Isoform specific functions of Dynamin 2
Zusammenfassung der Projektergebnisse
Clathrin mediated endocytosis (CME) is the major pathway for the internalization of cell surface components and involves the assembly of coat proteins into clathrin coated pits, which collect surface molecules, invaginate and pinch off to form clathrin coated vesicles. Dynamin (Dyn) 1 and 2 are neuronal and ubiquitously expressed isoforms, respectively, of the multidomain GTPase required for CME. The two dynamin isoforms are 79% identical, with the greatest diversity in their C-terminal Pro/Arg domain (PRD). Despite their high degree of identity, Dyn1 and Dyn2 are not fully functionally redundant. Dyn2 could only weakly rescue the specific defect in rapid synaptic vesicle uptake in neurons from Dyn1 knockout mice 1 ; whereas Dyn1 was less effective than Dyn2 at supporting CME in Dyn2-null mouse fibroblasts 2 . These reciprocal findings suggest a fundamental mechanistic difference between these two isoforms. Through the direct comparison of the biochemical activities of Dyn1 and Dyn2 on membrane templates of differing curvature, we have shown that Dyn1 is an efficient curvature generator, whereas Dyn2 is primarily a curvature sensor. Using Dyn1/2 chimera, we identified a single amino acid change in the membrane-binding pleckstrin homology (PH) domain as being responsible for their differential biochemical properties. Reconstitution of knockout mouse embryo fibroblasts 3 with Dyn1/2 chimeras demonstrated that both the PH and the Pro/Arg-rich domains are important determinants of isoform specific activity. These domains are specific to classical dynamins and are involved in regulating its activity. Our findings reveal opportunities for fundamental differences in the tissue specific regulation of Dyn1, which mediates rapid endocytosis at the synapse, versus Dyn2, which regulates early and late events in CME in nonneuronal cells. Interestingly, while mammals express three highly similar isoforms, less complex organisms like C. elegans and Drosophila express only one dynamin isoform. Sequence comparison of the dynamin PH domain from these organisms revealed more similarities to mammalian Dyn1. Thus mammals appear to express an evolutionarily conserved, ubiquitously expressed isoform (Dyn2) that exhibits greater curvature sensitivity and weakened ability to catalyze membrane fission from planar membranes than its evolutionary progenitor. These results beg the question: Why should Dyn2 have evolved to be more sensitive to membrane curvature than Dyn1 and why should the curvature sensing PH domain regulate dynamin function in vivo? We propose that these results reflect the differential requirements for the regulation of CME in neuronal vs. nonneuronal cells. We speculate that our findings represent an evolutionary adaptation of Dyn1 and Dyn2 to support the need for more complex, tissue-specific regulation of endocytosis in higher organisms in the presence of more complex pathways and cargo types. Dyn1 is a curvature generator due to its powerful PH domain. Therefore it can provide curvature independently of coat components and accessory factors, which could underly the rapid nature of synaptic vesicle recycling. This requires a negative regulation of Dyn1 at the synapse and indeed Dyn1 is a member of the dephosphins 5 , which are rapidly activated by dephosphorylation upon neuronal stimulation. On the contrary, Dyn2 acts as an ‘assembly switch’, perfectly designed for a dual role in endocytosis. Recruited to early stages of CME it does not contribute to curvature generation in nonneuronal cells but senses the curvature provided by other factors like BAR domain containing proteins. As a poor curvature generator, Dyn2 can localize to nascent CCPs without mediating a fission event and regulate the recruitment and assembly of other components. Only deeply invaginated and fully mature pits will present a sufficiently narrow neck, which resembles a suitable membrane template for Dyn2’s fission activity. We present an elegant example how the divergence of two highly similar isoforms provide intrinsic elements of regulation to support tissue specific functions. To gain further understanding of the components of the endocytic machinery it will be required to develop in vitro reconstitution systems and cell type and tissue specific assays in the future.
Projektbezogene Publikationen (Auswahl)
- PNAS Plus. Differential curvature sensing and generating activities of dynamin isoforms provide opportunities for tissue-specific regulation. PNAS 2011 108 (26) E234–E242
Ya-Wen Liu, Sylvia Neumann, Rajesh Ramachandran, Shawn M. Ferguson, Thomas J. Pucadyil, and Sandra L. Schmid
(Siehe online unter https://doi.org/10.1073/pnas.1102710108)
