| dc.description.abstracteng | The actin cytoskeleton is ubiquitously distributed among eukaryotic organisms and determines the shape and the movement of eukaryotic cells. In single migratory cells, rapid and
localized actin polymerization often follows an external stimulus, engaging migration towards or away from the stimulus. This process is termed chemotaxis and plays a crucial
role in several different eukaryotic processes, for instance the inflammatory response of
neutrophils and macrophages, cancer metastasis or the growth of axons. A suitable model
system to study the chemotactically induced response of the actin cytoskeleton is the social
amoeba Dictyostelium discoideum. Within its life cycle, it exists as an autonomously living
and proliferating single cell as well as a differentiated and multicellular organism. The developmental program to multicellularity is engaged under starvation conditions and serves
as a survival mechanism. Within this development, the cells become chemotactic towards
3’-5’ cyclic adenosine monophosphate (cAMP), which is emitted spontaneously by cells to
attract other amoebae. If a D. discoideum amoeba is exposed to a sudden upshift of external cAMP concentration, the actin cytoskeleton responds biphasically, i.e. an initial sharp
increase in filamentous actin concentration is followed by a broader and smaller maximum.
This biphasic behavior resembles a damped oscillation, if the input is shortened to a brief
pulse of cAMP.
Within this thesis, the oscillatory properties of the actin cytoskeleton and the chemotactic
signaling cascade, controlling the actin polymerization were probed by different periodic
input functions of externally administered cAMP. The pulses were created using the flow
photolysis method, in which chemically caged molecules are photochemically released in
the microfluidic flow. Pulse lengths down to 1.5 s are possible with concentration switching times below 1 s. The response of the D. discoideum actin cytoskeleton, which was labeled via LimE-GFP (a protein specifically associated with filamentous actin) suggests
a resonance of the actin cytoskeleton at input periods equal to 20 s. The second harmonic
frequency becomes apparent, above the resonance timescale. Furthermore, the response
to short periods suggest an onset of oscillatory behavior, i.e. the actin cytoskeleton follows the external forcing, above input periods of 8 s. This is considerably shorter than the
20 s timescale, previously estimated for the chemotactic signaling cascade. In conjunction
with these observations and the observation that a minor fraction of all cells showed selfsustained oscillations of the LimE-GFP fluorescence intensity, we proposed that the actin
cytoskeleton and/or the signal processing cascade operate close to an oscillatory instability.
A delay differential equation, which shows this type of Hopf bifurcation was successfully
used to model the principal observed temporal patterns. The delay was experimentally verified by measuring the delay between LimE-GFP, the polymerization marker and CoroninGFP as well as Aip1-GFP, two labels of the actin depolymerization process.
Interestingly self-sustained oscillations of the LimE-GFP label have been reported previously in a D. discoideum knockout mutant of the Arp2/3 regulatory SCAR-complex. One
could speculate that, within this mutant, a larger fraction of cells passed the instability criterion, explaining the higher rate of observed oscillations. The oscillatory patterns, obtained
by confocal laser scanning microscopy (CLSM), lacked verification by other microscopy
techniques and a possible light dependence of the oscillations was supposed. Additional
CLSM experiments were conducted within this thesis and such light dependence was not
observed. However, it was verified that the oscillations occur globally within the cell. Assuming that the constant polymerization and depolymerization cycles act directly on the cell
membrane gave rise to the possibility to read out the height of a cell as a non-optical parameter. Atomic force microscopy (AFM) height measurements over time characterized the
difference as stronger fluctuations of the cell height within the SCAR(-)/PIR121(-) knockout mutant.
To study the actin cytoskeleton on the timescales of polymerizing filamentous actin bundles other microscopy techniques than CLSM need to be applied. Most prominently the
total internal reflection fluorescence microscopy (TIRF) images solely the lower boundary
of the specimen on a high time resolution. One way to improve the quality of TIRF imaging
is to compress the cell and therefore to confine its lower membrane to the glass surface,
increasing the observable parts of the cell. Previous approaches involved overlaying techniques, which lack precise control and stability. Here, easy-to-handle microfluidic-based
flattening techniques were developed and characterized. Microfluidic compression devices
greatly improved the degree of control over flattening and duration of the experiments, but
are nevertheless generable within one to two hours. | de |