Molecular mechanism of Fast Endophilin-Mediated Endocytosis

Abstract

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

Antidepressants stimulate lipoprotein(a) macropinocytosis via serotonin-enhanced cell surface binding

“…While EIPA predominantly inhibits macropinocytosis, it is also capable of inhibiting fast endophilin mediated endocytosis (Boucrot et al, 2015). Fast endophilin mediated endocytosis strictly requires growth factors present in serum (Boucrot et al, 2015; Casamento and Boucrot, 2020) therefore to minimise any potential contribution of this alternate endocytic pathway, all experiments were performed in serum free media following a minimum of 1 hour serum starvation. Specific inhibition of macropinocytosis by addition of EIPA in serum free media lead to robust inhibition of Lp(a) uptake (Figure 1C,D) confirming macropinocytosis is an important endocytic mechanism for Lp(a) internalisation.…”

“…Clathrin-dependent and fast endophilin mediated endocytosis represent mechanisms of receptor-mediated endocytosis. In both cases, cargo/ligand binding to the receptor leads to the assembly of adaptor and coat proteins around the forming endosome which promote receptor and cargo internalisation (Boucrot et al, 2015; Casamento and Boucrot, 2020; Haucke, 2006; Traub and Bonifacino, 2013). In contrast, macropinocytosis is a calcium-dependent endocytic pathway where cargo is internalised via membrane remodelling events as opposed to receptor-mediated initiation of endocytosis.…”

“…Macropinocytosis requires extracellular calcium to initiate an intracellular signalling cascade which stimulates characteristic actin remodelling required for engulfment of cargoes (Canton et al, 2016). To date a requirement for extracellular calcium has not been documented for other forms of endocytosis in non-neuronal cell types (Casamento and Boucrot, 2020; Kumari and Mayor, 2008; Lundmark et al, 2008; Sathe et al, 2018). Macropinocytosis is not defined by specific protein or cellular markers, although large dextran polymers (70 kDa and above) have been shown to be bona fide macropinocytosis cargo and thus often used as a marker of the process (Li et al, 2015).…”

Endophilin Recruitment via GPCR Interactions Enables Membrane Curvature Generation in the Absence of Anionic Lipids

“…Endophilin plays a central role in FEME by driving cargo recruitment, membrane curvature generation, and membrane scission (14,15). It has been suggested that molecular interactions occur between the endophilin SH3 domain and the third intracellular loop (TIL) of several GPCR family members during their internalization through the FEME pathway (10,16,17). Still, it has remained unclear how membrane remodeling by endophilin is functionally coupled with protein-protein interactions mediated by the SH3 domain.…”

Endophilin recruitment drives membrane curvature generation through coincidence detection of GPCR loop interactions and negative lipid charge

“…Endophilin plays a central role in FEME by driving cargo recruitment, membrane curvature generation, and membrane scission ( 14 , 15 ). It has been suggested that molecular interactions occur between the endophilin SH3 domain and the third intracellular loop (TIL) of several GPCR family members during their internalization through the FEME pathway ( 10 , 16 , 17 ). Still, it has remained unclear how membrane remodeling by endophilin is functionally coupled with protein–protein interactions mediated by the SH3 domain.…”