in-vitro-polymerization-of-f-actin-on-early-endosomes

<em>In Vitro</em> Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

University of Geneva

Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)-positive sorting endosomes that promotes the efficient recycling of low-density lipoprotein receptor-related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1-positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17-binding domain, we generated chimeric proteins in which the SNX17-binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non-polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin-Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17-binding receptors and the restricted function of SNX17 in the BSE.

A sorting nexin 17-binding domain within the LRP1 cytoplasmic tail mediates receptor recycling through the basolateral sorting endosome.

ApoER2 and the neurotrophin receptors Trk and p75(NTR) are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75(NTR) are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75(NTR) mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors.

Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Wnt directs the endosomal flux of LDL-derived cholesterol and lipid droplet homeostasis.

The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

Jorge Larios

Department of Biochemistry

University of Geneva

In Vitro Polymerization of F-actin on Early Endosomes

Jorge Larios

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry

University of Geneva

In Vitro Polymerization of F-actin on Early Endosomes

Department of Biochemistry