BioMEMS for Cell Biology - Introduction to BioMEMS - page 307

nearly constant integrin-ECM focal adhesion area. his inding indicates that cell shape, and
not cell-ECM contact area, determines cell fate.
Using immunoluorescence microscopy, a team led by Martin Bastmeyer, then at the
University of Konstanz (Germany), performed in 2004 a detailed molecular study of the focal
adhesions that cells establish with the ECM during cell adhesion, spreading, and migration.
Following the Whitesides-Ingber protocol (see Figure 2.27 ), this group used microstamping of
alkanethiols on gold followed by PEG-thiol SAM formation on the background to create ECM-
adhesive regions on the stamped areas; ECM only adsorbed on the alkanethiol-stamped areas,
so cells attached on these ECM islands and they were unable to form contacts with the PEG-
thiol background ( Figure 6.5 ). Cells cultured on these substrata adhere to and spread on ECM
regions as small as 0.1 μm 2 , when spacing between dots was less than 5 μm. Spacing of 5 to 25 μm
induces a cell to adapt its shape to the ECM pattern. he ability to spread and migrate on 1 μm 2
dots ceases when the dot separation is 30 μm. he extent of cell spreading is directly correlated
to the total substratum coverage with ECM proteins, but irrespective of the geometrical pattern.
An optimal spreading extent is reached at a surface coating of more than 15%. On homogeneous
substrata, staining for molecules such as paxillin or vinculin are found in dot-like adhesions at
the periphery of the cell, with most vinculin-positive foci connected to actin bundles ( Figure
6.5a ). When cells were forced to make contacts on 0.6 μm 2 ibronectin dots, they expressed
vinculin normally (only on the ibronectin dots) and actin ibers terminated in the peripheral
adhesion sites ( Figure 6.5b ). On a patterned substrate of 1 μm 2 ibronectin dots, cells stained
focally for focal adhesion kinase ( Figure 6.5c ) and for phosphotyrosine ( Figure 6.5d ). On pat-
terned substrata of 0.6 μm 2 vitronectin dots, cells expressing β3-integrin-GFP stained for pax-
illin ( Figure 6.5e ) and for actin ( Figure 6.5f ). he cell in Figure 6.5f is growing at the border
between a uniform and a patterned substratum: note the redistribution of integrin receptors on
the patterned substratum.
Because cell shape inluences gene expression (protein secretion and cell proliferation)
through the cell's cytoskeleton, it is not surprising that the positioning of the cell division axis
itself (the spindle orientation) can be experimentally manipulated by micropatterning of the

a b c

Act

Vin
Act

Vin
FN

FAK
FN

10 µm

d f

e β3

β3

Pax

PT

Act

VN

FN VN

FIGURE 6.5 Molecular.composition.of.focal.adhesions.on.micropatterned.substrates..Abbreviations:.
Vin,. vinculin;. Act,. actin;. FN,. ibronectin;. FAK,. focal. adhesion. kinase;. PT,. phosphotyrosine;. Pax,.
paxillin;. β 3,. β 3-integrin-GFP..(From.Dirk.Lehnert,.Bernhard.Wehrle-Haller,.Christian.David,.Ulrich.
Weiland,.Christoph.Ballestrem,.Beat.A..Imhof,.and.Martin.Bastmeyer,.“Cell.behaviour.on.micropat-
terned.substrata:.Limits.of.extracellular.matrix.geometry.for.spreading.and.adhesion,”. J. Cell Sci. .
117,.41-52,.2004..Figure.contributed.by.Martin.Bastmeyer.)