Abstract
Cytoskeletal dynamics are pivotal to memory, learning, and stress physiology, and thus psychiatric diseases. Downregulated in renal cell carcinoma 1 (DRR1) protein was characterized as the link between stress, actin dynamics, neuronal function, and cognition. To elucidate the underlying molecular mechanisms, we undertook a domain analysis of DRR1 and probed the effects on actin binding, polymerization, and bundling, as well as on actin-dependent cellular processes. Methods: DRR1 domains were cloned and expressed as recombinant proteins to perform in vitro analysis of actin dynamics (binding, bundling, polymerization, and nucleation). Cellular actin-dependent processes were analyzed in transfected HeLa cells with fluorescence recovery after photobleaching (FRAP) and confocal microscopy. Results: DRR1 features an actin binding site at each terminus, separated by a coiled coil domain. DRR1 enhances actin bundling, the cellular F-actin content, and serum response factor (SRF)-dependent transcription, while it diminishes actin filament elongation, cell spreading, and actin treadmilling. We also provide evidence for a nucleation effect of DRR1. Blocking of pointed end elongation by addition of profilin indicates DRR1 as a novel barbed end capping factor. Conclusions: DRR1 impacts actin dynamics in several ways with implications for cytoskeletal dynamics in stress physiology and pathophysiology.
Highlights
Stress is a risk factor for several pathologies, including mental disorders such as psychiatric diseases [1,2]
Downregulated in renal cell carcinoma 1 (DRR1) significantly slowed down actin polymerization to less than 20% control (Figure 4C)
Pathway-based methods to genetic data have been suggested to blend biological information with the power of –omics approaches [83]; we propose that the stress- and glucocorticoid-regulated DRR1 [22,23,47,88] should be included when analyzing the role of the actin cytoskeleton in physiology and pathology, in stress-related processes
Summary
Stress is a risk factor for several pathologies, including mental disorders such as psychiatric diseases [1,2]. For the co-sedimentation assays, purified G-actin from rabbit skeletal muscle was polymerized and incubated with purified wt and mutant DRR1 proteins (tagged with maltose binding protein (MBP)) followed by high speed centrifugation and analysis of the total (T), supernatant (S), and pellet (P) fractions by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining (Figure 1D). The mutant d7Cof 30 showed a mild reduction of filament elongation, which was not significant This finding indicates that the reduction of single filament elongation by DRR1 is independent of actin bundling, but both was strongly inhibited in comparison to the control (Figure 4A). Addition of profilin strongly enhanced DRR1’s inhibitory effect on actin polymerization supporting the notion of DRR1 as a novel capping protein at the barbed end. This capping activity of dC was less pronounced than for DRR1 wt (Figure 5C)
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