Endosome | EPFL Graph Search

Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation or can be recycled back to the cell membrane in the endocytic cycle. Molecules are also transported to endosomes from the trans Golgi network and either continue to lysosomes or recycle back to the Golgi apparatus. Endosomes can be classified as early, sorting, or late depending on their stage post internalization. Endosomes represent a major sorting compartment of the endomembrane system in cells. Function Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome. For example, low-density lipoprotein (LDL) is taken into the cell by binding to the LDL receptor at the cell surface. Upon reaching early endosom

Novel compartment-specific biosensors reveal a complementary subcellular distribution of bioactive Furin and PC7

Pierpaolo Ginefra

Proprotein Convertases (PCs) constitute a family of serine proteases that activate various hormones, growth factors and cell adhesion molecules by mediating endoproteolytic cleavage of their secreted inactive precursors during their transit in the secretory pathway. Their physiological roles in most tissues, in cancer and other diseases have remained nowadays poorly defined, partly because of technical hurdles in clearly distinguishing functionally overlapping PC activities from each other. Related to this, a major unsolved question is where, among the different subcellular compartments of the secretory pathway, substrate cleavage takes place. To address these questions, we mapped intracellular PC activities of two unrelated cell lines (HEK293T and B16F1 melanoma) by ratiometric and Förster Resonance Energy Transfer (FRET)-based imaging of the PC-specific biosensors, called cell-linked indicator of proteolysis (CLIP)v3 and CLIPv4 respectively, which are sorted to different subcellular compartments by specific trafficking signals. Until now, PCs have been thought to cleave the majority of the substrates in the trans-Golgi network (TGN). By contrast, our analysis of compartment-specific CLIPv3 and CLIPv4 variants in cultured cells suggested that PC activities are much higher in post-exocytic compartments. Furthermore, imaging in B16F1 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-edited clones for endogenously expressed Furin or PC7, revealed an enrichment of Furin activity in endosomes compared to TGN, whereas PC7 activity was confined to a distinct compartment resistant to the pan-PC inhibitor CMK, demonstrating that endogenous Furin and PC7 are biologically active in distinct compartments. In addition, we also demonstrated that substrate and protease localization are rate-limiting of how efficiently a given substrate is processed. PC inhibition represses human primary melanoma cells motility and migration in vitro and proliferation in other skin cancer cells both in vitro and in vivo. To elucidate the roles of PC activities in melanoma progression and differentiation, we evaluated relative contribution of Furin and PC7 in B16F1 tumor grafts. Both proteins were required for tumor pigmentation, whereas Furin deletion reduced tumor growth, but its loss may be rescued by PC7 deletion. Altogether, these findings will be important to inform future therapeutic approaches and strategies for targeting PCs to preferentially block oncogenic activities.

EPFL2018

Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice

Marc Moniatte, Jonathan Paz Montoya

Most cells acquire cholesterol by endocytosis of circulating low-density lipoproteins (LDLs). After cholesteryl ester de-esterification in endosomes, free cholesterol is redistributed to intracellular membranes via unclear mechanisms. Our previous work suggested that the unconventional phospholipid lysobisphosphatidic acid (LBPA) may play a role in modulating the cholesterol flux through endosomes. In this study, we used the Prestwick library of FDA-approved compounds in a high-content, image-based screen of the endosomal lipids, lysobisphosphatidic acid and LDL-derived cholesterol. We report that thioperamide maleate, an inverse agonist of the histamine H3 receptor HRH3, increases highly selectively the levels of lysobisphosphatidic acid, without affecting any endosomal protein or function that we tested. Our data also show that thioperamide significantly reduces the endosome cholesterol overload in fibroblasts from patients with the cholesterol storage disorder Niemann-Pick type C (NPC), as well as in liver of Npc1(-/-) mice. We conclude that LBPA controls endosomal cholesterol mobilization and export to cellular destinations, perhaps by fluidifying or buffering cholesterol in endosomal membranes, and that thioperamide has repurposing potential for the treatment of NPC.

2019

Cripto localizes nodal at the limiting membrane of early endosomes

Stéphane Baflast, Daniel Constam

Cripto is a glycosylphosphatidylinositol (GPI)-anchored co-receptor of Nodal and several other transforming growth factor-beta (TGF-beta) family ligands. It contains an epidermal growth factor (EGF)-like motif and a Cripto-FRL1-Cryptic (CFC) domain, which are conserved in a family of EGF-CFC proteins. The EGF domain is thought to recruit Nodal, whereas the CFC domain mediates binding to activin receptor-like kinase 4 (ALK4). We found that the EGF-like motif of Cripto was not essential for its binding to Nodal. However, through residues phenylalanine 78 and glycine 71, Cripto enriched Nodal at the limiting membrane of early endosomes. Similarly, residues in the CFC domain that mediate binding of Cripto to ALK4 were required to attenuate sequestration of Nodal in the endosomal lumen. Thus, we propose that Cripto stimulates Nodal activity by localizing it at the interface of endosomes with cytoplasmic effectors. To our knowledge, Cripto is the first GPI-anchored protein shown to control intraendosomal sorting of its associated cargo.

2008

Novel compartment-specific biosensors reveal a complementary subcellular distribution of bioactive Furin and PC7

Pierpaolo Ginefra

EPFL2018