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Seminario <b>jueves</b> 5 de marzo, <b>13 hs</b>. Aula de seminarios del
LFBM<br>
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<br>
<font face="Arial, Helvetica">Mercedes Dragovits y Claudio Rolli<br>
<br>
DescriptionMax-Planck-Institute for Metals Research<br>
Dept. of New Materials and Biosystems<br>
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and<br>
<br>
University of Heidelberg<br>
Dept. of Biophysical Chemistry<br>
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<br>
<br>
<b>Claudio Rolli<br>
</b>Study of cell migration through micro-size channels.<br>
<br>
Abstract:<br>
Cell migration plays a crucial role for a variety of pathological and
non-pathological processes of every living species. A better
understanding of the signalling pathways which trigger cell motility and
the mechanisms of cell migration on a molecular level is therefore of
great importance for biologists, chemists and physicists. The aim of this
thesis was to design and fabricate a micro?uidic-based device which would
allow the study of the invasive migration behavior of cells through
micro-sized channels on a single-cell level. Migration chips were
fabricated with different channel sizes using photolithographic processes
and molding techniques. The inorganic glass and iloxane-based
surfaces con?ning the geometry of the channels were bio-functionalized to
enhance and allow cell adhesion. The migration chips allowed to follow
and visualize the migration process via live-cell imaging microscopy.
Cell experiments were carried out with human pancreatic epithelium tumor
cells (Panc-1). A critical channel size was found, where the cells were
able to migrate through the channel only after adding a 10 µM
concentration of the bioactive lipid sphingosylphosphorylcholine (SPC).
SPC induces a reorganization of the cell´s cytoskeleton structure and
thereby changes the viscoelastic properties of the cell. The channels of
critical size were 150 µm long, 7 µm wide and 11 µm high. In addition,
?uorescently labeled cells were used to reveal the cell´s cytoskeleton
dynamics while crawling through the channels. As a proof of principle it
could be shown that the fabricated migration chip has the ability to
serve as a versatile and powerful tool to study cell migration dynamics
of single cells in a three-dimensionally de?ned environment on a
molecular level.<br>
<br>
<b>Mercedes Dragovits<br>
<br>
</b>Fibronectin secretion and assembly on surfaces functionalized with
integrin-ligands.<br>
<br>
Abstract:<br>
Transmembrane integrin receptors promote cell adhesion to the
rovided substrate. We want to control the integrin lateral clustering on
the ventral side of the fibroblast cell membrane to analyze how
fibronectin (FN) production and assembly are affected. <br>
To set up a baseline for cell adhesion and FN assembly we coated glass
slides with either cellular FN, plasma FN or super FN, which are
recognized by
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integrins, or a homogeneous layer of RGD peptides targeting mainly
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integrins, which cluster in focal adhesions. We compared fibroblast
spreading, focal and fibrillar adhesion formation, FN production and
polymerization on the different substrates. The maximal cell area was
reached first on cellular FN, followed by super FN, the RGD coating and
plasma FN. A few hours after seeding, cells grown on cellular FN
deposited FN fibrils mainly at the cell periphery and presented a more
elongated shape than on RGD, plasma FN or super FN, where they had a more
rounded shape presenting thinner and fewer FN fibrils.To control integrin
lateral clustering we developed nanopatterned biofunctionalized
interfaces presenting a defined and tunable inter-ligand distance. Rat
embryonic fibroblasts (REF) grown on substrates presenting an
inter-ligand distance of 58 nm were able to form stable focal adhesions
(FAs) and actin stress fibers and to deposit FN fibrils within the first
24 hr after seeding, whereas the formation of FAs failed on samples with
a lower peptide density (73 nm or 110 nm distance) and endogenous FN was
only detected as small dots. On a homogeneous RGD-coating FN fibrils were
present, but they were thinner and fewer than on the 58 nm patterns,
indicating that this is a critical
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-ligand distance that enhances not only cell adhesion but also affects FN
assembly. <br>
We showed how substrate functionalization with distinct types of FN or
specific integrin ligands affects differentially cell adhesion, spreading
and FN deposition. Fibroblasts that formed FAs and actin stress fibers on
all tested substrates were able to deposit and assemble FN into fibrils.
Our results reinforce the correlation between FA and fibrillar adhesion
(FB) formation, indicating that we can regulate FN assembly by
controlling cell adhesion.<br>
A deeper investigation is needed to elucidate the mechanisms underlying
the adaption of fibroblasts to different types of adhesive artificial
matrices. We will test the role of further proteins, such as
</font><font face="Times New Roman Greek, Times">â</font><font face="Arial, Helvetica">3-integrins,
vinculin, zyxin and collagen, involved in the process of adhesion and
extracellular matrix production and assembly.<br>
<br>
<br>
Host: Alejandro Colman Lerner<br>
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