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Growth cone response to ephrin gradients produced by microfluidic networks.

ABSTRACT: A microfluidic network (microFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (microCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 microm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5.

SUBMITTER: Lang S

PROVIDER: S-EPMC2755754 | biostudies-other | 2008 Feb

REPOSITORIES: biostudies-other

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Project description:F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps.

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Growth cone response to ephrin gradients produced by microfluidic networks.

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