Publications by authors named "Gert-Jan Kremers"

28 Publications

  • Page 1 of 1

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease.

Sci Rep 2020 12 14;10(1):21900. Epub 2020 Dec 14.

Department of Surgery, Erasmus MC-University Medical Center, Wytemaweg 80, 3015 CN, Rotterdam, The Netherlands.

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.
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http://dx.doi.org/10.1038/s41598-020-79082-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736890PMC
December 2020

Ciliary Tip Signaling Compartment Is Formed and Maintained by Intraflagellar Transport.

Curr Biol 2020 Nov 10;30(21):4299-4306.e5. Epub 2020 Sep 10.

Department of Cell Biology, Erasmus University Medical Centre, Dr. Molewaterplein 40, 3015 GD Rotterdam, the Netherlands. Electronic address:

Primary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. Within cilia, subcompartments are established to support signal transduction pathways, including Hedgehog signaling. How these compartments are formed and maintained remains largely unknown. Cilia use two mechanisms, a trafficking system and a diffusion barrier, to regulate the trafficking of proteins into, within, and out of cilia. The main ciliary trafficking machinery, intraflagellar transport (IFT), facilitates bidirectional transport of cargo, including signaling proteins, from the base (basal body) to the tip of the axoneme [1]. Anterograde IFT to the tip relies on kinesins, and cytoplasmic dynein enables retrograde transport back [2, 3]. To help confine proteins to cilia, a subdomain immediately distal to the basal body, called the transition zone (TZ), acts as a diffusion barrier for both membrane and soluble proteins [4-6]. Here, we show that in Caenorhabditis elegans a salt-sensing receptor-type guanylate cyclase, GCY-22, accumulates at a high concentration within a subcompartment at the distal region of the cilium. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the IFT system. Disruption of the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation and maintenance of the ciliary tip compartment. Structure-function studies uncovered GCY-22 protein domains needed for entry and tip localization. Together, our findings provide mechanistic insights into the formation and maintenance of a novel subdomain at the cilium tip that contributes to the behavioral response to NaCl.
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http://dx.doi.org/10.1016/j.cub.2020.08.032DOI Listing
November 2020

Guide-free Cas9 from pathogenic bacteria causes severe damage to DNA.

Sci Adv 2020 Jun 17;6(25):eaaz4849. Epub 2020 Jun 17.

Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.
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http://dx.doi.org/10.1126/sciadv.aaz4849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299616PMC
June 2020

A microcarrier-based spheroid 3D invasion assay to monitor dynamic cell movement in extracellular matrix.

Biol Proced Online 2020 1;22. Epub 2020 Feb 1.

1Laboratory of Experimental Oncology, Department of Pathology, Erasmus Medical Center, Rotterdam, the Netherlands.

Background: Cell invasion through extracellular matrix (ECM) is a critical step in tumor metastasis. To study cell invasion in vitro, the internal microenvironment can be simulated via the application of 3D models.

Results: This study presents a method for 3D invasion examination using microcarrier-based spheroids. Cell invasiveness can be evaluated by quantifying cell dispersion in matrices or tracking cell movement through time-lapse imaging. It allows measuring of cell invasion and monitoring of dynamic cell behavior in three dimensions. Here we show different invasive capacities of several cell types using this method. The content and concentration of matrices can influence cell invasion, which should be optimized before large scale experiments. We also introduce further analysis methods of this 3D invasion assay, including manual measurements and homemade semi-automatic quantification. Finally, our results indicate that the position of spheroids in a matrix has a strong impact on cell moving paths, which may be easily overlooked by researchers and may generate false invasion results.

Conclusions: In all, the microcarrier-based spheroids 3D model allows exploration of adherent cell invasion in a fast and highly reproducible way, and provides informative results on dynamic cell behavior in vitro.
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http://dx.doi.org/10.1186/s12575-019-0114-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995242PMC
February 2020

Inhibition of retinoic acid signaling induces aberrant pericyte coverage and differentiation resulting in vascular defects in congenital diaphragmatic hernia.

Am J Physiol Lung Cell Mol Physiol 2019 09 3;317(3):L317-L331. Epub 2019 Jul 3.

Department of Pediatric Surgery, Erasmus Medical Center, Sophia Children's Hospital, Rotterdam, The Netherlands.

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.
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http://dx.doi.org/10.1152/ajplung.00104.2018DOI Listing
September 2019

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Brain 2019 04;142(4):867-884

Department of Clinical Genetics, Erasmus University Medical Center (Erasmus MC), CA Rotterdam, The Netherlands.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.
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http://dx.doi.org/10.1093/brain/awz045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439326PMC
April 2019

Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFβ signaling.

Cell Signal 2019 06 4;58:65-78. Epub 2019 Mar 4.

Department of Molecular Genetics, Erasmus University Medical Center, Rotterdam, the Netherlands; Department of Vascular Surgery, Erasmus University Medical Center, Rotterdam, the Netherlands; Department of Radiation Oncology, Erasmus University Medical Center, Rotterdam, the Netherlands. Electronic address:

Fibulin-4 is an extracellular matrix (ECM) protein essential for elastogenesis and mutations in this protein lead to aneurysm formation. In this study, we isolated vascular smooth muscle cells (VSMCs) from mice with reduced fibulin-4 protein expression (Fibulin-4) and from mice with a smooth muscle cell specific deletion of the Fibulin-4 gene (Fibulin-4/SM22Cre). We subsequently analyzed and compared the molecular consequences of reduced Fibulin-4 expression versus total ablation of Fibulin-4 expression with regard to effects on the SMC specific contractile machinery, cellular migration and TGFβ signaling. Analysis of the cytoskeleton showed that while Fibulin-4/SM22Cre VSMCs lack smooth muscle actin (SMA) fibers, Fibulin-4 VSMCs were able to form SMA fibers. Furthermore, Fibulin-4/SM22Cre VSMCs showed a decreased pCofilin to Cofilin ratio, suggesting increased actin depolymerization, while Fibulin-4 VSMCs did not display this decrease. Yet, both Fibulin-4 mutant VSMCs showed decreased migration. We found increased activation of TGFβ signaling in Fibulin-4 VSMCs. However, TGFβ signaling was not increased in Fibulin-4/SM22Cre VSMCs. From these results we conclude that both reduction and absence of Fibulin-4 leads to structural and functional impairment of the SMA cytoskeleton. However, while reduced levels of Fibulin-4 result in increased TGFβ activation, complete absence of Fibulin-4 does not result in increased TGFβ activation. Since both mouse models show thoracic aortic aneurysm formation, we conclude that not only hampered TGFβ signaling, but also SMA cytoskeleton dynamics play an important role in aortic aneurysmal disease.
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http://dx.doi.org/10.1016/j.cellsig.2019.02.008DOI Listing
June 2019

Three-dimensional architecture of common benign and precancerous prostate epithelial lesions.

Histopathology 2019 Jun 21;74(7):1036-1044. Epub 2019 Apr 21.

Department of Pathology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Aims: Many glandular lesions can mimic prostate cancer microscopically, including atrophic glands, adenosis and prostatic intraepithelial neoplasia. While the characteristic histopathological and immunohistochemical features of these lesions have been well established, little is known about their three-dimensional architecture. Our objective was to evaluate the three-dimensional organisation of common prostate epithelial lesions.

Methods And Results: 500 μm-thick punches (n = 42) were taken from radical prostatectomy specimens, and stained with antibodies targeting keratin 8-18 and keratin 5 for identification of luminal and basal cells, respectively. Tissue samples were optically cleared in benzyl alcohol:benzyl benzoate and imaged using a confocal laser scanning microscope. The three-dimensional architecture of peripheral and transition zone glands was acinar, composed of interconnecting and blind-ending saccular tubules. In simple atrophy, partial atrophy and post-atrophic hyperplasia, the acinar structure was attenuated with branching blind-ending tubules from parental tubular structures. Three-dimensional imaging revealed a novel variant of prostate atrophy characterised by large Golgi-like atrophic spaces parallel to the prostate surface, which were represented by thin, elongated tubular structures on haematoxylin and eosin (H&E) slides. Conversely, adenosis lacked acinar organisation, so that it closely mimicked low-grade prostate cancer. High-grade prostatic intraepithelial neoplasia displayed prominent papillary intraluminal protrusions but retained an acinar organisation, whereas intraductal carcinoma predominantly consisted of cribriform proliferations with either spheroid, ellipsoid or complex interconnecting lumens.

Conclusions: While various prostate epithelial lesions might mimic malignancy on H&E slides, their three-dimensional architecture is acinar and clearly different from the tubular structure of prostate cancer, with adenosis as an exception.
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http://dx.doi.org/10.1111/his.13848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849837PMC
June 2019

Three-dimensional analysis reveals two major architectural subgroups of prostate cancer growth patterns.

Mod Pathol 2019 07 8;32(7):1032-1041. Epub 2019 Feb 8.

Department of Pathology, University Medical Center Rotterdam, Rotterdam, The Netherlands.

The Gleason score is one of the most important parameters for therapeutic decision-making in prostate cancer patients. Gleason growth patterns are defined by their histological features on 4- to 5-µm cross sections, and little is known about their three-dimensional architecture. Our objective was to characterize the three-dimensional architecture of prostate cancer growth patterns. Intact tissue punches (n = 46) of representative Gleason growth patterns from radical prostatectomy specimens were fluorescently stained with antibodies targeting Keratin 8/18 and Keratin 5 for the detection of luminal and basal epithelial cells, respectively. Punches were optically cleared in benzyl alcohol-benzyl benzoate and imaged using a confocal laser scanning microscope up to a depth of 500 µm. Gleason pattern 3, poorly formed pattern 4, and cords pattern 5 all formed a continuum of interconnecting tubules in which the diameter of the structures and the lumen size decreased with higher grades. In fused pattern 4, the interconnections between the tubules were markedly closer together. In these patterns, all tumor cells were in direct contact with the surrounding stroma. In contrast, cribriform Gleason pattern 4 and solid pattern 5 demonstrated a three-dimensional continuum of contiguous tumor cells, in which the vast majority of cells had no contact with the surrounding stroma. Transitions between cribriform pattern 4 and solid pattern 5 were seen. There was a decrease in the number and size of intercellular lumens from cribriform to solid growth pattern. Glomeruloid pattern 4 formed an intermediate structure consisting of a tubular network with intraluminal epithelial protrusions close to the tubule splitting points. In conclusion, three-dimensional microscopy revealed two major architectural subgroups of prostate cancer growth patterns: (1) a tubular interconnecting network including Gleason pattern 3, poorly formed and fused Gleason pattern 4, and cords Gleason pattern 5, and (2) serpentine contiguous epithelial proliferations including cribriform Gleason pattern 4 and solid Gleason pattern 5.
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http://dx.doi.org/10.1038/s41379-019-0221-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760644PMC
July 2019

Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds.

Stem Cells Dev 2017 09 31;26(18):1304-1315. Epub 2017 Jul 31.

1 Department of Surgery, Erasmus MC-University Medical Center , Rotterdam, the Netherlands .

Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.
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http://dx.doi.org/10.1089/scd.2017.0095DOI Listing
September 2017

Xist and Tsix Transcription Dynamics Is Regulated by the X-to-Autosome Ratio and Semistable Transcriptional States.

Mol Cell Biol 2016 Nov 13;36(21):2656-2667. Epub 2016 Oct 13.

Department of Developmental Biology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions.
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http://dx.doi.org/10.1128/MCB.00183-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064214PMC
November 2016

Three-dimensional microscopic analysis of clinical prostate specimens.

Histopathology 2016 Dec 21;69(6):985-992. Epub 2016 Sep 21.

Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.

Aims: Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5-μm-thick tissue sections. Because cross-sections give a two-dimensional (2D) representation, little is known about the actual underlying three-dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue-clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue.

Methods And Results: Optical clearing of intact fresh and formalin-fixed paraffin-embedded (FFPE) clinical prostate specimens allowed us to visualize tissue structures up to a depth of 800 μm, whereas, in uncleared tissue, detection of fluorescence was only possible up to 70 μm. Fluorescent labelling with a general nuclear dye and antibodies against cytokeratin (CK) 5 and CK8-18 resulted in comprehensive 3D imaging of benign peripheral and transition prostate zones, as well as individual PCa growth patterns. After staining, clearing, and imaging, samples could still be processed for 2D (immuno)histochemical staining and DNA analysis, enabling additional molecular and diagnostic characterization of small tissue specimens.

Conclusions: In conclusion, the applicability of 3D imaging to archival FFPE and fresh clinical specimens offers unlimited opportunities to study clinical and biological topics of interest in their actual 3D context.
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http://dx.doi.org/10.1111/his.13022DOI Listing
December 2016

Quantitative assessment of fluorescent proteins.

Nat Methods 2016 07 30;13(7):557-62. Epub 2016 May 30.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
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http://dx.doi.org/10.1038/nmeth.3891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927352PMC
July 2016

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications.

PLoS One 2015 4;10(8):e0133888. Epub 2015 Aug 4.

Department of Viroscience, Erasmus Medical Centre, Rotterdam, the Netherlands.

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524686PMC
May 2016

Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its cellular distribution.

Mol Endocrinol 2013 May 2;27(5):801-13. Epub 2013 Apr 2.

Department of Internal Medicine, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.
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http://dx.doi.org/10.1210/me.2012-1356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416761PMC
May 2013

Amphiregulin exosomes increase cancer cell invasion.

Curr Biol 2011 May 21;21(9):779-86. Epub 2011 Apr 21.

Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.
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http://dx.doi.org/10.1016/j.cub.2011.03.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417320PMC
May 2011

An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.

PLoS One 2011 03 29;6(3):e17896. Epub 2011 Mar 29.

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017896PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3066204PMC
March 2011

Induction of antitumor immunity by dendritic cells loaded with membrane-translocating mucin 1 Peptide antigen.

Transl Oncol 2011 Feb 1;4(1):1-8. Epub 2011 Feb 1.

Institute of Imaging Science, Vanderbilt University, Nashville, TN, USA.

To investigate the role of enhanced antigen presentation in dendritic cell (DC)-based immunotherapy. Here, we describe the development of a cell-penetrating mucin 1 (MUC1) antigen and its immunotherapeutic potential against tumors. After animal groups received two immunizations of MUC1-MPA(11)P-pulsed DCs, we observed a marked tumor regression compared with the mice treated with DCs alone or DCs pulsed with MUC1 peptide. We confirmed the migration and homing of DCs in the popliteal lymph node using magnetic resonance imaging during the study. In summary, enhanced antigen uptake using an MPA(11)P delivery molecule improves cell therapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026404PMC
http://dx.doi.org/10.1593/tlo.10166DOI Listing
February 2011

Multimodal imaging of dendritic cells using a novel hybrid magneto-optical nanoprobe.

Nanomedicine 2011 Aug 5;7(4):489-96. Epub 2011 Jan 5.

Institute of Imaging Science, Vanderbilt University, Nashville, Tennessee, USA.

Unlabelled: A transfecting agent-coated hybrid imaging nanoprobe (HINP) composed of visible and near-infrared (NIR) light emitting quantum dots (QDs) tethered to superparamagnetic iron oxide (SPIO) nanoparticles was developed. The surface modification of QDs and SPIO particles and incorporation of dual QDs within the SPIO were characterized by dynamic light scattering (DLS), quartz crystal microbalance (QCM) analysis and atomic force microscopy (AFM). The optical contrasting properties of HINP were characterized by absorption and photoluminescence spectroscopy and fluorescence imaging. Multicolor HINP was used in imaging the migration of dendritic cells (DCs) by optical, two-photon and magnetic resonance imaging techniques.

From The Clinical Editor: The development of a transfecting agent-coated hybrid imaging nanoprobe (HINP) composed of visible and near-infrared light emitting quantum dots (QDs) tethered to superparamagnetic iron oxide nanoparticles is reported in this paper. Multicolor HINP was used in imaging the migration of dendritic cells by optical, two-photon and magnetic resonance imaging techniques.
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http://dx.doi.org/10.1016/j.nano.2010.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148276PMC
August 2011

Fluorescent proteins at a glance.

J Cell Sci 2011 Jan;124(Pt 2):157-60

Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, TN 37232-0615, USA.

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http://dx.doi.org/10.1242/jcs.072744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037093PMC
January 2011

Rapid extravasation and establishment of breast cancer micrometastases in the liver microenvironment.

Mol Cancer Res 2010 Oct 19;8(10):1319-27. Epub 2010 Aug 19.

Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA.

To examine the interplay between tumor cells and the microenvironment during early breast cancer metastasis, we developed a technique for ex vivo imaging of murine tissue explants using two-photon microscopy. Cancer cells in the liver and the lung were compared by imaging both organs at specific time points after the injection of the same polyomavirus middle T-initiated murine mammary tumor cell line. Extravasation was greatly reduced in the lung compared with the liver, with 56% of tumor cells in the liver having extravasated by 24 hours, compared with only 22% of tumor cells in the lung that have extravasated. In the liver, imaged cells continually transitioned from an intravascular location to an extravascular site, whereas in the lung, extravasation rates slowed after 6 hours. Within the liver microenvironment, the average size of the imaged micrometastatic lesions increased 4-fold between days 5 and 12. Histologic analysis of these lesions determined that by day 12, the micrometastases were heterogeneous, consisting of both tumor cells and von Willebrand factor-positive endothelial cells. Further analysis with intravenously administered lectin indicated that vessels within the micrometastatic tumor foci were patent by day 12. These data present the use of two-photon microscopy to directly compare extravasation times in metastatic sites using the same tumor cell line and highlight the differences in early events and metastatic patterns between two important secondary sites of breast cancer progression with implications for future therapy.
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http://dx.doi.org/10.1158/1541-7786.MCR-09-0551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981168PMC
October 2010

Photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells.

J Vis Exp 2010 Jun 26(40). Epub 2010 Jun 26.

Department of Molecular Physiology and Biophysics, Vanderbilt University, USA.

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.
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http://dx.doi.org/10.3791/1995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153906PMC
June 2010

Photoconversion in orange and red fluorescent proteins.

Nat Methods 2009 May 12;6(5):355-8. Epub 2009 Apr 12.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

We found that photoconversion is fairly common among orange and red fluorescent proteins, as in a screen of 12 proteins, 8 exhibited photoconversion. Specifically, three red fluorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all fluorescent proteins described to date.
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http://dx.doi.org/10.1038/nmeth.1319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675661PMC
May 2009

Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy.

Biophys J 2008 Jul 21;95(1):378-89. Epub 2008 Mar 21.

Section Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, NL-1098 SM, Amsterdam, The Netherlands.

Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins.
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http://dx.doi.org/10.1529/biophysj.107.125229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426628PMC
July 2008

Fluorescent protein FRET: the good, the bad and the ugly.

Trends Biochem Sci 2007 Sep 30;32(9):407-14. Epub 2007 Aug 30.

Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, TN 37232-0615, USA.

Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such interactions is Förster resonance energy transfer (FRET). When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. Because FRET signals are usually small, however, their measurement requires careful interpretation and several control experiments. Nevertheless, the use of FRET in cell biological experiments has exploded over the past few years. Here we describe the physical basis of FRET and the fluorescent proteins appropriate for these experiments. We also review the approaches that can be used to measure FRET, with particular emphasis on the potential artifacts associated with each approach.
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http://dx.doi.org/10.1016/j.tibs.2007.08.003DOI Listing
September 2007

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells.

Biochemistry 2007 Mar 27;46(12):3775-83. Epub 2007 Feb 27.

Section of Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.
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http://dx.doi.org/10.1021/bi0622874DOI Listing
March 2007

Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius.

Biochemistry 2006 May;45(21):6570-80

Section Molecular Cytology and Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands.

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.
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http://dx.doi.org/10.1021/bi0516273DOI Listing
May 2006