Alix regulates cortical actin and the spatial distribution of endosomes

Abstract

Alix/AIP1 is a proline-rich protein that has been implicated in apoptosis, endocytic membrane trafficking and viral budding. To further elucidate the functions of Alix, we used RNA interference to specifically suppress its expression. Depletion of Alix caused a striking redistribution of early endosomes from a peripheral to a perinuclear location. The redistribution of endosomes did not affect transferrin recycling or degradation of endocytosed epidermal growth factor receptors, although the uptake of transferrin was mildly reduced when Alix was downregulated. Quantitative immunoelectron microscopy showed that multivesicular endosomes of Alix-depleted cells contained normal amounts of CD63, whereas their levels of lysobisphosphatidic acid were reduced. Alix depletion also caused an accumulation of unusual actin structures that contained clathrin and cortactin, a protein that couples membrane dynamics to the cortical actin cytoskeleton. Our results suggest that Alix functions in the actin-dependent intracellular positioning of endosomes, but that it is not essential for endocytic recycling or for trafficking of membrane proteins between early and late endosomes in non-polarised cells.

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

“…These multitasking properties of Alix derive from the ability of the protein to interact with a plethora of partner proteins, which are themselves components of large oligomeric complexes. By interacting with the endosomal sorting complexes required for transport, ESCRT I and III, Alix synergistically coordinates endocytosis and recycling of mem-brane receptors, viral budding, and cytokinesis (13)(14)(15)(16)(17)(18)(19)(20). Relevant to this study are the findings that Alix also participates in cytoskeleton remodeling by binding with F-actin, ␣-actinin, cortactin, and focal adhesion kinase (14,21).…”

“…More recent reports have implicated Alix in other basic cellular processes, which all hinge on membrane trafficking, endosomal sorting, and remodeling of the actin cytoskeleton (12)(13)(14). These multitasking properties of Alix derive from the ability of the protein to interact with a plethora of partner proteins, which are themselves components of large oligomeric complexes.…”

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

“…An alternative explanation comes from the observations that multivesicular endosomes contain more than one type of lumenal vesicles (Gillooly et al, 2000;Kobayashi et al, 2002;Sobo et al, 2007a), raising the possibility that some are involved in the transport of signaling receptors to the lysosomes, whereas others undergo fusion at the limiting membrane. If so, intralumenal mechanisms must control the fate of intralumenal vesicles, back-fusion or degradation, including perhaps LBPA via its effector Alix, because Alix knockdown inhibits back-fusion (Abrami et al, 2004;Le Blanc et al, 2005) but not EGF receptor degradation (Schmidt et al, 2004;Cabezas et al, 2005). In addition, it cannot be excluded that some signaling receptors are returned to the endosomelimiting membrane via back-fusion and then recaptured within newly formed vesicles, perhaps like the EGF receptor in our assay, a mechanism that may contribute to explain MAPK signaling from late endosomes (Teis et al, 2002;Taub et al, 2007).…”

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

“…In trafficking events, cortactin has been involved in clathrin-mediated endocyto-sis as well as in pinocytosis, regulation of epidermal growth factor receptor (EGFR) degradation, or endosomal positioning (McNiven et al, 2000;Cao et al, 2003Cao et al, , 2005Lynch et al, 2003;Cabezas et al, 2005;Merrifield et al, 2005;Sauvonnet et al, 2005;Timpson et al, 2005;Zhu et al, 2005;Mettlen et al, 2006;Le Clainche et al, 2007).…”