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In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway.
In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes.
The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses.
G-protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the “light receptor” rhodopsin has been shown to activate with a re-arrangement of the transmembrane helix bundle within ≈1 ms, while all other receptors are thought to become activated in subsecond range at saturating concentrations. Here we investigate activation kinetics of a dimeric GPCR, the metabotropic glutamate receptor-1 (mGluR1), and several class A GPCRs, as muscarinic receptor 3 (M3R), adrenergic (α2aAR and β1R) and opioid (µOR) receptors. We first used UV-light-triggered uncaging of glutamate in intact cells. Sub-millisecond Förster resonance energy transfer recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1≈1-2 ms. These rapid changes were followed by significantly slower conformational changes in the transmembrane domain (τ2≈20 ms). We further characterized novel photoswitchable negative allosteric modulators for mGluR1, which bind to its transmembrane core and block the conformational change as well as the downstream signaling. Effects of the compounds were quantified in pharmacological cell assays in the dark and using UV and green light illumination. We finally develop a framework for image-based kinetic analysis of GPCRs which allowed us to measure activation kinetics of several prototypical class A GPCRs and to discover membrane heterogeneities of GPCR activation. It appears that GPCR activation signal is not only dependent on the amount of activated receptors, but also has some level of correlation with the local density of activated receptors.
In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood.
Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation.
Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood.
Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury.
G-protein- coupled receptors (GPCRs) are the largest family of membrane confined receptors and they transduce ligand binding to downstream effects. Almost 40% of the drugs in the world target GPCRs due to their function, albeit knowing less about their activation. Understanding their dynamic behaviour in basal and activated state could prove key to drug development in the future. GPCRs are known to exhibit complex molecular mobility patterns. A plethora of studies have been and are being conducted to understand the mobility of GPCRs. Due to limitations of imaging and spectroscopic techniques commonly used, the relevant timescales are hard to access. The most commonly used techniques are electron paramagnetic resonance or double electronelectron resonance, nuclear magnetic resonance, time-resolved fluorescence, single particle tracking and fluorescence recovery after photobleaching. Among these techniques only fluorescence has the potential to probe live cells. In this thesis, I use different time-resolved fluorescence spectroscopic techniques to quantify diffusion dynamics / molecular mobility of β2-adrenergic receptor (β2-AR) in live cells. The thesis shows that β2-AR exhibits mobility over an exceptionally broad temporal range (nanosecond to second) that can be linked to its respective physiological scenario. I explain how β2-AR possesses surprisingly fast lateral mobility (~10 μm²/s) associated with vesicular transport in contrast to the prior reports of it originating from fluorophore photophysics and free fluorophores in the cytosol. In addition, β2-AR has rotational mobility (~100 μs) that makes it conform to the Saffman-Delbrück model of membrane diffusion unlike earlier studies. These contrasts are due to the limitations of the methodologies used. The limitations are overcome in this thesis by using different time-resolved fluorescence techniques of fluorescence correlation spectroscopy (FCS), time-resolved anisotropy (TRA) and polarisation resolved fullFCS (fullFCS). FCS is limited to microsecond to the second range and TRA is limited to the nanosecond range. fullFCS complements the two techniques by covering the blind spot of FCS and TRA in the microsecond range. Finally, I show how ligand stimulation causes a decrease in lateral mobility which could be a hint at cluster formation due to internalisation and how β2-AR possesses a basal oligomerisation that does not change on activation. Thus, through this thesis, I show how different complementary fluorescence techniques are necessary to overcome limitations of each technique and to thereby elucidate functional dynamics of GPCR activation and how it orchestrates downstream signalling.
Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane.
Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand.
Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.
Platelets play an important role in the body, since they are part of the hemostasis
system, preventing and stopping blood loss. Nevertheless, when platelet or
coagulation system function are impaired, uncontrolled bleedings but also irreversible
vessel occlusion followed by ischemic tissue damage can occur. Therefore,
understanding platelet function and activation, mechanisms which are controlled by a
variety of platelet membrane receptors and other factors is important to advance out
knowledge of hemostasis and platelet malfunction. For a complete picture of platelet
function and their modulating behavior it is desired to be able to quantify receptor
distributions and interactions of these densely packed molecular ensembles in the
membrane. This challenges scientists for several reasons. Most importantly, platelets
are microscopically small objects, challenging the spatial resolution of conventional
light microscopy. Moreover, platelet receptors are highly abundant on the membrane
so even super-resolution microscopy struggles with quantitative receptor imaging on
platelets.
With Expansion microscopy (ExM), a new super-resolution technique was introduced,
allowing resolutions to achieve super-resolution without using a super-resolution
microscope, but by combining a conventional confocal microscopy with a highly
processed sample that has been expanded physically. In this doctoral thesis, I
evaluated the potential of this technique for super-resolution platelet imaging by
optimizing the sample preparation process and establishing an imaging and image
processing pipeline for dual-color 3D images of different membrane receptors. The
analysis of receptor colocalization using ExM demonstrated a clear superiority
compared to conventional microscopy. Furthermore, I identified a library of
fluorescently labeled antibodies against different platelet receptors compatible with
ExM and showed the possibility of staining membrane receptors and parts of the
cytoskeleton at the same time.
Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the “gold standard” gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes.
Die Rolle des Immunsystems nach MI hat innerhalb der letzten Jahrzehnte immer mehr Aufmerksamkeit erfahren, trotzdem herrschen weiterhin einige Unklarheiten. Daher war es Ziel dieser Arbeit, das Verhalten der T-Zellen nach MI im Mausmodell näher zu betrachten und zu analysieren. Dafür wurde einerseits mittels Durchflusszytometrie die T-Zell-Immunantwort im Herzen und in verschiedenen lymphatischen Organen mit Fokus auf pro- und antiinflammatorische Zytokine und deren Transkriptionsfaktoren genauer analysiert und andererseits ein Protokoll etabliert, um die T-Zellen im Herzen und in den Lymphknoten mittels Lichtblattmikroskopie sichtbar zu machen.
Dabei konnte festgestellt werden, dass die Expression von LAP, welches nicht-kovalent an das antiinflammatorische Zytokin TGF-ß1 gebunden ist und das wichtig für eine ausgeglichene Immunantwort ist, indem es überschießende Entzündungsreaktionen verhindert, in T-Zellen im Herzen nach MI im Vergleich zu naiven und scheinoperierten Mäusen signifikant hochreguliert war. Dieses Ergebnis konnte nur im Herzen und in keinem anderen der untersuchten Organe erzielt werden, weshalb es sich somit um eine lokale Immunreaktion handeln muss, die nur im Herzen nach MI stattfindet. Eine weitere Besonderheit war, dass die Häufigkeit des Vorkommens an Foxp3+ Treg im Herzen im Vergleich zu den anderen untersuchten Organen durchgehend am höchsten war, sowohl bei den Mäusen nach MI als auch bei naiven und scheinoperierten Mäusen. Dies unterstreicht, dass Foxp3+ Treg im Herzen eine wichtige Rolle spielen.
Dank der Verbesserung des Protokolls zur bildlichen Darstellung von T-Zellen im Herzen konnte gezeigt werden, dass sich diese nach MI insbesondere im Infarktgewebe befinden und dort relativ gleichmäßig verteilt sind. Außerdem konnten die mediastinalen Lymphknoten im Ganzen dargestellt und die einzelnen T-Zellen sichtbar gemacht werden.
Insgesamt lässt sich sagen, dass durch die vorliegende Arbeit neue Erkenntnisse zur Charakterisierung der T-Zell-Immunantwort nach MI im Mausmodell hinzugewonnen werden konnten. Die LAP+ T-Zellen scheinen nach MI im Herzen eine wichtige Rolle zu spielen, weshalb die Funktion dieser Zellen im Reparaturprozess nach MI in zukünftigen Versuchen genauer betrachtet werden sollte. Außerdem wurde der Grundstein zur Anfärbung und Darstellung von T-Zellen in Herzen und in Lymphknoten mittels Lichtblattmikroskopie gelegt, weshalb daran weitergearbeitet werden sollte, um auch andere Immunzellen neben den T-Zellen zeigen zu können. Dadurch können weitere Hinweise auf das Zusammenspiel der Immunzellen nach MI erhalten werden, um die immunologischen Vorgänge immer besser verstehen zu können.
Megakaryocytes (MKs) are the largest cells of the hematopoietic system and the precursor cells of platelets. During proplatelet formation (PPF) bone marrow (BM) MKs extent large cytoplasmic protrusions into the lumen of sinusoidal blood vessels. Under homeostatic conditions PPF occurs exclusively in the direction of the sinusoid, while platelet generation into the marrow cavity is prevented. So far, the mechanisms regulating this process in vivo are still not completely understood, especially when PPF is deregulated during disease. This thesis investigated the mechanisms of PPF in native BM and after myeloablation by total body irradiation (TBI).
First, we have identified a specialized type of BM stromal cells, so called CXCL12-abundant reticular (CAR) cells, as novel possible regulators of PPF. By using complementary high-resolution microscopy techniques, we have studied the morphogenetic events at the MK/vessel wall interface in new detail, demonstrating that PPF formation preferentially occurs at CAR cell-free sites at the endothelium.
In the second part of this thesis, we analyzed the processes leading to BM remodeling in response to myeloablation by TBI. We used confocal laser scanning microscopy (CLSM) to study the kinetic of radiation-triggered vasodilation and mapped extracellular matrix (ECM) proteins after TBI. We could demonstrate that collagen type IV and laminin α5 are specifically degraded at BM sinusoids. At the radiation-injured vessel wall we observed ectopic release of platelet-like particles into the marrow cavity concomitantly to aberrant CAR cell morphology, suggesting that the balance of factors regulating PPF is disturbed after TBI. ECM proteolysis is predominantly mediated by the matrix metalloproteinase MMP9, as revealed by gelatin-zymography and by a newly established BM in situ zymography technique. In transgenic mice lacking MMP9 vascular recovery was delayed, hinting towards a role of MMP9 in vessel reconstitution after myeloablation.
In a third series of experiments, we studied the irradiated BM in the context of hematopoietic stem cell transplantation (HSCT). By using mice as BM donors that ubiquitously express the fluorescent reporter protein dsRed we tracked engraftment of donor cells and especially MKs in the recipient BM. We found a distinct engraftment pattern and cluster formation for MKs, which is different from other blood cell lineages.
Finally, we assessed platelet function after TBI and HSCT and were the first to demonstrate that platelets become massively hyporeactive, particularly upon stimulation of the collagen receptor GPVI.
In summary, our findings shed light on the processes of PPF during health and disease which will help to develop treatments for aberrant thrombopoiesis.
Every year, stroke affects over 100 million people worldwide and the number of cases continues to grow. Ischemic stroke is the most prevalent form of stroke and rapid restoration of blood flow is the primary therapeutic aim. However, recanalization might fail or reperfusion itself induces detrimental processes leading to infarct progression. Previous studies identified platelets and immune cells as drivers of this so-called ischemia/reperfusion (I/R) injury, establishing the concept of ischemic stroke as thrombo-inflammatory disease. Reduced cerebral blood flow despite recanalization promoted the hypothesis that thrombus formation within the cerebral microcirculation induces further tissue damage. The results presented in this thesis refute this: using complementary methodologies, it was shown that infarct growth precedes the occurrence of thrombi excluding them as I/R injury-underlying cause. Blood brain barrier disruption is one of the hallmarks of ischemic stroke pathology and was confirmed as early event during reperfusion injury in the second part of this study. Abolished platelet α-granule release protects mice from vascular leakage in the early reperfusion phase resulting in smaller infarcts. Using in vitro assays, platelet α-granule-derived PDGF-AB was identified as one factor contributing to blood-brain barrier disruption.
In vivo visualization of platelet activation would provide important insights in the spatio-temporal context of platelet activation in stroke pathology. As platelet signaling results in elevated intracellular Ca2+ levels, this is an ideal readout. To overcome the limitations of chemical calcium indicators, a mouse line expressing an endogenous calcium reporter specifically in platelets and megakaryocytes was generated. Presence of the reporter did not interfere with platelet function, consequently these mice were characterized in in vivo and ex vivo models.
Upon ischemic stroke, neutrophils are among the first cells that are recruited to the brain. Since for neutrophils both, beneficial and detrimental effects are described, their role was investigated within this thesis. Neither neutrophil depletion nor absence of NADPH-dependent ROS production (Ncf-/- mice) affected stroke outcome. In contrast, abolished NET-formation in Pad4-/- mice resulted in reduced infarct sizes, revealing detrimental effects of NETosis in the context of ischemic stroke, which might become a potential therapeutic target.
Cerebral venous (sinus) thrombosis, CV(S)T is a rare type of stroke with mainly idiopathic onset. Whereas for arterial thrombosis a critical contribution of platelets is known and widely accepted, for venous thrombosis this is less clear but considered more and more. In the last part of this thesis, it was shown that fab-fragments of the anti-CLEC-2 antibody INU1 trigger pathological platelet activation in vivo, resulting in foudroyant CVT accompanied by heavy neurological symptoms. Using this novel animal model for CVT, cooperative signaling of the two platelet receptors CLEC-2 and GPIIb/IIIa was revealed as major trigger of CVT and potential target for treatment.