Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate

Abstract

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P 2 breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P 2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P 2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrincoated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P 2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.actin ͉ dynamin ͉ epsin ͉ phosphoinositides ͉ rapamycin P hosphorylation-dephosphorylation of the inositol ring of inositol phospholipids plays a major role in cell regulation. Their phosphorylated head groups interact with a variety of cytosolic protein modules and thus mediate the recruitment and regulation of proteins at the membrane-cytosol interface (1-7). A direct interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], a phosphoinositide concentrated at the plasma membrane, has been demonstrated for all of the endocytic clathrin adaptors and for several of their accessory factors, for the GTPase dynamin, and for many actin regulatory proteins (8-16). The physiological significance of these interactions has been supported by cell biological (9, 15, 17-21) and pharmacological (22) studies in living cells and by forward and reverse genetics. For example, mutations in genes encoding inositol 5-phosphatases, primarily synaptojanin family members, were shown to cause endocytic and actin defects in a variety of organisms (16,(23)(24)(25)(26)(27)(28). However, genetic manipulations that result in long-lasting changes in cellular levels of PI(4,5)P 2 may act indirectly or trigger compensatory mechanisms that can complicate the interpretation of results.Conclusive evidence for the importance of PI(4,5)P 2 in events that occur at the plasma membrane will come from experiments involving the acute and specific manipulation of its levels. Recently, a technique for the rapid, conditional recruitment of proteins to membranes has been used to deliver 5-phosphatase modules to the cell surface (20,(29)(30)(31)(32). In this method, the genes encoding two proteins that can be cross-linked by the add...

A phosphoinositide conversion mechanism for exit from endosomes

“…Besides being a precursor for PI(4,5)P 2 , recent studies indicate that PI(4)P contributes to the polyanionic lipid pool that defines the PM, additionally to, but independent of PI(4,5)P 2 (Hammond, Fischer et al 2012). Clathrin coated pit (CCP) nucleation, although dependent on the general PM PI(4,5)P 2 content (Zoncu, Perera et al 2007), is driven by local synthesis of PI(4,5)P 2 by PIP5K type I. The most prominent PI(4,5)P 2 -specific clathrin adaptor is the tetrameric adaptor protein AP-2 and cargo-AP-2-complexes stimulate PIP5K activity (Krauss, Kukhtina et al 2006).…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…Dynamin binds PtdIns(4,5)P 2 through its PH domain; AP180, epsin, endophilin, and amphiphysin do so through their ANTH, ENTH, and BAR domains, respectively. Acute elimination of PtdIns(4,5)P 2 from the plasma membrane, either by recruitment of a phosphatase (using a small-molecule dimerizer) or by treatment with butanol (which inhibits PtdIns(4,5)P 2 production), results in nearly complete loss of clathrin-coated structures Zoncu et al 2007). Clathrin assembly at the plasma membrane thus requires both recruitment by lipid-associated adaptor proteins and cooperativity of lattice interactions.…”

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…In a typical experiment the disappearance or increase in mobility of CCPs labelled with GFP or RFP clathrin light chain was used as an indicator for endocytosis. This approach was used to characterise the recruitment dynamics of AP2 [3,28,41,80], Hip1R [5], epsin [4,6], actin [18], endophilin2 [37], synaptojanin1 [37], APPL1 [59,60], OCRL1 [59], FBP17 [6], N-WASP [47], Arp3 [47,80], GAK [15,16], and dynamin1/2 [3,15,18,41] to endocytic sites. This enabled CCP/CCV components to be divided into those that are recruited at early stages (orange time course in [A]) or transiently at late stages (yellow time course in [A]).…”