Abstract
BackgroundCysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified.ResultsCRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET) microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts.ConclusionCRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.
Highlights
Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton
We show that CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles
Expression of CRP1 increases F-actin bundling in REFs To determine the influence of CRP1 on the actin cytoskeleton, rat embryonic fibroblasts (REFs) were transfected with increasing concentrations of DNA encoding CFPCRP1 resulting in increasing levels of expression of the fluorescent fusion protein (Fig. 1A)
Summary
Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. Stress fibers are bundles of actin microfilaments formed in cells following integrin-mediated attachment and spreading [1] Regulation of these contractile fibers is critical for cell adhesion and motility. One important discovery was the periodic and alternating association of myosin and α-actinin which is clearly visualized as a beaded pattern along stress fibers in cells stained for immunofluoresence microscopy [2]. It is not understood how this alternating association of myosin and α-actinin with the microfilaments is regulated, it is critical for the contractility of the stress fiber. Determining the function of these ancillary proteins is important for understanding the regulation of stress fibers
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