Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation

Loerke, Dinah; Mettlen, Marcel; Yarar, Defne; Jaqaman, Khuloud; Jaqaman, Henry; Danuser, Gaudenz; Schmid, Sandra L.

doi:10.1371/journal.pbio.1000057

Cited by 395 publications

(719 citation statements)

References 35 publications

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“…This was similar to previous estimates in BSC1 cells, wherein productive CCSs were defined as spot-like CCSs having lifetimes anywhere from tens to hundreds of seconds (average 87 s) [3,27,28]. Because the size and lifetimes of scission-detected CCSs were so variable ( Figure 1H and 1I), in our subsequent investigation of late events in CME we made measurements over a time window of 680 s, centred on scission.…”

Section: Author Summarysupporting

confidence: 81%

“…Therefore, scission events defined a class of larger, longer-lived CCSs. The shorter-lived scission-undetected CCSs most likely correspond to the ''abortive'' CCSs described previously [3,27,28], although some of these structures may have represented endosomal clathrin.…”

Section: Author Summarymentioning

confidence: 59%

“…Previous studies defined different populations of CCSs on the basis of size (i.e., spot-like CCSs versus larger CCSs) and lifetime or whether CCSs disappeared following scission (terminal events) or persisted (non-terminal events) [10,23,28,61]. Detailed mechanistic inferences have been based on these types of dynamic classification [23].…”

Section: The Relationship Between the Dynamic Characteristics Of Ccssmentioning

confidence: 99%

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

Perrais²,

2011

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Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ,2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/ 2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2b1, and syndapin2. For each protein we aligned ,1,000 recruitment profiles to their respective scission events and constructed characteristic ''recruitment signatures'' that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

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Section: Author Summarysupporting

confidence: 81%

Section: Author Summarymentioning

confidence: 59%

Section: The Relationship Between the Dynamic Characteristics Of Ccssmentioning

confidence: 99%

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

Perrais²,

2011

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“…S5 A-N). With respect to DYNAMIN, which is known to complete vesicle formation through membrane fission (44), previous studies have shown it is also present and functionally important at early stages of CLATHRIN-mediated endocytosis (45,46). AZ localization of endocytic proteins might suggest a mechanism of release site clearance involving rapid formation of CLATHRIN-coated vesicles directly from the AZ; however, this mechanism is not favored, primarily because ultrastructural studies indicate that CLATHRIN-coated vesicles form at PAZ, rather than AZ, regions of the plasma membrane (47,48) (Fig.…”

Section: Rapid Endocytic Mechanisms Maintaining Neurotransmitter Releasementioning

confidence: 99%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activitydependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.I ntensive study of chemical synaptic transmission has led to detailed molecular models of synaptic vesicle endocytosis (1-4). Ongoing efforts seek to define the underlying molecular components and interactions further as well as their spatial organization with respect to neurotransmitter release sites. The present study reveals novel molecular mechanisms and interactions in synaptic vesicle endocytosis involving a member of the DISABLED family of Phosphotyrosine Binding (PTB) domain proteins. In Drosophila, the disabled (dab) gene, the founding member of the dab gene family, was first identified in a screen for genetic modifiers of abelson (5). Although putative dab mutations were subsequently mapped to a different gene (6), recent studies have established functional interactions of Drosophila DISABLED (dDAB) and ABELSON (7). Proteins closely related to dDAB, including mouse DAB-2 (mDAB-2) and the single Caenorhabditis elegans family member ceDAB-1, function as CLATHRIN-associated adaptors in endocytic trafficking of cell surface receptors (8-10). These DABs have been shown to interact with CLATHRIN, the α-ADAPTIN (AP-2) adaptor complex, and other components of the endocytic machinery through conserved binding motifs (8-10) (Fig. 1A). Notably, mDAB-2, ceDAB-1, and dDAB share a PTB/DAB Homology (DH) domain near the N terminus (Fig. 1B), which interacts with specific vesicle cargo proteins and phosphatidylinositol-4,5-diphosphate (11, 12). Neither DAB proteins nor PTB domain interactions have been previously implicated in synaptic vesicle trafficking.The present study reveals a role for DAB proteins in synaptic vesicle endocytosis and extends previous work on interactions of endocytic and exocytic mechanisms in neurotransmitter release. Here, we use a glutamatergic neuromuscular synapse of the Drosophila adult as a model for genetic analysis of molecular mechanisms determining conserved properties of glutamatergic synapse function (13,14). Previous analysis i...

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“…L'initiation de la formation de vésicu-les de clathrine est une étape difficile à étudier : les puits recouverts de clathrine se forment et deviennent des vésicules relativement rapidement (20 à 90 secondes), sont trop petits (environ 100 nm) pour être observés directement par microscopie optique et se forment aléatoirement sur la surface des cellules [19,20]. L'adaptateur AP2 a longtemps été considéré comme responsable de la nucléation des puits de clathrine.…”

Section: Induction Des Vésicules De Clathrine : Le Rôle Controversé Dunclassified

Initiation de l’endocytose par vésicules de clathrine

Boucrot¹,

McMahon²

2011

Med Sci (Paris)

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Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation

Cited by 395 publications

References 35 publications

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

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

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Initiation de l’endocytose par vésicules de clathrine

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