Fascin Discussion

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The multiple alignment of fascin 1 supports research conducted by Wulfkuhle (1993), concluding the fascin contains strong sequence similarities between human, sea urchin, and Drosophilia. Furthermore, Fascin 1 contains 4 conserved PKC phosphorylation sites across the Eukaryotes tested. However despite the findings of Mitchison (2004), Fascin 1 was found not to be closely related to Protozoa, and their use of actin in actin-dependant propulsion.

The multiple allignment also revealed 2 regions that seemed to be more highly conserved than the already highly conserved sequence. These were IAMHPQV (residues 136-143) and LINRPIIVFRGEHGVICCRK (residues 380 to 400). Similar residue conservation was seen in an analysis of Drosophila and sea urchin fascin molecules with overlap in the 380-400 residue region (Kant K & Cooley L, 1996) further confirming this region as important in the molecule.

Despite Mitchison (2004) findings, phylogenetic researched conducted did not support his findings that some Protozoa contain fascin homolog regions. Two trees were created to prove/disprove his conclusion, the first was created straight from a multiple alignment of 100 sequences, with multiple alignments from the same organism deleted. The second however was a selective tree, with PKC phosphorylation sites viewed and unconserved or gapped sequences cleared.

No Bacteria or Archea were found in the broad tree, therefore it can be concluded that Fascin 1 is a Eukaryote specific actin-binding protein. Across the Eukaryotes, Fascin 1 was highly conserved, particularly at the PKC phosphorylation sites. This shows that the sites contain important regions for functional and structural integrity.

The phylogenetic tree further illustrates the conservation of Facin 1 throughout the Eukaryotes. Although distant, the bootstrap values indicate that the Insecta and Animalia branches are closely related and supports Wulfkuhle (1993) conclusion that Drosophilia, Homo sapien and Strongylocentrotus purpuratus contain a fascin homolog. Fascin binds beta-catenin, a sub-unit of the cadherin protein complex and is important in Wnt signalling pathway and therefore is highly conserved throughout Eukaryotes.

In understanding the structure of the protein, the cleft analysis using the online service CASTp (Dundas J et. al. 2006) showed many different pocket regions apart from the actin-binding regiosn on the surface of the protein to which many other interacting proteins can bind such as Thromospondin-1 (TS1) and fibronectin (FN) which induce distinct distinct intracellular signals with regard to the activation of small GTPases and PKCa, the latter being the mediator of fascin phosphorylation in FNadherent cells. It is now clear that regulation of fascin is not only conducted from the ECM. Insulin-like growth factor I (IGF-I) and nerve growth factor (NGF) also act as inducers of fascin protrusions (Adams 2004) which can be seen in figure 1 in the "how does it work" link on the fascin function page.

Through integration of results from evolutioinary analysis showing conserved regions, research into its function and structural analysis including pocket analysis and electrostatic surface models allowed for the identification of possible actin-binding regions in the N-terminal region and the C-terminal half of fascin region which is a hydrphobic conserved domain. the addition of negatively charged glutamic acid was shown to disrupt the teritiary structure of the C-terminal domain that interacts with actin (Cant and Cooley 1995).

The electrostatic analysis showed that the two actin-binding regions circled have a negatively charged centre which is encapsulated within a positively charged region. Structure analysis also confers with functional research in that fascin attaches itself to two actin filaments when producing its bundling affects with the dynamic cross-linking property of fascin (Aratyn et al. 2007).

From the hydrophobicity plot, it was concluded that the fascin protein overall is hydrophobic making it fat soluble and this is essential for fascin transport in the cells. An advantage of the dynamic cross-links is that dynamic binding would enhance the availability of fascin molecules for newly polymerized actin filaments at the filopodial tips by decreasing the diffusion distance. This would ensure timely filament bundling during filopodia elongation. If fascin was stably bound to actin filaments in filopodia and released only with filament disassembly at the rear, free fascin would need to diffuse through the whole filopodial length, which is on the order of micrometers, to reach sites of growth (Vignjevic et al. 2006).

Abstract | Introduction | Methods | Results | Discussion | Conclusion | References
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