Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.

Quantitative evaluation of adhesion of osteosarcoma cells to hydrophobic polymer substrate with tunable elasticity.

We investigated a potential application of hydrophobic poly(n-butyl acrylate) networks (cPnBA) as substrates with tunable elasticity for culturing, maintenance, and regulation of human osteosarcoma cells (U2OS). Nanoindentation experiments with an atomic force microscope revealed that the mechanical properties of cPnBA films are maintained under aqueous conditions, confirming that the substrate elasticity can be controlled simply by the degree of cross-linking, independent from the culture medium. We found that the adhesion U2OS cells to cPnBA substrates could be improved by surface treatments such as oxgen plasma and serum proteins. To determine the strength of cell adhesion, the critical pressure to detach cells from cPnBA substrates was measured using a shock wave induced by an intensive picosecond laser pulse. A monotonic increase in the cell adhesion strength in accordance with the substrate elasticity demonstrated the potential of intrinsically hydrophobic cPnBA as a new class of substrate material with tunable mechanical properties that are not influenced by the culture medium.