Cell adhesion, spreading and migration require the dynamic formation and dispersal of contacts with the extracellular matrix (ECM). In vivo, the number, availability and distribution of ECM binding sites dictate the shape of a cell and determine its mobility. To analyse the geometrical limits of ECM binding sites required for cell attachment and spreading, we used microcontact printing to produce regular patterns of ECM protein dots of defined size separated by nonadhesive regions. Cells cultured on these substrata adhere to and spread on ECM regions as small as 0.1 microm2, when spacing between dots is less than 5 microm. Spacing of 5-25 microm induces a cell to adapt its shape to the ECM pattern. The ability to spread and migrate on dots > or =1 microm2 ceases when the dot separation is > or =30 microm. The 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 above 15%. Knowledge of these geometrical limits is essential for an understanding of cell adhesion and migration, and for the design of artificial surfaces that optimally interact with cells in a living tissue

We have developed a new method for observing cell/substrate contacts of living cells in culture based on the optical excitation of surface plasmons. Surface plasmons are quanta of an electromagnetic wave that travel along the interface between a metal and a dielectric layer. The evanescent field associated with this excitation decays exponentially perpendicular to the interface, on the order of some hundreds of nanometers. Cells were cultured on an aluminum-coated glass prism and illuminated from below with a laser beam. Because the cells interfere with the evanescent field, the intensity of the reflected light, which is projected onto a camera chip, correlates with the cell/substrate distance. Contacts between the cell membrane and the substrate can thus be visualized at high contrast with a vertical resolution in the nanometer range. The lateral resolution along the propagation direction of surface plasmons is given by their lateral momentum, whereas perpendicular to it, the resolution is determined by the optical diffraction limit. For quantitative analysis of cell/substrate distances, cells were imaged at various angles of incidence to obtain locally resolved resonance curves. By comparing our experimental data with theoretical surface plasmon curves we obtained a cell/substrate distance of 160 +/- 10 nm for most parts of the cells. Peripheral lamellipodia, in contrast, formed contacts with a cell substrate/distance of 25 +/- 10 nm.

E587 antigen, an L1-related cell adhesion molecule, is expressed by growing axons and has previously been shown to enhance axon growth and to mediate fasciculation of axons from newborn retinal ganglion cells in goldfish. In zebrafish, the monoclonal antibody E17 against E587 antigen stains all axons in the primary tracts and commissures from 17 h postfertilization (pf) onward and axons which are added subsequently to this scaffold. Moreover, Fab fragments of an E587 antiserum (E587 Fabs) injected into the ventricle of 30-h pf zebrafish embryos caused a marked defasciculation of distinct axon bundles in the posterior commissure, in hindbrain commissures, and in longitudinal tracts of the hindbrain, where they also caused increased crossings between fascicles. The regulated expression of E587 antigen by all developing axons and the effects caused by E587 Fabs show that E587 antigen contributes to the formation of tight and orderly fascicles in the developing CNS.

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