[Todos] Seminarios conjuntos DFBMC-IFIByNE- Mercedes Dragovits y Claudio Rolli- RECORDATORIO

[Paula Felman]

Seminario jueves 5 de marzo, 13 hs. Aula de seminarios del LFBM


Mercedes Dragovits y Claudio Rolli

DescriptionMax-Planck-Institute for Metals Research
Dept. of New Materials and Biosystems
http://www.mf.mpg.de/de/abteilungen/spatz/ and

University of Heidelberg
Dept. of Biophysical Chemistry
http://www.pci.uni-heidelberg.de/bpc/biophysik.html

Claudio Rolli
Study of cell migration through micro-size channels.

Abstract:
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.

Mercedes Dragovits

Fibronectin secretion and assembly on surfaces functionalized with 
integrin-ligands.

Abstract:
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.
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 á5â1 integrins, or a homogeneous layer of RGD peptides targeting mainly 
ávâ3  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 inter-ávâ3 -ligand distance that enhances not only cell adhesion 
but also affects FN assembly.
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.
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 â3-integrins, vinculin, 
zyxin and collagen, involved in the process of adhesion and extracellular 
matrix production and assembly.


Host: Alejandro Colman Lerner



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