translation of NTP-based treatments.

scholarly journals Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Prerna Vohra ◽  
Christina Vrettou ◽  
Jayne C. Hope ◽  
John Hopkins ◽  
Mark P. Stevens
Keyword(s):  
Lymph Nodes ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Large Animal ◽  
Ileal Mucosa ◽  
Zoonotic Infections ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Salmonella Enterica Serovar Dublin

AbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

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