Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

BACKGROUND: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. METHODS: A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. RESULTS: N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. CONCLUSIONS: Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway.

BACKGROUND: Cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. This process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. The mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation. RESULTS: In this study, we evaluated the role of the actin nucleation factor, Arp2/3 complex, during cytokinesis. We found that the Arp2/3 complex is recruited late to the cleavage furrow suggesting a potential involvement of Arp2/3 complex during this process. Furthermore, wiskostatin a potent inhibitor of N-WASP activity towards the Arp2/3 complex blocked cytokinesis without affecting mitosis. Nonetheless, this inhibition could not be reproduced using alternative approaches targeting the N-WASP/Arp2/3 complex pathway. CONCLUSION: We conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the N-WASP/Arp2/3 pathway. Wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to N-WASP could affect the activity of other factors involved in cytokinesis.

Chemical inhibition through conformational stabilization of Rho GTPase effectors.

The Rho family of small GTP-binding proteins can activate a large number of downstream effectors and participate in a wide variety of biological processes, including cell motility, membrane trafficking, cell polarity, gene transcription, and mitosis. Specific small-molecule inhibitors of individual effector proteins downstream of Rho GTPases would be powerful tools to elucidate the contributions of particular effectors to these processes. In this chapter we describe the identification of a chemical inhibitor of a Rho effector and scaffolding protein neural-Wiskott-Aldrich syndrome protein (N-WASP), and the discovery of its novel mechanism of action, stabilization of N-WASP's native autoinhibited conformation. Inasmuch as several other Rho GTPase effectors are regulated by autoinhibition, we discuss how this regulatory mechanism could be exploited by small molecules to develop highly specific inhibitors of other Rho GTPase effectors. We illustrate this concept with the Rac/Cdc42 effector p21-activated kinase (Pak1) and the Rho effector mammalian diaphanous-related formin (mDia1).