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Abstract
Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgment
We are grateful to Drs. Nickolas Ktistakis, Tamotsu Yoshimori, Sharon Tooze, Noburu Mitsushima, Sergio Grinstein, Phillipe Chavrier, Mark R. Phillips and Beth Levine for the material provided. We also thank Alejandra Medero for technical assistance with tissue culture, Marcelo Furlan for technical assistance with electron microscopy sample preparation and Graciela Gutierrez for technical assistance. This work was partly supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2005 38420 and PICT 2008 0192) and from SeCTyP (Universidad Nacional de Cuyo) to M.I.C.
Supplemental Materials
Supplemental materials can be found at: www.landesbioscience.com/journals/autophagy/article/21459