Lipid bilayers form the basis of the membranes that serve as a barrier between a cell and its physiological environment. Their physical properties make them ideally suited for this role: they are extremely soft with respect to bending but essentially incompressible under lateral tension, and they are quite permeable to water but essentially impermeable to ions which allows the rapid establishment of the osmotic gradients. The function of membrane proteins, which are vital for tasks ranging from signal transduction to energy conversion, depends on their interactions with the lipid environment. Because of the complexity of natural membranes, model systems consisting of simpler lipid mixtures have become indispensable tools in the study of membrane biophysics. The objective of the work reported here is to develop a deeper understanding of the underlying physics of lipid bilayers through nanoscale measurements of the mechanical properties of mixed lipid systems including cholesterol, a key ingredient of cell membranes. Atomic force microscopy (AFM) has been used extensively to measure the topographical and elastic properties of supported lipid bilayers displaying complex phase behaviour and containing mixtures of important PC, PE lipids and cholesterol. Phase transformations have been investigated varying the membrane temperature, and the effects of cholesterol in controlling membrane fluidity, phase, and energetics have been studied. Elastic modulus measurements were correlated with phase behaviour observations. To aid in the nanoscale probing of lipid bilayers, AFM probes with a high aspect ratio and tip radii of $sim$4~nm were fabricated and characterised. These probes were used to investigate the phase boundary in binary and ternary lipid systems, leading to the discovery of a raised region at the boundary which has implications for the localisation of reconstituted proteins as well as the role of natural domains or lipid rafts. The electrical properties of the probes were examined to assess their potential application for combined structural and electrical measurements in liquid. A novel technique was developed to aid in the study of the physical properties of lipid bilayers. Membrane budding was induced above microfabricated substrates through osmotic pressure. Modification of the adhesion energy of the bilayer through biotin-avidin linking was successful in modulating budding behaviour of liquid disordered bilayers. The free energy of the system was modelled to allow quantitative information to be extracted from the data.

Die mechanischen Eigenschaften von Zellen charakterisieren und beeinflussen deren Zustand. Die vorliegende Arbeit zielt auf ein besseres Verständnis der biomechanischen Eigenschaften von Zellen ab. Der Fokus lag dabei auf der Biegesteifigkeit von Zellmembranen und der Deformierbarkeit der Zellen. Es werden drei Studien vorgestellt in der diese Materialparameter untersucht wurden. Die erste Studie befasst sich mit der Temperaturabhängigkeit der mechanischen Eigenschaften. Hierbei wurden acht verschieden Zelllienien bei jeweils fünf Temperaturen rheologisch vermessen. Zur Messung wurde der sog. \"optical stretcher\" verwendet der gleichzeitig die Zellen deformieren und aufheizen kann. Die Versuche zeigen, dass eine Zeit-Temperatur superposition dabei nicht für alle Zelltypen funktioniert. In der zweiten Studie wurden die Membransteifigkeit von Gewebeproben von Brust- und Gebärmutterhalskrebspatienten untersucht. Als Kontrollsystem wurde gutartiges Gewebe aus dem Umfeld des Tumors verwendet. Es konnte gezeigt werden, dass die Zellmembranen von Tumorzellen weicher waren als von gesundem Vergleichsgewebe. Die Änderung der Membrankomposition wurde dabei als mögliche Ursache massenspektroskopisch Untersucht und verschieden Ursachen der weichen Membrane diskutiert. Für die dritte Studie wurde der chemische Wirkstoff Soraphen A eingesetzt um die Membransteifigkeit von zwei Zelllienien zu erhöhen. Dies zeigte eine Verringerung von Zellbeweglichkeit und Invasivität.

No description available.

During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the sPLA2 active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting increased lipid spacing. Second, laurdan detected increased solvation of the lower head group region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it was proposed that the apoptotic membranes become susceptible to sPLA2 through a reduction in lipid-neighbor interactions which facilitates migration of phospholipids into the enzyme active site. This proposal was then examined in T-lymphocytes treated with glucocorticoid, a more physiologically relevant apoptotic stimulant, using similar techniques. The following observations corresponded to induction of membrane susceptibility: increased merocyanine 540 intercalation; phosphatidylserine flip-flop, detected by annexin binding; and alterations in laurdan fluorescence properties. These observations implied a relationship among sPLA2 susceptibility, lipid spacing, and phosphatidylserine exposure. To clarify this relationship, additional assays were also performed using dibutyryl-cAMP to induce apoptosis, a drug reported to induce apoptosis in S49 cells without the typical translocation of phosphatidylserine. Our results indicated that in cells treated with dibutyryl-cAMP, the merocyanine 540 response and its correlation with sPLA2 susceptibility was similar to that observed with dex-treated samples. This suggests that the underlying mechanisms which promote sPLA2 hydrolysis lead to alterations that may be facilitated by but do not require phosphatidylserine exposure. Taken together, all of the results suggest that direct regulation of the biophysical microenvironment of the membrane is the mode of control of membrane susceptibility to the hydrolytic activity of sPLA2.

Indiana University-Purdue University Indianapolis (IUPUI) / Omega-3 polyunsaturated fatty acids (n-3 PUFAs) relieve the symptoms of a wide variety of chronic inflammatory disorders. Typically, they must be obtained in the diet from sources such as fish oils. Docosahexaenoic acid (DHA) is one of these n-3 PUFAs. As yet the structural mechanism responsible for the health benefits within the body is not completely understood. One model that has emerged from biochemical and imaging studies of cells suggests that n-3 PUFAs are taken up into phospholipids in the plasma membrane. Thus the focus here is on the plasma membrane as a site of potential structural modification by DHA. Within cellular membranes, the huge variety of molecules (called lipids) which constitute the membrane suggest inhomogeneous mixing, thus domain formation. One potential domain of interest is called the lipid raft, which is primarily composed of sphingomyelin (SM) and cholesterol (chol). Here the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane was examined by solid state 2H NMR spectroscopy. Solid state 2H NMR spectroscopy extracts details of molecular orientation and anisotropy of molecular reorientation by analysis of the lineshape. This essentially non-invasive technique allows for a direct measurement of dynamics in bulk materials which has been extensively applied to biological materials. It is a niche area of NMR for which standard software often lack necessary features. Two software programs, “EchoNMR processor” and “EchoNMR simulator”, collectively known as “EchoNMR tools”, that were developed to quickly process and analyze one-dimensional solid-state NMR data, will be described along with some theoretical background of the techniques used. EchoNMR tools has been designed with a focus on usability and the open-source mindset. This is achieved in the in the MATLAB® programming environment which allows for the development of the graphical user interfaces and runs as an interpreter which allows the code to be open-source. The research described here on model membranes demonstrates the utility of the software. The NMR spectra for PSM-d31 in mixtures with PDPC or POPC with cholesterol were interpreted in terms of the presence of nano-sized SM-rich/chol-rich (raft-like) and PC-rich/chol-poor (non-raft) domains that become larger when POPC was replaced by PDPC. An increase in the differential in order and/or thickness between the two types of domains is responsible. The observation of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), attributed to the raft-like and non-raft domains enabled the determination of the composition of the domains. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain. There was also a substantial amount of PDPC (70%) in the raft-like domain that appears to have minimal effect on the order of SM. PDPC molecules sequestering into small groups to minimize the contact of DHA chains with cholesterol is one possible explanation that would also have implications on raft continuity. These results refine the understanding of how DHA may modulate the structure of raft domains in membranes.

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.

The nucleus of eukaryotic cells is a vitally important organelle that sequesters the genetic information of the cell, and protects it with the help of two highly evolved structures, the nuclear envelope (NE) and nuclear pore complexes (NPCs). Together, these two structures mediate the bidirectional trafficking of molecules between the nucleus and cytoplasm by forming a barrier. NE transmembrane proteins (NETs) embedded in either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM) play crucial roles in both nuclear structure and functions, including: genome architecture, epigenetics, transcription, splicing, DNA replication, nuclear structure, organization and positioning. Furthermore, numerous human diseases are associated with mutations and mislocalization of NETs on the NE. There are still many fundamental questions that are unresolved with NETs, but we focused on two major questions: First, the localization and transport rate of NETs, and second, the transport route taken by NETs to reach the INM. Since NETs are involved with many of the mechanisms used to maintain cellular homeostasis, it is important to quantitatively determine the spatial locations of NETs along the NE to fully understand their role in these vital processes. However, there are limited available approaches for this task, and moreover, these methods provide no information about the translocation rates of NETs between the two membranes. Furthermore, while the trafficking of soluble proteins between the cytoplasm and the nucleus has been well studied over the years, the path taken by NETs into the nucleus remains in dispute. At least four distinct models have been proposed to suggest how transmembrane proteins destined for the INM cross the NE through NPC-dependent or NPC-independent mechanisms, based on specific features found on the soluble domains of INM proteins. In order to resolve these two major questions, it is necessary to employ techniques with the capabilities to observe these dynamics at the nanoscale. Current experimental techniques are unable to break the temporal and spatial resolution barriers required to study these phenomena. Therefore, we developed and modified single-molecule techniques to answer these questions. First, to study the distribution of NETs on the NE, we developed a new single-molecule microscopy method called single-point single-molecule fluorescence recovery after photobleaching (smFRAP), which is able to provide spatial resolution <10 nm and, furthermore, provide previously unattainable information about NET translocation rates from the ONM to INM. Secondly, to examine the transport route used by NETs destined for the INM, we used a single-molecule microscopy technique previously developed in our lab called single-point edge-excitation sub-diffraction (SPEED) microscopy, which provides spatio-temporal resolution of <10 nm precision and 0.4 ms detection time. The major findings from my doctoral research work can be classified into two categories: (i) Technical developments to study NETs in vivo, and (ii) biological findings from employing these microscopy techniques. In regards to technical contributions, we created and validated of a new single-molecule microscopy method, smFRAP, to accurately determine the localization and distribution ratios of NETs on both the ONM and INM in live cells. Second, we adapted SPEED microscopy to study transmembrane protein translocation in vivo. My work has also contributed four main biological findings to the field: first, we determined the in vivo translocation rates for lamin-B receptor (LBR), a major INM protein found in the nucleus of cells. Second, we verified the existence of peripheral channels in the scaffolding of NPCs and, for the first time, directly observed the transit of INM proteins through these channels in live cells. Third, our research has elucidated the roles that both the nuclear localization signal (NLS) and intrinsically disordered (ID) domains play in INM protein transport. Finally, my work has elucidated which transport routes are used by NETs destined to localize in the INM. / Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.

Zellmembranen sind hochspezialisierte Mehrkomponentenlegierungen, welche sowohl die Zelle selbst als auch ihre Organellen umgeben. Sie spielen eine entscheidende Rolle bei vielen biologisch relevanten Prozessen wie die Signaltransduktion und die Zellbewegung. Aus diesem Grund ist eine genaue Charakterisierung ihrer Eigenschaften der Schlüssel zum Verständnis der Bausteine des Lebens sowie ihrer Erkrankungen. Besonders Krebs steht im engen Zusammenhang mit Veränderungen der biomechanischen Eigenschaften vom Gewebe, Zellen und ihren Organellen. Während Veränderungen des Zytoskeletts von Krebszellen im Fokus vieler Biophysiker stehen, ist die Bedeutung der Biomechanik von Zellmembran weitgehend unklar. Zellmembranen faszinieren Wissenschaftler jedoch nicht nur wegen ihrer biomechanischen Eigenschaften. Sie sind auch Beispiele für eine selbstorganisierte und heterogene Landschaft, in der Prozesse fernab des Gleichgewichtes, wie z.B. räumliche und zeitliche Musterbildungen, auftreten. Die vorgelegte Dissertation untersucht erstmals umfassend die zentrale Rolle der Zellmembran und ihrer molekularen Architektur für die Signalübertragung, die Biomechanik und die Zellmigration. Hierfür werden einfache Modellmembranen aber auch komplexere Vesikel und ganze Zellen mittels etablierter physikalischer Methoden analysiert. Diese reichen von Fourier- Analysen zur Charakterisierung von thermisch angeregten Membranundulationen über Massenspektrometrie und ‘Optical Stretcher’ Messungen von ganzen Zellen bis hin zur Filmwaagentechnik. Des Weiteren wird ein Modellsystem vorgestellt, welches sowohl einen experimentellen als auch einen mathematischen Zugang zum ‘ME-switch’ ermöglicht. Die vorgelegte Dissertation bietet neue Einblicke in wichtige Funktionen von Zellmembranen und zeigt neue therapeutische Perspektiven in der Membran- und Krebsforschung auf.

Zellmembranen sind hochspezialisierte Mehrkomponentenlegierungen, welche sowohl die Zelle selbst als auch ihre Organellen umgeben. Sie spielen eine entscheidende Rolle bei vielen biologisch relevanten Prozessen wie die Signaltransduktion und die Zellbewegung. Aus diesem Grund ist eine genaue Charakterisierung ihrer Eigenschaften der Schlüssel zum Verständnis der Bausteine des Lebens sowie ihrer Erkrankungen. Besonders Krebs steht im engen Zusammenhang mit Veränderungen der biomechanischen Eigenschaften vom Gewebe, Zellen und ihren Organellen. Während Veränderungen des Zytoskeletts von Krebszellen im Fokus vieler Biophysiker stehen, ist die Bedeutung der Biomechanik von Zellmembran weitgehend unklar. Zellmembranen faszinieren Wissenschaftler jedoch nicht nur wegen ihrer biomechanischen Eigenschaften. Sie sind auch Beispiele für eine selbstorganisierte und heterogene Landschaft, in der Prozesse fernab des Gleichgewichtes, wie z.B. räumliche und zeitliche Musterbildungen, auftreten. Die vorgelegte Dissertation untersucht erstmals umfassend die zentrale Rolle der Zellmembran und ihrer molekularen Architektur für die Signalübertragung, die Biomechanik und die Zellmigration. Hierfür werden einfache Modellmembranen aber auch komplexere Vesikel und ganze Zellen mittels etablierter physikalischer Methoden analysiert. Diese reichen von Fourier- Analysen zur Charakterisierung von thermisch angeregten Membranundulationen über Massenspektrometrie und ‘Optical Stretcher’ Messungen von ganzen Zellen bis hin zur Filmwaagentechnik. Des Weiteren wird ein Modellsystem vorgestellt, welches sowohl einen experimentellen als auch einen mathematischen Zugang zum ‘ME-switch’ ermöglicht. Die vorgelegte Dissertation bietet neue Einblicke in wichtige Funktionen von Zellmembranen und zeigt neue therapeutische Perspektiven in der Membran- und Krebsforschung auf.:1 Introduction 2 Background 2.1 The Cell Membrane 2.1.1 Lipids in Cell Membranes 2.1.2 Membrane Proteins 2.1.3 An Overview on Membrane Models 2.1.4 Lipid Rafts 2.2 Model Membranes – An Experimental Access to Cell Membranes 2.2.1 Surface Tension and Thermodynamic Equilibrium 2.2.2 Langmuir Monolayer 2.2.3 The Polymorphism of Langmuir Monolayers 2.2.4 Membrane Vesicles 2.3 Biological Membranes as Semiflexible Shells 2.3.1 Elasticity of Soft Shells 2.3.2 Helfrichs Theory About Bending Deformations 2.3.3 Membrane Undulation 2.4 Membranes in Cell Signaling 2.4.1 Signal Transduction Fundamentals 2.4.2 Phosphoinositides 2.4.3 Phosphatidylinositol Signaling Pathway 2.4.4 The Myristoyl-Electrostatic Switch 2.5 Reaction-Diffusion Systems 2.5.1 Diffusion 2.5.2 Michaelis-Menten Kinetics 2.5.3 Reaction-Diffusion Systems 3 Methods, Materials and Theory 3.1 Optical Microscopy 3.1.1 Fluorescence Microscopy 3.1.2 Phase Contrast Microscopy 3.2 Cell Culture and GPMV Formation 3.2.1 Tumor Dissociation and Cell Culturing of Primary Cells 3.2.2 Cell Lines and Cell Culturing 3.2.3 Preparation of Giant Plasma Membrane Vesicles 3.3 Optical Stretcher 3.4 Fourier Analysis of Thermally Excited Membrane Fluctuations 3.4.1 The Quasi-Spherical Model – Membrane Fluctuations 3.4.2 Determination of the Bending Rigidity 3.5 Mass Spectrometry 3.5.1 MALDI-TOF Mass Spectrometry 3.5.2 ESI Mass Spectrometry 3.6 Migration, Invasion and Cell Death Assays 3.7 Langmuir-Blodgett Technique 3.7.1 Langmuir Troughs and Film Balances 3.7.2 Experimental Setup and Monolayer Preperation 3.7.3 Phospholipids, Dyes and Buffer Solutions 4 Fluctuations in Cell Membranes 4.1 Cell Membrane Softening in Human Breast and Cervical Cancer Cells 4.1.1 Bending Rigidity of Human Beast and Cervical Cell Membranes 4.1.2 MALDI-TOF Analysis of Lipid Composition 4.1.3 Summary and Discussion 4.2 Targeting of Membrane Rigidity – Implications on Migration 4.2.1 ESI Tandem Analysis of Lipid Composition 4.2.2 Biomechanical Behavior of Whole Cells and Membranes 4.2.3 Migration and Invasion Behavior 4.2.4 Summary and Discussion 5 Oscillations in Cell Membranes 5.1 Mimicking the ME-switch 5.1.1 DPPC/PIP2 monolayers at the presence of MARCKS 5.1.2 Lateral organization of PIP2 in DPPC/PIP2 monolayers 5.1.3 Translocation of MARCKS 5.1.4 Phosphorylation of MARCKS by PKC 5.1.5 Summary and Discussion 5.2 Dynamic Membrane Structure Induces Temporal Pattern Formation 5.2.1 Mechanism of the Oscillation 5.2.2 Modeling the ME-switch 5.2.3 Time Evolution 5.2.4 Phase Diagrams and Open Systems 5.2.5 Summary and Discussion 6 Conclusion and Outlook Appendix Bibliography List of Figures List of Abbreviations Acknowledgement