Compartmental signal modulation: Endosomal phosphatidylinositol 3-phosphate controls endosome morphology and selective cargo sorting

Abstract

It is increasingly recognized that the compartmental organization of signaling processes has a profound influence on cellular behavior. However, our inability to influence these compartmental events in a spatially restricted and acute manner limits our understanding of causation. To determine whether local compartmental loss of a phosphoinositide disrupts the normal traffic of specific cargoes through endosomes, we developed the use of a regulated dimerization device, here designed to compartmentally modify the phosphoinositide content of Rab5-positive endosomes. This modification is effected through the specific regulated recruitment of the 3-phosphatase myotubularin to endosomal membranes in intact cells. The selective manipulation of endosomal phosphatidylinositols (PIs) demonstrates that it is the phosphatidylinositol 3-phosphate (PtdIns3P) or its metabolite PtdIns(3,5)P2 within this compartment that determines the normal maturation of the endosomal compartment and the flux of receptors through it. On local loss of PtdIns3P͞PtdIns(3,5)P2, the endosomal compartment itself fails to continue its normal maturation process, leading to the microtubule-dependent tubularization of the endosomal network. Furthermore, it is shown that endosomal PtdIns3P͞PtdIns(3,5)P2 is necessary for transferrin receptor traffic through this compartment while having an effect on EGF receptor (EGFR) entry into and sorting from this endosome compartment. The ability to acutely and selectively influence compartmental behavior as exemplified here for endomsomes clearly illustrates the power of the approach used to dissect the role of localized signals and events.

A phosphoinositide conversion mechanism for exit from endosomes

“…Recruitment and PI turnover is thought to be regulated by Vps34 ( Figure 4) (Cao, Laporte et al 2007) and by the phosphatase-inactive MTMR12 (Nandurkar, Layton et al 2003). Targeted recruitment of MTM1 to early endosomes and thereby PI(3)P depletion enhances microtubule-dependent endosome tubulation (Fili, Calleja et al 2006) during endosomal recycling. However, based on other studies local PI(3)P-turnover was suggested to be a crucial determinant for tubule formation (Carpentier, N'Kuli et al 2013).…”

A Small Molecule Mimicking a Phosphatidylinositol (4,5)-Bisphosphate Binding Pleckstrin Homology Domain

“…Currently, altering cellular PI(4,5)P 2 levels requires the use of chemical dimerizers (40)(41)(42)(43), biological lipid recognition domains or lipid metabolizing enzymes, which are prone to interference by protein-protein interactions. In addition, response times are subject to biological restrains, since the cell has to express sufficient amounts of protein prior to any observable effects.…”

Manipulating signal delivery – plasma-membrane ERK activation in aPKC-dependent migration

“…Plasmids used as templates containing the tandem FK506-binding protein domain (2xFKBP) and the mutant FRB domain (FRB*) were previously constructed from components of the ARGENT Regulated Heterodimerization Kit provided by ARIAD (Fili et al, 2006). To generate the target fusion constructs, EGFP-2xFKBP was amplified by standard PCR using the GFP-Rab5a-target plasmid as template and the product subcloned into AgeI-BamHI sites of pIRESpuro2 (Clonetech).…”

“…To address the question of what is sufficient to suppress the aPKC dependence of signal delivery, under conditions where aPKC function is blocked, we have triggered delivery independently using the dimerization agent rapalogue (Fili et al, 2006;Inoue et al, 2005;Varnai et al, 2006). Exploiting this approach we show that the localized activation of ERK at the leading edge and not the related JNK can indeed partially rescue the loss of aPKC action, restoring focal adhesion dynamics.…”