Jacques Blümmel

Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability, and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and the signaling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 mum when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization and initiate cell migration and signaling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.

Cell interactions with hierarchically structured nano-patterned adhesive surfaces

The activation of well-defined numbers of integrin molecules in predefined areas by adhesion of tissue cells to biofunctionalized micro-nanopatterned surfaces was used to determine the minimum number of activated integrins necessary to stimulate focal adhesion formation. This was realized by combining micellar and conventional e-beam lithography, which enabled deposition of 6 nm large gold nanoparticles on predefined geometries. Patterns with a lateral spacing of 58 nm and a number of gold nanoparticles, ranging from 6 to 3000 per adhesive patch, were used. For α(v) β(3)-integrin activation, gold nanoparticles were coated with c(-RGDfK-)-thiol peptides, and the remaining glass surface was passivated to prevent non-specific protein adsorption and cell adhesion. Results show that focal adhesion formation is dictated by the underlying hierarchical nanopattern. Adhesive patches with side lengths of 3000 nm and separated by 3000 nm, or with side lengths of 1000 nm and separated by 1000 nm, containing approximately 3007 ± 193 or 335 ± 65 adhesive gold nanoparticles, respectively, induced the formation of actin-associated, paxillin-rich focal adhesions, comparable in size and shape to classical focal adhesions. In contrast, adhesive patches with side lengths of 500, 250 or 100 nm, and separated from adjacent adhesive patches by their respective side lengths, containing 83 ± 11, 30 ± 4, or 6 ± 1 adhesive gold nanoparticles, respectively, showed a significant increase in paxillin domain length, caused by bridging the pattern gap through an actin bundle in order to mechanically, synergistically strengthen each single adhesion site. Neither paxillin accumulation nor adhesion formation was induced if less than 6 c(-RGDfK-)-thiol functionalised gold nanoparticles per adhesion site were presented to cells.

Polymeric substrates with tunable elasticity and nanoscopically controlled biomolecule presentation

Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Youngs moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidine-tagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.

Block copolymer micelle nanolithography on non-conductive substrates

A new lithographic technique has been developed and applied to cell adhesion studies and electro-optical material development. Attachment of 6 nm Au particles, in periodic and non-periodic pattern, onto non-conductive substrates has been achieved. This was performed via a combination of diblock copolymer self-assembly and electron beam lithographic techniques. To optimize e-beam resolution on non-conductive materials, an additional carbon layer was thread-coated onto the substrates. This carbon coating and the diblock copolymer used in the self-assembly step were simultaneously removed by a final hydrogen plasma treatment to reveal Au nanodot patterns of unprecedented pattern quality. These optically transparent substrates (glass cover slips) were bio-functionalized via the Au-dot patterns to yield a platform for unique cell adhesion studies. The same Au-dot patterning technique was applied to sapphire substrates, which were subsequently employed to nucleate electro-optically active ZnO nanopost growth.

Actin-cytoskeleton dynamics in non-monotonic cell spreading

The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from these regions. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds.

MOTOR PROTEIN DRIVEN MICROTUBULE TRANSPORT ON GOLD PARTICLE NANOPATTERNS

Novel glass surfaces with quasi-hexagonally arranged gold nanoparticles to control motor protein immobilisation and motor protein dependent microtubule transport were applied. We first show that the kinesin-like motor protein Eg5 adsorbs efficiently and selectively to gold nanodots comprising a molecular motor nanopattern, while the glass surface between nanodots is passivated by a layer of polyethylene glycol in order to reduce non-specific interactions of individual motors with the substrate. We show that the motor nanopattern and density is indeed controlled by the gold nanodot density. We then use these motor protein arrays to investigate the kinetics of microtubule transport and find that the characteristics of the molecular motor nanopattern influence the characteristics of microtubule transport. This finding describes new biomimetic surfaces of molecularly controlled motor protein surface densities.