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Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 04/05 Podcast
CHEMOTACTIC GRADIENT GENERATOR - A microfluidic Approach on how D. discoideum change direction

CHEMOTACTIC GRADIENT GENERATOR - A microfluidic Approach on how D. discoideum change direction

Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 04/05 vor 14 Jahren

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Chemotaxis, the ability of cells to detect and migrate directly
towards a source of a chemically active agent, is the result of a
sophisticated interplay of proteins within a complex regulatory
network. However, partially redundant pathways that simultaneously
mediate chemotaxis and dynamic protein distributions complicate the
experimental identication of distinct signaling cascades and their
inuence on chemotactic migration. Yet, increasingly precise
generation and rapid modication of chemotactic stimuliin microuidic
devices promise further insight into the basic principles of
cellular feedback signaling. I developed a Chemotactic Gradient
Generator (CGG) for the exposure of living cells to chemotactic
gradient elds with alternating gradient direction based on a double
T-junction microuidic chamber. A large extension of the
concentration gradients enables the parallel exposure of several
dozens of cells to identical chemotactic stimuli, allowing for a
reliable quantitative analysis of the chemotactic migration
behavior. Two pressure pumps and a syringe pump facilitate accurate
control of the inow velocities at the individual ow chamber inlets,
pivotal for precise manipulation of the chemotactic stimuli. The
CGG combines homogeneous gradients over a width of up to 300 µm and
rapid alterations of gradient direction with switching frequencies
up to 0.7 Hz. Fast gradient switching in our experimental design
facilitates cell stimulation at the intrinsic time scales of their
chemotactic response as demonstrated by a gradual increase in the
switching frequency of the gradient direction. We eventually
observe a "chemotactically trapped" state of Dictyostelium
discoideum (D. discoideum) cells at a switching rate of 0.01 Hz.
Here, gradient switching proves too fast for the cells to respond
to the altered gradient direction by migration. In contrast, we
observe oscillatory runs at switching frequencies of less than 0.02
Hz. We distinguish between re-polymerizing cells that exhibit an
internal re-organization of the actin cortex in response to
chemotactic stimulation and stably polarized cells that gradually
adjust their leading edge when the gradient is switched. To
experimentally characterize both response types, we record cell
shape and the intracellular distribution of actin polymerization
activity. Cell shape is readily described by the eccentricity of
the cell and to record F-actin polymerization dynamics we introduce
a fluorescence distribution moment (FDM). Accurate description of
the migratory response behavior facilitates a quantitative analysis
of the inuence of both the experimental boundary conditions such as
gradient shape, ongoing starvation of the cells, and in particular
the inuence of distinct signaling cascades on chemotactic
migration. Here, we demonstrate this ability of the GCC by
inhibition of PI3-Kinase with LY 294002. PI3-Kinase initiates the
formation of fresh pseudopods in the direction of the chemotactic
gradient and therefore is one of the key signaling pathways
mediating the chemotactic response. In shallow gradients and with
ongoing starvation of the cells, we find a decreased ratio of
re-polymerizing cells, pointing towards a diminished influence of
PI3-Kinase. After inhibition of PI3-Kinase, cell re-polymerization
in response to a switch in gradient direction is hindered at 5h of
starvation, whereas at 7h of starvation evidence is found that
chemotactic migration is more efficient. We observe the astonishing
result that in dependency of the boundary conditions of the
experiment inhibition of PI3-Kinase promotes an effective
chemotactic response. Thus, the CGG for the rst time facilitates a
quantitative analysis of the starvation time dependent effect of
PI3-Kinase inhibition on D. discoideum chemotaxis.