Michael Prummer

Three-Dimensional High-Resolution Particle Tracking for Optical Tweezers by Forward Scattered Light

A quadrant photodiode placed in the back‐focal plane of the microscope of a laser trap provides a high‐resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three‐dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a < 0.2λ) for displacements up to the focal dimensions. The cross‐talk between the signals in the three directions is explained and it is shown that this cross‐talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three‐dimensional single‐particle tracking over conventional video‐tracking are shown through the example of the diffusion of the GPI‐anchored membrane protein Thy1.1 on a neurite. Microsc. Res. Tech. 44:378–386, 1999.

Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

White-to-brown metabolic conversion of human adipocytes by JAK inhibition

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK–STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+‐translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization‐resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0‐specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.

Orientation dependence of fluorescence lifetimes near an interface

The fluorescence lifetime of single DiI-dye molecules in a 20 nm polymer film on glass is measured as a function of the orientation of the absorption dipole moment. A strong dependence of the lifetime on the orientation of the dye molecules relative to the polymer/air interface is found. Molecules with a dipole moment perpendicular to the interface exhibit a lifetime which is by a factor of 2.1±0.1 longer than the lifetime of molecules with parallel dipole moments. The general trend of the results is in good agreement with theoretical predictions. However there are significant deviations which are attributed to varying molecular environments.

Three-dimensional optical polarization tomography of single molecules

We apply the concept of tomography to polarization-sensitive optical microscopy of single fluorophores to determine the three-dimensional orientation of molecular absorption dipoles with isotropic sensitivity. Wide-field microscopy provides the opportunity to monitor simultaneously three-dimensional rotation and two-dimensional translation of many molecules in parallel. For orientation determination the molecules are illuminated from different directions of incidence with linearly polarized light. In each exposure the excitation along a particular projection of the absorption dipole on the electric field leads to a distinct fluorescence intensity. Five exposures are sufficient to determine the full orientation of the fluorophores. To demonstrate the potential of the method we determine the orientation and position of individual immobilized lipid membrane markers. The shot-noise-limited isotropic angular resolution is 2°. For time-resolved studies the bandwidth can be expanded up to 200 Hz.

Combined mutation in Vhl, Trp53 and Rb1 causes clear cell renal cell carcinoma in mice

Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel–Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-α transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-α inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Post-translational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells

Keywords: Fluorescent probes ; Membrane proteins ; Neurokinin-1 receptor ; Signal transduction ; Single-molecule studies Reference LCPPM-ARTICLE-2007-003doi:10.1002/cbic.200500477View record in Web of Science Record created on 2007-04-22, modified on 2017-05-12