The levels of twinfilin-1 protein do not seem to change significantly in twinfilin-2a Lesinurad knockout mouse tissues. Furthermore, qRT-PCR experiments did not reveal significant changes in the twinfilin-1 or twinfilin-2b mRNA expression levels in twinfilin-2a knockout mice, suggesting that depletion of twinfilin-2a is not compensated by upregulation of the two other isoforms. Genetic studies on budding yeast and Drosophila revealed that twinfilin plays a central role in actin dynamics in lower eukaryotes. However, the function of twinfilin in vertebrate development and physiology has not been reported. Studies on vertebrate twinfilins are Methicillin sodium salt further complicated by the presence of three twinfilin isoforms with partially overlapping expression patterns and similar biochemical activities. Here, we report that inactivation of one mouse twinfilin isoform, does not result in gross abnormalities in mouse development or physiology. We speculate that the depletion of twinfilin-2a may be compensated by the presence of twinfilin, which is typically co-expressed in the same non-muscle tissues and cell-types with twinfilin-2a. Similar functional redundancy has been previously reported also for isoforms of other central actin regulators such as VASP family proteins. However, despite the biochemical similarities, the localizations of twinfilin-1 and twinfilin-2a in cells appear to be regulated through different pathways. Whereas twinfilin-1 localizes to the lamellipodial actin network and cell-cell contacts upon expression of dominant active Rac and Cdc42, respectively, these small GTPases do not appear to regulate the sub-cellular localization of twinfilin-2a. It is therefore possible that twinfilin knockout mice will display more subtle defects in physiological processes, which require specific regulation of the different twinfilin isoforms. Previous studies have shown that both twinfilin-1 and twinfilin contribute to endocytosis in cultured mammalian cells. Furthermore, twinfilin-2a was identified in an RNAi screen as a central protein promoting neuronal outgrowth. Surprisingly, analysis of the knockout mice suggests that in intact animals the presence of twinfilin-2a is not required for central cell biological processes such as receptor-mediated endocytosis. Furthermore, the lack of detectable behavioral abnormalities or histological alterations in the brain of knockout mice does not support a central role for twinfilin-2a in neuronal outgrowth.