Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells

We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway

WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies

The WRAP53 protein regulates the formation and maintenance of Cajal bodies (nuclear sub-organelles), as well as directs the recruitment of nuclear factors to Cajal bodies

Visualizing Interacting Biomolecules In Situ

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures

Association of the Protein-Tyrosine Phosphatase DEP-1 with Its Substrate FLT3 Visualized by In Situ Proximity Ligation Assay

Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation

Pedagogical facilities of the family as contemporary problem in the preschool

Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation

Simultaneous detection of individual phosphorylated PDGFRβ and <i>DUSP6</i> mRNA molecules in BJhTert cells.

<p>Cells were stimulated for (Ai) 5 min or (Aii) 180 min with PDGF-BB. <i>In situ</i> PLA signals, derived from PDGFRβ phosphorylation, are shown as red dots while <i>DUSP6</i> molecules are shown as green dots. The nuclei are shown in grey. Inserts represent magnified views over random cells. Scalebars represent 20 µm. (B) Simultaneous detection of individual phosphorylated PDGFRβ and <i>DUSP6</i> mRNA molecules cells treated with Gleevec or 5-Iodotubercidin at different time points after PDGF-BB stimulation. (Bi) Schematic illustration of the different drug target sites in PDGFRβ pathway and the consequences for signaling and expected RCPs. (Bii) Simultaneous detection of individual phosphorylated PDGFRβ and <i>DUSP6</i> mRNA molecules in BJhTert cells treated with Gleevec or 5-Iodotubercidin, at different time points after PDGF-BB stimulation. Black circles represent the numbers of RCPs detected per cell (in total ∼100–140 cells per condition), the red bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments. The result is presented in separate figures for PDGFRβ phosphorylation and <i>DUSP6</i> expression, although recorded together.</p

Method overview.

<p>(A) Upon stimulation with PDGF-BB the PDGFRβ becomes autophosphorylated at various sites. This promotes downstream signaling to ERK activation. Phosphorylated ERK translocates into the nucleus and enhances <i>DUSP6</i> expression. Upon fixation of the cells <i>DUSP6</i> mRNA is reverse transcribed utilizing an LNA-modified primer. Subsequently the cDNA is made accessible through RNase H digestion. The single standed cDNA stays attached to the mRNA-primer by the 5′-end of the primer that contains the LNA bases, which is not recognized by RNase H – to allow hybridization of padlock probes. (B) After ligation of the padlock probe to a circular DNA molecule, primary antibodies directed against the PDGFRβ and phosphorylated tyrosines, followed by addition of secondary PLA probes, are applied for detection of the phosphorylated PDGFRβ. If the antibodies and probes are bound in close proximity two circularization oligonucleotides can hybridize to the PLA probes. (C) This molecule can subsequently also be joined by ligation, giving rise to another circular DNA molecule. (D) Both DNA circles are then simultaneously amplified by RCA, primed from either the cDNA or the oligonucleotide attached to the PLA probe. The resulting bundles of DNA (amplified ∼1000 times) are still attached to the place where the RCA was primed and can be detected by hybridization of fluorescence labeled oligonucleotides (different fluorophores (green and pink stars) for the two types of circles), resulting in bright spots easily distinguishable from background.</p

Similar kinetics of FLT3-DEP-1 complex formation and FLT3 autophosphorylation.

<p>Association of DEP-1 with FLT3 was measured by <i>in situ</i> PLA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone-0062871-g001" target="_blank">Figure 1</a>. (<b>A</b>) Example images; DEP-1/FLT3 complexes as RCPs are shown in red, nuclei are depicted in blue and scale bars represent 20 µm for the overview images and 5 µm for the insets. (<b>B</b>) Cumulated data from 7 independent experiments (means ± SD, *p<0.05, **p<0.01 for difference from unstimulated sample by one-way ANOVA). (<b>C</b>) FLT3 was immunoprecipitated from THP-1 cells stimulated for the indicated timepoints and autophosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibodies (pY100). FLT3 amounts were detected by reblot with anti-FLT3 antibodies. Note that for the FLT3 reblot only the section above the very abundant IgG band (star) is depicted. Two FLT3 species of 130 kDa and 150 kDa represent the immature high-mannose form and the mature, complex glycosylated form, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone.0062871-SchmidtArras2" target="_blank">[31]</a>. Only the 150 kDa form (arrows) becomes phosphorylated in a ligand-dependent manner. Weak bands of even higher molecular mass in the stimulated samples represent ubiquitinylated FLT3. Right panel: Quantification of multiple (n = 5) independent experiments. The pFLT3 signals were normalized to FLT3 amounts of the stripped and reprobed blots (means ± SD, *p<0.05, **p<0.01 for difference from unstimulated sample by one-way ANOVA).</p