Intracellular micromechanics of the syncytial Drosophila embryo
by Daniel Rene Alok Weßel
Date of Examination:2015-03-23
Date of issue:2015-09-28
Advisor:Prof. Dr. Christoph F. Schmidt
Referee:Prof. Dr. Christoph F. Schmidt
Referee:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Detlev Schild
Persistent Address:
http://dx.doi.org/10.53846/goediss-5276
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Abstract
English
A developing embryo is a striking and fascinating example of self-organization and collective behavior of biological matter. In the presented work, I investigated the cytoplasmic interior of Drosophila melanogaster embryos. In its early stage the embryo forms a syncytium, i.e. multiplying nuclei are not yet separated by cell membranes, but are interconnected by cytoskeletal polymer networks consisting of actin and microtubules. Between division cycles 9 and 13, nuclei form a 2D cortical layer with the cytoskeleton associated to it. To probe the mechanical properties and dynamics of this self-organizing "pre-tissue", I measured shear moduli in the embryo by high-speed video microrheology. Therefore, I built a multi-color fluores- cence microscope for simultaneously imaging at high speeds with frame rates of up to several kHz and at normal video rates in order to access an extended frequency range. I recorded position fluctuations of injected micron-sized fluorescent beads and characterized the viscoelasticity of the embryo in different locations. Thermal fluctuations dominated over non-equilibrium activity for frequencies between 0.3 and 1000 Hz. Between nuclear layer and central yolk the cytoplasm was homogeneous and viscously-dominated, with a viscosity three orders of magnitude higher than that of water. Close to the nuclear layer, particularly close to the cortex, I found an increase of the elastic and viscous moduli consistent with an increased microtubule density. Mechanical response near the nuclear layer is likely to be caused by loosely entangled microtubule networks, whereas in the interior, towards the central yolk, it is due to a macromolecular solution. Drug-interference experiments showed that microtubules contribute to the measured viscoelasticity inside the embryo, whereas actin only plays a minor role in the regions I probed with the micron sized beads, i.e. outside of the actin caps and cortex. Measurements at different stages of the nuclear division cycle showed little variation. During nuclear separation at anaphase I found directed motion of probe particles, the only measurable sign for non-equilibrium ac- tivity, so far. Secondly I investigated single-walled carbon nanotubes (CNT) as fluorescent and trappable probes by means of the custom-built setup incorporating near-infrared imaging and spectroscopy instruments as well as multiple trapping lasers and an interferometric detection system. CNTs have an intrinsic near-infrared fluorescence, emitting within a wavelength window almost free of autofluorescence in biological tissue. Hence, imaging of single CNTs injected into the whole living fly embryo by wide-field microscopy was possible. Mean squared displacements of tracked CNTs within the embryonic cytoplasm showed diffusive and subdiffusive motion as well as directed movements, some of them correlating with nuclear motion. In vitro experi- ments characterized CNTs as fluorescent probes, lacking fluorescence intermittency, exhibiting short fluorescence cycle lifetimes and high photostability (though lower than in previous findings). I optically trapped CNTs, simultaneously confirmed by fluorescence microscopy and interferometric detection. The shape of a position power spectral density of a trapped CNT was close to a Lorentzian and the mea- surement of the position variance allowed a determination of the number of trapped particles. No clear signs for resonance effects on trapping efficiency were observed.
Keywords: Syncytial; Drosophila; Microrheology; Carbon Nanotubes; Intracellular; Viscoelastic; Rheology; Optical Trapping; High-speed; Video Microrheology