Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 6 (2017): e30867, doi:10.7554/eLife.30867.The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.DWG has received funding from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 241548 (MitoSys) and no. 258068 (Systems Microscopy), an ERC Starting Grant under agreement no. 281198 (DIVIMAGE), and from the Austrian Science Fund (FWF) project no. SFB F34-06 (Chromosome Dynamics). FS has received funding from an EMBO long-term fellowship (ALTF 1447–2012). SM has received funding from Human Frontier Science Program cross-disciplinary fellowship (LT000096/2011)

Radaufhängung für angetriebene Hinterachsen von geländegängigen Radfahrzeugen.

Gegenstand dieser Arbeit war die Entwicklung einer Hinterachse für ein allradgetriebenes, geländegängiges (6x6) Fahrzeug, mit der Zielsetzung diese raum- und massesparend auszulegen. Weiterhin sollten sich über den Verlauf des Radhubes die gewünschten kinematischen Änderungen wie Vorspur, Sturz und Spurweitenänderung einstellen. Vor Beginn der Entwicklung der Halbachse erfolgte eine Untersuchung verschiedener Achskonzepte, die herausstellen sollte, welche Radführung die oben genannten Kriterien am besten erfüllt. Aus dieser Bewertung ging die Schräglenkerachse als Vorzugsvariante hervor. Danach folgte die kinematische Grundauslegung dieser Radführung. Darin wurden unter anderem die Spurweite und die Kinematikpunkte festgelegt. Daran anschliessend erfolgte die konstruktive Ausarbeitung der Schräglenkerachse unter Berücksichtigung der Randbedingungen von Antrieb, Bremse, Federung und Dämpfung. Mittels Finite- ELemente- Methode wurden zwei definierte Lastfälle berechnet, um so die statische Festigkeit der Halbachse nachzuweisen. Abschliessend wurde, unter Verwendung o.g. Kriterien, ein Vergleich mit einer Doppelquerlenkachse, welche ebenfalls für dieses Fahrzeug entwickelt wurde, durchgeführt

High-throughput rheological measurements with an optical stretcher

The cytoskeleton is a major determinant of the mechanical strength and morphology of most cells. The composition and assembly state of this intracellular polymer network evolve during the differentiation of cells, and the structure is involved in many cellular functions and is characteristically altered in many diseases, including cancer. Here we exploit the deformability of the cytoskeleton as a link between molecular structure and biological function, to distinguish between cells in different states by using a laser-based optical stretcher (OS) coupled with microfluidic handling of cells. An OS is a cell-slized, dual-beam laser trap designed to nondestructively test the deformability of single suspended cells. Combined with microfluidic delivery, many cells can be measured serially in a short amount of time. With this tool it could be shown that optical deformability is sensitive enough to monitor subtle changes during the progression of cells from normal to cancerous and even a metastatic state. Stem cells can also be distinguished from more differentiated cells. The surprisingly low number of cells required for this assay reflects the tight regulation of the cytoskeleton by the cell. This suggests the possibility of using optical deformability as an inherent cell marker for basic cell biological investigation, diagnosis of disease, and sorting of stem cells from heterogeneous populations, obviating the need for external markers or special preparation. Many additional biological assays can be easily adapted to utilize this innovative physical method. This chapter details the setup and use of the microfluidic OS, the analysis and interpretation of data, and the results of a typical experiment

Feeling for cells with light

In an optical stretcher, infrared laser light is used to exert surface stress on biological cells, causing an elongation of the trapped cell body along the laser beam axis. These optically induced deformations characterize individual cells and cell lines. When integrated within a microfluidic chamber with high throughput, this enables diagnosis of diseases, on a cellular level, that are associated with cytoskeletal processes. Additionally, it allows sorting of cells with high accuracy in a non-contact manner. To determine the surface stress on the cell, ray optics calculations as well as the system transfer operator (T-matrix) approach with an appropriate incident field are used. The latter approach allows a more accurate modeling of the cell in the optical stretcher and reveals a more detailed stress profile acting on the cell surface. Analyzing the deformation behavior of normal and malignantly transformed fibroblasts, significant differences in axial elongation even for sample sizes as low as 30 cells are already measurable on a time scale of 0.1s. Here, malignant transformation of cells is discussed as an example of how any process that affects the cell's optical or mechanical properties allows classification with the optical stretcher

Oral Cancer Diagnosis by Mechanical Phenotyping

Oral squamous cell carcinomas are among the 10 most common cancers and have a 50% lethality rate after 5 years. Despite easy access to the oral cavity for cancer screening, the main limitations to successful treatment are uncertain prognostic criteria for (pre-)malignant lesions. Identifying a functional cellular marker may represent a significant improvement for diagnosis and treatment. Toward this goal, mechanical phenotyping of individual cells is a novel approach to detect cytoskeletal changes, which are diagnostic for malignant change. The compliance of cells from cell lines and primary samples of healthy donors and cancer patients was measured using a microfluidic optical stretcher. Cancer cells showed significantly different mechanical behavior, with a higher mean deformability and increased variance. Cancer cells (n approximate to 30 cells measured from each patient) were on average 3.5 times more compliant than those of healthy donors [D-normal = (4.43 +/- 0.68) 10(-3) Pa-1; D-cancer = (15.8 +/- 1.5) 10(-3) Pa-1; p < 0.01]. The diagnosis results of the patient samples were confirmed by standard histopathology. The generality of these findings was supported by measurements of two normal and four cancer oral epithelial cell lines. Our results indicate that mechanical phenotyping is a sensible, label-free approach for classifying cancer cells to enable broad screening of suspicious lesions in the oral cavity. It could in principle be applied to any cancer to aid conventional diagnostic procedures. [Cancer Res 2009;69(5):1728-32

Modelling the structural response of an eukaryotic cell in the optical stretcher

The cytoskeleton of an eukaryotic cell is a composite polymer material with unique structural (mechanical) properties. To investigate the role of individual cytoskeletal polymers in the deformation response of a cell to an external force (stress), we created two structural models - a thick shell model for the actin cortex, and a three-layered model for the whole cell. These structural models for a cell are based on data obtained by deforming suspended cells, where each cell is stretched between two counter-propagating laser beams using an optical stretcher. Our models, with the data, suggest that the outer actin cortex is the main determinant of the structural response of the cell