BIOADHESION MEETS FUNCTIONAL GENOMICS IN FLATWORMS

Adhesives are today employed in many areas of industry and medicine. Medical adhesives are mainly used for implants, tissue bonding, dentistry, and wound closure. These adhesives are not biocompatible and they can release cytotoxic substances into the body to cause dermatological, allergic and respiratory problems. Therefore, biocompatibility is one critical challenge for novel medical adhesives. Novel biomimetic adhesives based on natural glues of plants or animals can lead to the development of long-sought novel medical adhesives that have improved biocompatibility. However, most organisms that exhibit adhesive features usually produce little amounts of the adhesive substance. This limited availability often impedes identifications and biochemical analyses. In addition, these organisms are non-model systems that are not amenable for a powerful functional genomic approach. In the present project we aim identifying novel molecules that are involved in biological adhesion and release in the flatworm Macrostomum lignano. M. lignano harbors several features that render this species as particularly well suited to study biological adhesion including the very rapid adhesion-release mechanism, the state- of-the-art methodological toolbox, and the excellent knowledge of the morphology of the tail plate, which harbours the duo-gland adhesive system. In this project we will gradually narrow down the number of candidate genes that are involved in M. lignano adhesion and release. First, we will generate a highly tail-plate specific transcriptome database followed by a whole mount in situ hybridization screen of all tail specific genes. Next, knock-down of selected candidate genes will be performed using RNA interference which will result in a non-adhesive or non-release phenotype. Those phenotypes will then be analyzed by semi-thin sectioning- and ultrastructural analyses. This will allow selecting candidate genes to generate polyclonal antibodies. The antibodies will be used to pull-down the adhesive and release proteins which will then be analyzed by Mass Spectrometry to obtain an overview on post-translational protein modifications which are known to play important roles in adhesion. The carbohydrate components involved in adhesion or release vesicles will additionally be studied using lectin staining. In a proof of concept we have already screened 50 genes and were able to show that the proposed tools and methods can indeed be used to identify genes involved in M. lignano adhesion. We have already identified a non- adhesive phenotype by RNAi affecting a tail-plate specific intermediate filament gene, providing evidence for the suitability of the proposed approach. With the suggested functional genomics approach the project is at the forefront of the exciting research area of biological adhesion. In summary, we take advantage of the biology of the animals combined with the available methods for M. lignano to lay the basis for a future development of a novel glue-detachment system based on the M. lignano adhesion-release mechanism.

Man-made adhesives contain hazardous components which are toxic and cause skin irritations, respiratory problems or they are suspected carcinogens. Furthermore, these adhesives perform poorly in wet environments. In contrast, biological adhesives produced by animals can be considered as non-toxic, tissue compatible, and they are able to function under wet conditions. However, little is known about the mechanisms underlying biological adhesives. The free-living flatworm Macrostomum lignano can attach and release several times within a second on any substrate in seawater. It was the goal of this project to analyze the adhesion mechanism and the components involved in Macrostomum attachment and release. We have performed a detailed analysis of the Macrostomum duo-gland system which consists of an adhesive-, and a releasing gland cell, and the anchor cell. We have identified adhesive proteins using transcriptomics, differential gene expression, Mass Spectrometry, In situ Hybridization screening, Lectin staining and pull-down, specific antibodies, and light- and electron microscopy. We now have identified two key adhesive proteins which result in a non-adhesive phenotype upon RNAi knock-down. We aim for understanding the fundamental mechanisms that mediate adhesion and release in Macrotomum with the goal to generate a flatworm-derived biomimetic glue that can be applied in biomedicine and industry.