25 research outputs found
Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles
The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10nm and 5ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion
Controlled Grafting Expansion Microscopy
Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM
The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models
Background/Aims The therapeutic options for metastatic neuroendocrine tumors
(NETs) are limited. As PI3K signaling is often activated in NETs, we have
assessed the effects of selective PI3Kp110α inhibition by the novel agent
BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling
pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and
pulmonary (H727) NET cell lines. Methods Cell viability was investigated by
WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7
assay, the cell cycle by FACS, cell signaling by Western blot analysis,
expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and
chromogranin A secretion by ELISA. Results BYL719 dose-dependently decreased
cell viability and colony formation with the highest sensitivity in BON-1,
followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced
apoptosis and G0/G1 cell cycle arrest associated with increased p27
expression. Western blots showed inhibition of PI3K downstream targets to a
varying degree in the different cell lines, but IGF1R activation. The most
sensitive BON-1 cells displayed a significant, and H727 cells a non-
significant, GSK3 inhibition after BYL719 treatment, but these effects do not
appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1
cells showed no GSK3 inhibition, but a modest activation, which would
partially counteract the other anti-proliferative effects. Accordingly, BYL719
enhanced neuroendocrine differentiation with the strongest effect in BON-1,
followed by H727 cells indicated by induction of chromogranin A and
somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and
QGP-1 cells, the BYL719/everolimus combination was synergistic through
simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin
receptor 2 transcription compared to each drug separately. Conclusion Our
results suggest that the agent BYL719 could be a novel therapeutic approach to
the treatment of NETs that may sensitize NET cells to somatostatin analogs,
and that if there is resistance to its action this may be overcome by
combination with everolimus
An efficient GUI-based clustering software for simulation and Bayesian cluster analysis of single-molecule localization microscopy data
Ligand binding of membrane proteins triggers many important cellular signaling events by the
lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma
membrane. This local clustering can lead to the co-enrichment of molecules that create an
intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium
towards the execution of a signaling event. In this way, clustering can serve as a cellular switch.
The underlying uneven distribution and local enrichment of the signaling cluster’s constituting
membrane proteins can be used as a functional readout. This information is obtained by combining
single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly
distinguish clusters from fluctuations in the background noise to generate quantitative data on
this complex process.
Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative
field, and several algorithms and software solutions have been put forward. However, in most
cases, such cluster algorithms require multiple analysis parameters to be defined by the user,
which may lead to biased results. Furthermore, most cluster algorithms neglect the individual
localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it
has entailed high computational cost, increasing runtime drastically. Finally, most software is
challenging to use as they require advanced technical knowledge to operate.
Here we combined three advanced cluster algorithms with the Bayesian approach and
parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing
than for previous approaches. Our work will simplify access to a well-controlled analysis of
clustering data generated by SMLM and significantly accelerate data processing. The inclusion
of a simulation mode aids in the design of well-controlled experimental assays
Synapsin condensation controls synaptic vesicle sequestering and dynamics
Neuronal transmission relies on the regulated secretion of neurotransmitters, which are packed in synaptic vesicles (SVs). Hundreds of SVs accumulate at synaptic boutons. Despite being held together, SVs are highly mobile, so that they can be recruited to the plasma membrane for their rapid release during neuronal activity. However, how such confinement of SVs corroborates with their motility remains unclear. To bridge this gap, we employ ultrafast single-molecule tracking (SMT) in the reconstituted system of native SVs and in living neurons. SVs and synapsin 1, the most highly abundant synaptic protein, form condensates with liquid-like properties. In these condensates, synapsin 1 movement is slowed in both at short (i.e., 60-nm) and long (i.e., several hundred-nm) ranges, suggesting that the SV-synapsin 1 interaction raises the overall packing of the condensate. Furthermore, two-color SMT and super-resolution imaging in living axons demonstrate that synapsin 1 drives the accumulation of SVs in boutons. Even the short intrinsically-disordered fragment of synapsin 1 was sufficient to restore the native SV motility pattern in synapsin triple knock-out animals. Thus, synapsin 1 condensation is sufficient to guarantee reliable confinement and motility of SVs, allowing for the formation of mesoscale domains of SVs at synapses in vivo
The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models
BACKGROUND/AIMS The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110\textgreeka inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. METHODS Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. RESULTS BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. CONCLUSION Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus
Daytime variation of perioperative myocardial injury in non-cardiac surgery and effect on outcome
Recently, daytime variation in perioperative myocardial injury (PMI) has been observed in patients undergoing cardiac surgery. We aim at investigating whether daytime variation also occurs in patients undergoing non-cardiac surgery.; In a prospective diagnostic study, we evaluated the presence of daytime variation in PMI in patients at increased cardiovascular risk undergoing non-cardiac surgery, as well as its possible impact on the incidence of acute myocardial infarction (AMI), and death during 1-year follow-up in a propensity score-matched cohort. PMI was defined as an absolute increase in high-sensitivity cardiac troponin T (hs-cTnT) concentration of ≥14 ng/L from preoperative to postoperative measurements.; Of 1641 patients, propensity score matching defined 630 with similar baseline characteristics, half undergoing non-cardiac surgery in the morning (starting from 8:00 to 11:00) and half in the afternoon (starting from 14:00 to 17:00). There was no difference in PMI incidence between both groups (morning: 50, 15.8% (95% CI 12.3 to 20.3); afternoon: 52, 16.4% (95% CI 12.7 to 20.9), p=0.94), nor if analysing hs-cTnT release as a quantitative variable (median morning group: 3 ng/L (95% CI 1 to 7 ng/L); median afternoon group: 2 ng/L (95% CI 0 to 7 ng/L; p=0.16). During 1-year follow-up, the incidence of AMI was 1.2% (95% CI 0.4% to 3.2%) among morning surgeries versus 4.1% (95% CI 2.3% to 6.9%) among the afternoon surgeries (corrected HR for afternoon surgery 3.44, bootstrapped 95% CI 1.33 to 10.49, p log-rank=0.03), whereas no difference in mortality emerged (p=0.70).; Although there is no daytime variation in PMI in patients undergoing non-cardiac surgery, the incidence of AMI during follow-up is increased in afternoon surgeries and requires further study.; NCT02573532;Results
Daytime variation of perioperative myocardial injury in non-cardiac surgery and effect on outcome
Recently, daytime variation in perioperative myocardial injury (PMI) has been observed in patients undergoing cardiac surgery. We aim at investigating whether daytime variation also occurs in patients undergoing non-cardiac surgery.; In a prospective diagnostic study, we evaluated the presence of daytime variation in PMI in patients at increased cardiovascular risk undergoing non-cardiac surgery, as well as its possible impact on the incidence of acute myocardial infarction (AMI), and death during 1-year follow-up in a propensity score-matched cohort. PMI was defined as an absolute increase in high-sensitivity cardiac troponin T (hs-cTnT) concentration of ≥14 ng/L from preoperative to postoperative measurements.; Of 1641 patients, propensity score matching defined 630 with similar baseline characteristics, half undergoing non-cardiac surgery in the morning (starting from 8:00 to 11:00) and half in the afternoon (starting from 14:00 to 17:00). There was no difference in PMI incidence between both groups (morning: 50, 15.8% (95% CI 12.3 to 20.3); afternoon: 52, 16.4% (95% CI 12.7 to 20.9), p=0.94), nor if analysing hs-cTnT release as a quantitative variable (median morning group: 3 ng/L (95% CI 1 to 7 ng/L); median afternoon group: 2 ng/L (95% CI 0 to 7 ng/L; p=0.16). During 1-year follow-up, the incidence of AMI was 1.2% (95% CI 0.4% to 3.2%) among morning surgeries versus 4.1% (95% CI 2.3% to 6.9%) among the afternoon surgeries (corrected HR for afternoon surgery 3.44, bootstrapped 95% CI 1.33 to 10.49, p log-rank=0.03), whereas no difference in mortality emerged (p=0.70).; Although there is no daytime variation in PMI in patients undergoing non-cardiac surgery, the incidence of AMI during follow-up is increased in afternoon surgeries and requires further study.; NCT02573532;Results
Actomyosin Contractility in the Generation and Plasticity of Axons and Dendritic Spines
Actin and non-muscle myosins have long been known to play important roles in growth cone steering and neurite outgrowth. More recently, novel functions for non-muscle myosin have been described in axons and dendritic spines. Consequently, possible roles of actomyosin contraction in organizing and maintaining structural properties of dendritic spines, the size and location of axon initial segment and axonal diameter are emerging research topics. In this review, we aim to summarize recent findings involving myosin localization and function in these compartments and to discuss possible roles for actomyosin in their function and the signaling pathways that control them.Deutsche ForschungsgemeinschaftPeer Reviewe
Die Stimme Gottes im Feuer : Eine Predigt ; am 20. p. Trinit. über das ordentliche SonntagsEvangelium bey dem trauervollen Andenken der, am 2ten October a. c. zu Schöten, einem in das Herzogl. Sachsen-Weimarische Amt Heußdorf gehörigen Dorfe, entstandenen heftigen Feuers-Brunst, wodurch innerhalb zwoer Stunden fast dieser ganze Ort ... in einen Stein und Aschenhaufen verwandelt worden, vor einer sehr zahlreichen Versammlung, unter freyen Himmel gehalten und ... dem Drucke übergeben
von M. Joh. Adolph Jacob Rentsch, Pfarrer zu Heußdorf, Nauendorf und Schöten, der latein. Gesellschaft zu Jena EhrenMitgliedeVorlageform des Erscheinungsvermerks: Jena, in der Gollner und Rudolphischen Buchhandlung. 1780