Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small(„5% deformation) and large strains(>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.

Die Metaphasenspindel ist eine selbstorganisierende molekulare Maschine, die die entscheidende Funktion erfüllt, das Genom während der Zellteilung gleichmäßig zu trennen. Spindellänge und -form sind emergente Eigenschaften, die durch komplexe Wechselwirkungsnetzwerke zwischen Molekülen hervorgerufen werden. Obwohl erhebliche Fortschritte beim Verständnis der einzelnen molekularen Akteure erzielt wurden, die ihre Länge und Form beeinflussen, haben wir erst kürzlich damit begonnen, die Zusammenhänge zwischen Spindelmorphologie, Dynamik und Materialeigenschaften zu untersuchen. In dieser Arbeit untersuchte ich zunächst quantitativ die Rolle zweier molekularer Kraftgeneratoren - Kinesin-5 und Dynein - bei der Regulierung der Spindelform von Xenopus-Eiextrakt. Eine Störung ihrer Aktivität veränderte die Spindelmorphologie, ohne die Gesamtmasse der Mikrotubuli zu beeinflussen. Um die Spindelform physikalisch zu stören, wurde ein Optical Stretcher (OS) -Aufbau entwickelt. Obwohl das OS Vesikel in Extrakten verformen könnte, konnte keine Kraft auf Spindeln ausgeübt werden. Die Untersuchung des Brechungsindex der Struktur mittels optischer Beugungstomographie (ODT) ergab, dass es keinen Unterschied zwischen Spindel und Zytoplasma gab. Korrelative Fluoreszenz- und ODT-Bildgebung zeigten, wie sich die Materialeigenschaften innerhalb verschiedener Biomoleküle räumlich unterschieden. Die Gesamttrockenmasse der Spindel skalierte mit der Länge, während die Gesamtdichte konstant blieb. Interessanterweise waren die Spindeln in HeLa-Zellen dichter als das Zytoplasma. Schließlich deckte eine störende Mikrotubulusdichte auf, wie die Gesamttubulinkonzentration die Spindelgröße, die Gesamtmasse und die Materialeigenschaften regulierte. Insgesamt bietet diese Studie eine grundlegende Charakterisierung der physikalischen Eigenschaften der Spindel und hilft dabei, Zusammenhänge zwischen der Biochemie und der Biophysik einer aktiven Form weicher Materie zu beleuchten. / The metaphase spindle is a self-organising molecular machine that performs the critical function of segregating the genome equally during cell division. Spindle length and shape are emergent properties brought about by complex networks of interactions between molecules. Although significant progress has been made in understanding the individual molecular players influencing its length and shape, we have only recently started exploring the links between spindle morphology, dynamics, and material properties. A thorough analysis of spindle material properties is essential if we are to comprehend how such a dynamic structure responds to forces, and maintains its steady-state length and shape. In this work, I first quantitatively investigated the role of two molecular force generators– Kinesin-5 and Dynein in regulating Xenopus egg extract spindle shape. Perturbing their activity altered spindle morphology without impacting total microtubule mass. To physically perturb spindle shape, an Optical Stretcher (OS) setup was developed. Although the OS could deform vesicles in extracts, force could not be exerted on spindles. Investigating the structure’s refractive index using Optical Diffraction Tomography (ODT) revealed that there was no difference between the spindle and cytoplasm. Correlative fluorescence and ODT imaging revealed how material properties varied spatially within different biomolecules. Additionally, spindle mass density and the microtubule density were correlated. The total dry mass of the spindle scaled with length while overall density remained constant. Interestingly, spindles in HeLa cells were denser than the cytoplasm. Finally, perturbing microtubule density uncovered how total tubulin concentration regulated spindle size, overall mass and material properties. Overall, this study provides a fundamental characterisation of the spindle’s physical properties and helps illuminate links between the biochemistry and biophysics of an active form of soft matter.

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

No description available.

The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.