CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature

Abstract

SummaryThe size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

Vesicular Synaptobrevin/VAMP2 Levels Guarded by AP180 Control Efficient Neurotransmission

“…Instead, our data are most consistent with a model according to which clathrin/AP180 depending on the frequency of firing reform Syb2-containing SVs either directly from the plasma membrane via conventional CME or from ELVs following clathrin-independent membrane retrieval Kononenko et al, 2014), possibly involving ultrafast (Watanabe et al, 2013b;Watanabe et al, 2014) or other forms of bulk endocytosis (Cheung and Cousin, 2013). Genetic manipulation of endocytic proteins in this model would lead to alterations in SV size and shape due to defects in membrane deformation and/or coat assembly as reported for several endocytic protein mutants (Dittman and Ryan, 2009;Ferguson et al, 2007;Milosevic et al, 2011;Nonet et al, 1999;Saheki and De Camilli, 2012;Zhang et al, 1998), acute perturbation of AP180 function in squid axons (Morgan et al, 1999), and endocytic vesicles in non-neuronal cells depleted of the clathrin-associated AP180 paralog CALM (Meyerholz et al, 2005;Miller et al, 2015).…”

Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis

“…Indeed, preincubation of cmd1-3 cells on ice stimulated LY uptake into cmd1-3 cells (Figure S3B,C). Notably, there is precedent for temperature shift-induced rescue of endocytic defects in mammalian cells (Miller et al, 2015). By another constant temperature assay, cmd1-3 cells are also less sensitive than WT cells to K28 killer toxin, which requires endocytosis for toxicity (Carroll et al, 2009) (Figure S3D).…”

Zero Tolerance: Amphipathic Helices in Endocytosis

“…In contrast to the supporting role of the Sla2 ANTH domain, Miller et al (2015) show in this issue of Developmental Cell that the ANTH domain of the mammalian clathrin adaptor CALM/ PICALM directly influences membrane curvature via its own amphipathic Helix 0. Just like epsin, CALM's Helix 0-actually an extension of Helix 1-was unseen in previous crystal structures (Ford et al, 2001;Miller et al, 2011).…”

“…Skruzny et al (2015) and Miller et al (2015) now shed light on endocytic proteins that bear a ''Helix 0'' and on the proteins' role in the struggle to make clathrin-coated vesicles.…”