Live-cell Studies Research Articles

Hepatocellular carcinoma stem cells: origins and roles in hepatocarcinogenesis and disease progression

Hepatocellular carcinoma (HCC) is a treatment-resistant malignancy with an increasing incidence worldwide. More than 500,000 individuals suffer from this disease annually. Risk factors for human HCC include hepatitis B and C infections, dietary aflatoxin, alcohol abuse, smoking, and oral contraceptive use. Accumulating evidence suggests that liver stem cells play a critical role in HCC development and progression. Dedifferentiated hepatocytes, hepatic oval cells and bone marrow cells are the three major types of liver stem cells, and CD133, CD90, and EpCAM are identified as specific antigenic markers for HCC stem cells. Wnt, Hedgehog, and the angiogenic signalings are main pathways that regulate the HCC stem cell self-renewal and pluripotential, and may be potential targets for novel therapeutic strategies of this malignancy. This review article provides an update in the studies of live and HCC stem cells.

Role of the Cytoskeleton in the Regulation of Weibel-Palade Body Exocytosis

Calcium-driven secretion of the haemostatic protein Von Willebrand factor (VWF) is mediated through exocytosis of specialized endothelial cell (EC)-specific secretory organelles called Weibel-Palade bodies (WPBs). Biochemical studies indicate that VWF secretion is regulated by the cytoskeleton and that the Rab27a-MyRIP-myosinVa complex plays a role in this process, however, little is known about their contribution to the underlying kinetics and extent of WPB exocytosis. To address this we have combined biochemical approaches with simultaneous high speed imaging of intracellular free calcium and exocytosis of fluorescent WPBs in living human umbilical vein endothelial cells (HUVEC) stimulated with ionomycin (1μM) or histamine (100μM). Cytochalasin-D disruption of actin had a subtle effect on the kinetics and extent of ionomycin-evoked WPB exocytosis; delays to the first fusion event and maximal rate of WPB exocytosis were not altered. There was a small increase in the extent of fluorescent WPB degranulation, from 61.1±11.5% (±SD, n=19 cells) in control cells to 71.0±15.9% (±SD, n=23 cells) (p<0.03), although in separate biochemical experiments a small (10-15%) but non-significant increase in VWF secretion was seen. RNAi knockdown MyRIP resulted in complete loss of WPB associated MyRIP-immunoreactivity and a modest (15-20%) but significant (P<0.007) increase in VWF secretion. Disruption of microtubules with Nocodazole substantially decreased (30-40%) VWF secretion and in live cell studies significantly increased the delay and decreased both the rate and extent of ionomycin-evoked WPB exocytosis. Together the data suggests that microtubules play the dominant role in shaping the kinetics and extent of WPB exocytosis, while the actin cytoskeleton and MyRIP play a minor role.

Single-Molecule In Vitro and Live-Cell Studies of Allostery through DNA

Often in nature, a macromolecule undergoes conformational changes upon binding of a ligand in order to modify its affinity for another ligand at a distant binding site. This phenomenon, called “allostery”, is a fundamental mechanism for dynamic regulation of macromolecular properties. Although allostery is well-documented in proteins, it is less recognized for DNA-protein interactions, in which DNA has been often considered a mere template providing recognition sequences for proteins. Here we investigate the allosteric interactions through DNA both in single molecule experiments in vitro and in live cells. In the in vitro experiments, we demonstrate that when two proteins specifically bind to DNA within tens of base pairs, the binding affinity of one protein is altered by the other. We prove that this is not due to protein-protein interactions but to allostery through DNA. As the distance between the two proteins is varied, this allosteric coupling oscillates between positive and negative cooperativity with a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA. The allostery through DNA is explained in terms of the free energy associated with the overall conformation of the ternary complex. We also demonstrate that such allostery affects gene expression in live E. coli cells, suggesting its physiological relevance.

Abstract A203: Identification of FoxM1/Bub1b signaling pathway as a molecular target in rhabdomyosarcoma.

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Intensive multi-modal therapeutic regimens have increased overall survival in the last 25 years, but 25% of patients still succumb to disease, thus necessitating the identification of novel targets for therapy. In this study, we identified that Bub1b is necessary for the growth and survival of both ARMS and ERMS cells using a loss of function high-throughput shRNA screen. Using many additional Bub1b shRNA sequences, we were able to confirm that human RMS cells were more sensitive to Bub1b knockdown compared to human fibroblasts or human ovarian cancer cells. Knockdown of Bub1b also resulted in significant suppression of RMS xenografts growth in vivo. Mechanistically, flow cytometry analysis of knockdown Bub1b cells had an increase in &amp;gt;4N DNA content. Live-cell time-lapse microscopy studies provided direct evidence that knockdown of Bub1b promotes endoreduplication, eventually causing mitotic catastrophe. Further, ChIP assay demonstrated that Forkhead Box M1 (FoxM1), an essential transcriptional factor for G1/S transition and mitotic progression, directly binds to the Bub1b promoter. Luciferase reporter assay showed that FoxM1-binding region is critical for the activation of the Bub1b promoter. Suppression of FoxM1, either by shRNA or the inhibitor siomycin A, led to reduction of Bub1b expression and inhibition of cell growth and survival in vitro. These findings indicate that the FoxM1-Bub1b axis is important for the growth and survival of RMS cells and may be a target for RMS therapy in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A203.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein–protein interaction.

On-chip modulation of evanescent illumination and live-cell imaging with polymer waveguides

Imaging of live cells was carried out using evanescent-wave excitation on a polymer waveguide chip. Integrated waveguide-based interferometric light modulators were fabricated in order to demonstrate on-chip control of excitation light, e.g., for time-lapse fluorescence microscopy. When combined with a sensitive high-resolution imaging system, the integrated waveguide-excitation platform provides an ideal method of near-surface studies of live cells, where photobleaching and/or phototoxicity effects are of critical concern.

Improved molecular toolkit for cAMP studies in live cells

BackgroundcAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations.FindingsWe have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα) that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments.ConclusionsThe modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.

Abstract 4070: Treatment with the MEK inhibitor U0126 induces increased lactate production in prostate and breast cancer cell lines

Abstract Introduction. In this study we investigated the metabolic consequences of U0126, a highly selective MEK1/2 inhibitor, in human prostate and breast cancer cell lines using 1H and 13C magnetic resonance spectroscopy (MRS) and gene expression data. Methods. Prostate (PC3 and LNCaP) and breast (MCF7) human cancer cells were treated for 48 h with the IC50 dose of U0126. 1H MRS data were acquired from cell extracts and repeated univariate statistical testing with false discovery rate correction was performed to determine significant changes in metabolite levels. The rate of glycolysis was determined from dynamic studies in live cells using 13C MRS to monitor the metabolism of 1-13C-labeled glucose. Protein and gene expression levels were measured using Western blotting and microarray analysis. Results and Discussion. The effect of U0126 was confirmed by western blot analysis demonstrating a drop in p-ERK. The univariate statistical analysis revealed that the 48 h drug treatment induced several metabolic changes including depletion of intracellular aspartate, succinate and phosphocholine in the prostate cells. However, one of the most significant metabolic change in all three cell lines was a substantial increase in intracellular lactate levels following treatment, with lactate up 134±16% (p=0.002), 149±20% (p=0.001) and 213±43% (n=2) of control in PC3, LNCaP and MCF7 cells respectively. Dynamic studies in live MCF7 cells were in line with the extract data and showed that the rate of lactate production increased following treatment 169±43% from 260±98 fmol/cell/hour to 439±114 fmol/cell/hour (p=0.03). Preliminary studies in PC3 cells showed a similar trend. Consistent with the metabolic observations, overexpression of genes associated with glycolysis including PFKFB3, PDK1, HK2, and GLUT1 was observed in PC3 cells pointing to an increase in glycolytic enzymes induced by U0126 treatment. In an effort to explain this observation we assessed the expression of other signaling proteins and found that although U0126 inhibits its target kinase, increased Akt phosphorylation was also observed in the PC3 cells. Importantly Akt activation was previously reported to mediate an increase in glycolysis. We therefore hypothesize that the observed increases in lactate production and its intracellular accumulation following inhibition of MAPK kinase signaling result from activation of the PI3K/Akt pathway observed in our cells. Conclusion. Deregulation of the MAPK pathway is often involved in oncogenesis, and therefore inhibitors of the pathway are being investigated as targeted cancer therapeutics. Our results highlight the value of MRS in identifying metabolic consequences of treatment that may inform on molecular drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4070. doi:10.1158/1538-7445.AM2011-4070

Graphene and graphene oxide: biofunctionalization and applications in biotechnology

Graphene is the basic building block of 0D fullerene, 1D carbon nanotubes, and 3D graphite. Graphene has a unique planar structure, as well as novel electronic properties, which have attracted great interests from scientists. This review selectively analyzes current advances in the field of graphene bioapplications. In particular, the biofunctionalization of graphene for biological applications, fluorescence-resonance-energy-transfer-based biosensor development by using graphene or graphene-based nanomaterials, and the investigation of graphene or graphene-based nanomaterials for living cell studies are summarized in more detail. Future perspectives and possible challenges in this rapidly developing area are also discussed.

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

Biological Scanning Electrochemical Microscopy and Its Application to Live Cell Studies

ADVERTISEMENT RETURN TO ISSUEFeatureNEXTBiological Scanning Electrochemical Microscopy and Its Application to Live Cell StudiesProgress in instrumental design continues to pave the way for high-resolution, real-time electrochemical measurements with living cells.Isabelle Beaulieu, Sabine Kuss, Janine Mauzeroll, and Matthias GeisslerView Author Information University of Québec in Montréal (Canada) National Research Council of CanadaCite this: Anal. Chem. 2011, 83, 5, 1485–1492Publication Date (Web):January 7, 2011Publication History Published online7 January 2011Published inissue 1 March 2011https://doi.org/10.1021/ac101906aCopyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views4525Altmetric-Citations63LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-AlertsSUBJECTS:Diffusion,Electrochemical cells,Electrodes,Gold,Sensors Get e-Alerts

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

In this review, I stress the importance of direct data and accurate terminology when formulating and communicating conclusions on how the G2/M and metaphase/anaphase transitions are regulated. I argue that entry into mitosis (i.e., the G2/M transition) is guarded by several checkpoint control pathways that lose their ability to delay or stop further cell cycle progression once the cell becomes committed to divide, which in vertebrates occurs in the late stages of chromosome condensation. After this commitment, progress through mitosis is then mediated by a single Mad/Bub-based checkpoint that delays chromatid separation, and exit from mitosis (i.e., completion of the cell cycle) in the presence of unattached kinetochores. When cells cannot satisfy the mitotic checkpoint, e.g., when in concentrations of spindle poisons that prohibit the stable attachment of all kinetochores, they are delayed in mitosis for many hours. In normal cells, the duration of this delay depends on the organism and ranges from ∼4h in rodents to ∼22h in humans. Recent live cell studies reveal that under this condition, many cancer cells (including HeLa and U2OS) die in mitosis by apoptosis within ∼24h, which implies that biochemical studies on cancer cell populations harvested in mitosis after a prolonged mitotic arrest are contaminated with dead or dying cells.

A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes

Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.

Live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by approximately 40% in nontransformed human RPE-1, approximately 80% in PtK2 (rat kangaroo), and approximately 25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends.

4D Chromatin dynamics in cycling cells

This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements.

Raman Tweezers Spectroscopy of Live, Single Red and White Blood Cells

An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

4D Chromatin dynamics in cycling cells: Theodor Boveri's hypotheses revisited

This live cell study of chromatin dynamics in four dimensions (space and time) in cycling human cells provides direct evidence for three hypotheses first proposed by Theodor Boveri in seminal studies of fixed blastomeres from Parascaris equorum embryos: (I) Chromosome territory (CT) arrangements are stably maintained during interphase. (II) Chromosome proximity patterns change profoundly during prometaphase. (III) Similar CT proximity patterns in pairs of daughter nuclei reflect symmetrical chromosomal movements during anaphase and telophase, but differ substantially from the arrangement in mother cell nucleus. Hypothesis I could be confirmed for the majority of interphase cells. A minority, however, showed complex, rotational movements of CT assemblies with large-scale changes of CT proximity patterns, while radial nuclear arrangements were maintained. A new model of chromatin dynamics is proposed. It suggests that long-range DNA-DNA interactions in cell nuclei may depend on a combination of rotational CT movements and locally constrained chromatin movements.