Uncovering the molecular basis of compartmentalization as a principle of neuronal organization

Cells are faced with coordinating countless, simultaneous, partly antagonistic biochemical reactions. This is especially true for neurons that must orchestrate the complex task of neurotransmission. One solution to this problem is the formation of specialized compartments. To understand the molecular mechanisms of such compartments this thesis investigates two system in neuronal cells: i. the plasma membrane and its underlying cytoskeleton and ii. synaptic vesicle clusters inside synaptic boutons. Towards this end, a combinatorial approach of computational modeling, single particle tracking and super-resolution microscopy is employed. A periodic array of actin rings in the neuronal axon initial segment has been known to confine membrane protein motion. Still, a local enrichment of ion channels offers an alternative explanation. Using computational modeling this thesis now shows that ion channels, in contrast to actin rings, cannot mediate confinement. Furthermore, by employing single particle tracking and super-resolution microscopy, this work shows that actin rings are close to the plasma membrane and that actin rings confine membrane proteins in several neuronal cell types. Further, it is shown that actin ring disruption leads to a reduction of membrane compartmentalization. Synaptic boutons in the axon of neurons are the location of synaptic vesicle release. Synaptic vesicles form dense clusters inside boutons, that are essential for pre-synaptic function. In vitro experiments have suggested that the soluble phosphoprotein synapsin 1 controls synaptic clustering via liquid liquid phase separation. However, the in vivo mechanism remains elusive. This work now shows via two-color single molecule tracking in live neurons that synapsin 1 drives synaptic vesicle clustering. Furthermore, using a synapsin knock-out model it is shown that synapsin 1 controls the mobility of synaptic vesicles through its intrinsically disordered region, which is responsible for phase separation. By studying the dynamics of compartmentalized systems in neuronal cells this work uncovers two molecular mechanisms: actin rings form membrane diffusion barriers and synapsin 1 controls synaptic vesicle clustering and mobility through liquid liquid phase separation. Thus, this thesis makes important strides towards deepening the understanding of neuronal function by uncovering how compartmentalization operates in both the plasma membrane and the cytosol of neuronal cells.Zellen müssen unzählige, gleichzeitige, teilweise gegensätzliche biochemische Reaktionen koordinieren. Dies gilt insbesondere für Neuronen, die die komplexe Aufgabe der Neurotransmission koordinieren. Eine Lösung für dieses Problem ist die Bildung spezialisierter Kompartimente. Um die molekulare Funktionsweise solcher Kompartimente zu verstehen, werden in dieser Arbeit zwei Systeme in neuronalen Zellen untersucht: i. die Plasmamembran und das darunterliegende Zytoskelett und ii. synaptische Vesikel-Cluster in Boutons. Zu diesem Zweck, wurde ein kombinatorischer Ansatz aus Computermodellierung, Einzelpartikelverfolgung und superauflösender Mikroskopie verwendet. Periodische Aktinringe im neuronalen Axoninitialsegment schränken die Bewegung von Membranproteinen ein. Jedoch liefert eine lokale Anreicherung von Ionenkanälen eine alternative Erklärung. Durch Computermodellierung wird in dieser Arbeit nun gezeigt, dass Ionenkanäle keine Einschränkung der Membranmolekülbewegung bewirken. Darüber hinaus wird durch Einzelpartikelverfolgung und superauflösende Mikroskopie gezeigt, dass Aktinringe nahe der Plasmamembran sind und dass Aktinringe Membranproteine in verschiedenen neuronalen Zelltypen in ihrer Bewegung einschränken. Weiterhin wird gezeigt, dass die Zerstörung der Aktinringe Membrankompartmentalisierung reduziert. Synaptische Boutons im Axon sind der Ort der Freisetzung synaptischer Vesikel. Synaptische Vesikel bilden dichte Cluster in Boutons, welche für die Funktion der Präsynapse essenziell sind. In vitro Experimente haben gezeigt, dass das lösliche Phosphoprotein Synapsin 1 das Clustern durch Flüssig-Flüssig-Phasentrennung steuert, der Mechanismus in vivo ist jedoch unklar. Diese Arbeit zeigt nun mittels Zweifarben-Einzelmolekülverfolgung in lebenden Neuronen, dass Synapsin 1 das Clustern synaptischer Vesikel steuert. Anhand eines Synapsin-Knock-out-Modells wird gezeigt, dass Synapsin 1 die Mobilität synaptischer Vesikel durch seine intrinsisch ungeordnete Region kontrolliert, die für die Phasentrennung verantwortlich ist. Durch Untersuchungen der Dynamik kompartmentalisierter Systeme in neuronalen Zellen deckt diese Arbeit zwei molekulare Mechanismen auf: Aktinringe bilden Membrandiffusionsbarrieren und Synapsin 1 steuert Clustern und Mobilität synaptischer Vesikel durch Flüssig-Flüssig-Phasentrennung. Somit macht diese Arbeit wichtige Fortschritte zum Verständnis der Funktionsweise neuronaler Zellen, indem sie aufdeckt, wie die Kompartmentalisierung der Plasmamembran und des Zytosols gesteuert wird

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