Role In Clathrin-mediated Endocytosis Research Articles

The AP-2 complex interacts with γ-TuRC and regulates the proliferative capacity of neural progenitors.

Centrosomes are organelles that nucleate microtubules via the activity of gamma-tubulin ring complexes (γ-TuRC). In the developing brain, centrosome integrity is central to the progression of the neural progenitor cell cycle, and its loss leads to microcephaly. We show that NPCs maintain centrosome integrity via the endocytic adaptor protein complex-2 (AP-2). NPCs lacking AP-2 exhibit defects in centrosome formation and mitotic progression, accompanied by DNA damage and accumulation of p53. This function of AP-2 in regulating the proliferative capacity of NPCs is independent of its role in clathrin-mediated endocytosis and is coupled to its association with the GCP2, GCP3, and GCP4 components of γ-TuRC. We find that AP-2 maintains γ-TuRC organization and regulates centrosome function at the level of MT nucleation. Taken together, our data reveal a novel, noncanonical function of AP-2 in regulating the proliferative capacity of NPCs and open new avenues for the identification of novel therapeutic strategies for the treatment of neurodevelopmental and neurodegenerative disorders with AP-2 complex dysfunction.

Endocytic myosin-1 is a force-insensitive, power-generating motor.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.

Self-organizing actin networks drive sequential endocytic protein recruitment and vesicle release on synthetic lipid bilayers

Forces generated by actin assembly assist membrane invagination during clathrin-mediated endocytosis (CME). The sequential recruitment of core endocytic proteins and regulatory proteins, and assembly of the actin network, are well documented in live cells and are highly conserved from yeasts to humans. However, understanding of CME protein self-organization, as well as the biochemical and mechanical principles that underlie actin's role in CME, is lacking. Here, we show that supported lipid bilayers coated with purified yeast Wiskott Aldrich Syndrome Protein (WASP), an endocytic actin assembly regulator, and incubated in cytoplasmic yeast extracts, recruit downstream endocytic proteins and assemble actin networks. Time-lapse imaging of WASP-coated bilayers revealed sequential recruitment of proteins from different endocytic modules, faithfully replicating in vivo behavior. Reconstituted actin networks assemble in a WASP-dependent manner and deform lipid bilayers, as seen by electron microscopy. Time-lapse imaging revealed that vesicles are released from the lipid bilayers with a burst of actin assembly. Actin networks pushing on membranes have previously been reconstituted; here, we have reconstituted a biologically important variation of these actin networks that self-organize on bilayers and produce pulling forces sufficient to bud off membrane vesicles. We propose that actin-driven vesicle generation may represent an ancient evolutionary precursor to diverse vesicle forming processes adapted for a wide array of cellular environments and applications.

Evidence on differential role for alpha 1 and alpha 2 subtypes of AP-2 adaptin in the central nervous system

Two subtypes of alpha (α)subunits, α1and α2, belonging to AP-2 complex have been described in the central nervous system (CNS). The specific role of each subtype is still unclear. In this study, we evaluated the expression and interaction with cell membranes of both subtypes in the postnatal developing cerebral cortex and cerebellum in two rat strains that display distinct developmental features. We observed that α2 displays higher variations than α1 during development, and at lesser extent in the rats with delayed rate of development. Additionally, by in vitro binding assays we evaluated the interaction of α subunits with bovine brain membranes. Both subtypes displayed clear differences in their performance, maximum binding of α1 was higher and α2 reached it faster than α1. In addition, both subtypes displayed different binding to membranes when bivalent cations or nucleotides were added. We conclude that both subtypes interact differently with membranes and that they may play different roles in clathrin-mediated endocytosis in the CNS.

PICALM and Alzheimer's Disease: An Update and Perspectives.

Genome-wide association studies (GWAS) have identified the PICALM (Phosphatidylinositol binding clathrin-assembly protein) gene as the most significant genetic susceptibility locus after APOE and BIN1. PICALM is a clathrin-adaptor protein that plays a critical role in clathrin-mediated endocytosis and autophagy. Since the effects of genetic variants of PICALM as AD-susceptibility loci have been confirmed by independent genetic studies in several distinct cohorts, there has been a number of in vitro and in vivo studies attempting to elucidate the underlying mechanism by which PICALM modulates AD risk. While differential modulation of APP processing and Aβ transcytosis by PICALM has been reported, significant effects of PICALM modulation of tau pathology progression have also been evidenced in Alzheimer's disease models. In this review, we summarize the current knowledge about PICALM, its physiological functions, genetic variants, post-translational modifications and relevance to AD pathogenesis.

Nanodissected elastically loaded clathrin lattices relax to increased curvature.

Clathrin-mediated endocytosis (CME) is the major endocytosis pathway for the specific internalization of large compounds, growth factors, and receptors. Formation of internalized vesicles from the flat plasma membrane is accompanied by maturation of cytoplasmic clathrin coats. How clathrin coats mature and the mechanistic role of clathrin coats are still largely unknown. Maturation models proposed clathrin coats to mature at constant radius or constant area, driven by molecular actions or elastic energy. Here, combining high-speed atomic force microscopy (HS-AFM) imaging, HS-AFM nanodissection, and elasticity theory, we show that clathrin lattices deviating from the intrinsic curvature of clathrin form elastically loaded assemblies. Upon nanodissection of the clathrin network, the stored elastic energy in these lattices drives lattice relaxation to accommodate an ideal area-curvature ratio toward the formation of closed clathrin-coated vesicles. Our work supports that the release of elastic energy stored in curvature-frustrated clathrin lattices could play a major role in CME.

Dynamin-dependent vesicle twist at the final stage of clathrin-mediated endocytosis.

Dynamin plays an important role in clathrin-mediated endocytosis (CME) by cutting the neck of nascent vesicles from the cell membrane. Here through using gold nanorods as cargos to image dynamin action during live CME, we show that near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle drastically increases, accompanied with simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.

Role of sorting nexin adaptor proteins in GPCR regulation

Signaling by the G protein-coupled receptor CXCR4 is essential to a number of developmental and physiological processes, including stem cell mobilization, organ development, and immune responses. In addition, CXCR4 signaling has been linked to disease, including cancer and certain immune disorders. Despite this, the mechanisms governing CXCR4 signaling remain poorly defined. Typically, upon binding to its cognate ligand CXCL12, CXCR4 signaling occurs via G protein- and β-arrestin-dependent pathways. Although G protein-dependent CXCR4-mediated signaling is well understood, β-arrestin1-dependent signaling remains poorly understood. With this in mind, we performed proximity biotin labeling by ascorbic acid peroxidase (APEX2) fused to β-arrestin1 in order to identify proteins that are part of the β-arrestin1 interaction network downstream of CXCR4. Labeled proteins from cells treated with vehicle or CXCL12 for 5 or 60 minutes were selectively purified and identified by mass spectrometry. Several proteins known to be part of the b-arrestin network downstream of CXCR4 including STAM1 and FAK were identified. Several other proteins of biological relevance were identified that were enriched in cells treated with CXCL12 for 5 or 60 min. Orthogonal biochemical experiments verified that two of these proteins, WAVE2 and SNX9, interact with β-arrestin1. We selected SNX9 for further study of its functional role in CXCR4 signaling. SNX9 (sorting nexin 9), and related proteins SNX18 and SNX33, are multifunctional adaptor proteins best known for their role in clathrin-mediated endocytosis of multiple cell surface receptors. Whether they mediate endocytosis of GPCRs remains unknown. To address this, we transfected HEK293 cells stably expressing epitope-tagged CXCR4 with siRNA against SNX9, SNX18, and SNX33, and examined agonist-induced endocytosis of CXCR4 by whole cell ELISA. Simultaneous transfection with siRNA against SNX9 and SNX18 significantly reduced CXCR4 endocytosis when compared with control siRNA or when cells were transfected with individual siRNA against SNX9, SNX18 or SNX33. These data provide evidence for the first time for a role of members of the SNX9-family in GPCR endocytosis, likely by serving as adaptors for clathrin-mediated endocytosis. Additional work is needed to conclusively demonstrate that SNX9 functions as an adaptor protein for CXCR4 endocytosis and to determine the mechanism by which this occurs. To accomplish this we will use a combination of biochemical, molecular, imaging and biophysical approaches. In addition, we will investigate how broadly SNX9-family members impact CXCR4 endocytosis across multiple cell types and also address the impact of the SNX9-family on GPCR endocytosis in general.

Creation of a functional unc-11/PICALM GFP knock-in by CRISPR.

unc-11 is the only C. elegans ortholog of mammalian PICALM/AP180, paralogs that play an important role in Clathrin-Mediated Endocytosis (CME) and the recycling of a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs). In this publication we report the creation of a new unc-11 allele that is endogenously-tagged with GFP just upstream of the stop codon. Moreover, we demonstrate that the UNC-11::GFP fusion protein functions like wild type with an expression pattern similar to UNC-11 antibody staining described previously.

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omega-shaped CCSs. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition of a CCS and stabilizes its curvature at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment. Epsin’s binding to adaptor protein 2 and clathrin is critical for epsin’s association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and unstructured IDP region of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells.

Objective Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). Results We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Picalm reduction exacerbates tau pathology in a murine tauopathy model.

Genome-wide association studies (GWAS) have identified PICALM as one of the most significant susceptibility loci for late-onset Alzheimer's disease (AD) after APOE and BIN1. PICALM is a clathrin-adaptor protein and plays critical roles in clathrin-mediated endocytosis and in autophagy. PICALM modulates brain amyloid ß (Aß) pathology and tau accumulation. We have previously reported that soluble PICALM protein level is reduced in correlation with abnormalities of autophagy markers in the affected brain areas of neurodegenerative diseases including AD, sporadic tauopathies and familial cases of frontotemporal lobar degeneration with tau-immunoreactive inclusions (FTLD-tau) with mutations in the microtubule-associated protein tau (MAPT) gene. It remains unclarified whether in vivo PICALM reduction could either trigger or influence tau pathology progression in the brain. In this study, we confirmed a significant reduction of soluble PICALM protein and autophagy deficits in the post-mortem human brains of FTLD-tau-MAPT (P301L, S364S and L266V). We generated a novel transgenic mouse line named Tg30xPicalm+/-by crossing Tg30 tau transgenic mice with Picalm-haploinsufficient mice to test whether Picalm reduction may modulate tau pathology. While Picalm haploinsufficiency did not lead to any motor phenotype or detectable tau pathology in mouse brains, Tg30xPicalm+/- mice developed markedly more severe motor deficits than Tg30 by the age of 9months. Tg30xPicalm+/- had significantly higher pathological tau levels in the brain, an increased density of neurofibrillary tangles compared to Tg30 mice and increased abnormalities of autophagy markers. Our results demonstrate that Picalm haploinsufficiency in transgenic Tg30 mice significantly aggravated tau pathologies and tau-mediated neurodegeneration, supporting a role for changes in Picalm expression as a risk/sensitizing factor for development of tau pathology and as a mechanism underlying the AD risk associated to PICALM.

Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

AAGAB Controls AP2 Adaptor Assembly in Clathrin-Mediated Endocytosis

Multimeric adaptors are broadly involved in vesicle-mediated membrane trafficking. AP2 adaptor, in particular, plays a central role in clathrin-mediated endocytosis (CME) by recruiting cargo and clathrin to endocytic sites. It is generally thought that trafficking adaptors such as AP2 adaptor assemble spontaneously. In this work, however, we discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB), an uncharacterized factor identified in our genome-wide genetic screen of CME. AAGAB guides the sequential association of AP2 subunits and stabilizes assembly intermediates. Without the assistance of AAGAB, AP2 subunits fail to form the adaptor complex, leading to their degradation. The function of AAGAB is abrogated by a mutation that causes punctate palmoplantar keratoderma type 1 (PPKP1), a human skin disease. Since other multimeric trafficking adaptors operate in an analogous manner to AP2 adaptor, their assembly likely involves a similar regulatory mechanism.

Exon Inclusion Modulates Conformational Plasticity and Autoinhibition of the Intersectin 1 SH3A Domain

The scaffolding protein intersectin 1 plays important roles in clathrin-mediated endocytosis and in the replenishment of release-ready synaptic vesicles (SV). Two splice variants of intersectin's SH3A domain are expressed in the brain, and association of the neuron-specific variant with synapsin I has been shown to enable sustained neurotransmission and to be regulated by an adjacent C-terminal motif. Here, we demonstrate that the ubiquitously expressed short SH3A variant of intersectin 1 interacts with an N-terminal intramolecular sequence that operates synergistically with the C-terminal motif. NMR spectroscopic investigations show that the five-amino acid insertion into the β strand 2 of the neuronal SH3A variant introduces conformational plasticity incompatible with binding of the N-terminal sequence. The difference in the autoregulatory mechanism of the domain's variants differentially affects its synaptic binding partners, thereby establishing alternative splicing in conjunction with autoinhibitory motif variation as a mechanism to regulate protein interaction networks.

Plant AP180 N-Terminal Homolog Proteins Are Involved in Clathrin-Dependent Endocytosis during Pollen Tube Growth in Arabidopsis thaliana.

Polarized cell growth in plants is maintained under the strict control and exquisitely choreographed balance of exocytic and endocytic membrane trafficking. The pollen tube has become a model system for rapid polar growth in which delivery of cell wall material and membrane recycling are controlled by membrane trafficking. Endocytosis plays an important role that is poorly understood. The plant AP180 N-Terminal Homolog (ANTH) proteins are putative homologs of Epsin 1 that recruits clathrin to phosphatidylinositol 4, 5-bisphosphate (PIP2) containing membranes to facilitate vesicle budding during endocytosis. Two Arabidopsis ANTH encoded by the genes AtAP180 and AtECA2 are highly expressed in pollen tubes. Pollen tubes from T-DNA inserted knockout mutant lines display significant morphological defects and unique pectin deposition. Fluorescent tagging reveals organization into dynamic foci located at the lateral flanks of the pollen tube. This precisely defined subapical domain coincides which clathrin-mediated endocytosis (CME) and PIP2 localization. Using a liposome-protein binding test, we showed that AtECA2 protein and ANTH domain recombinant proteins have strong affinity to PIP2 and phosphatidic acid containing liposomes in vitro. Taken together these data suggest that Arabidopsis ANTH proteins may play an important role in CME, proper cell wall assembly and morphogenesis.

SGIP1 dimerizes via intermolecular disulfide bond in μHD domain during cellular endocytosis

Along with its homologs FCHo1 and FCHo2, SGIP1 plays an important role in clathrin-mediated endocytosis. The highly conserved C-terminal μHD domains in these proteins are the critical regions interacting with adapter molecules such as Eps15. The crystal structure of μHD domain of SGIP1 has been reported previously. In this study, we found that μHD domain of SGIP1 is capable of forming a stable dimer by an intermolecular disulfide bond formed by C632 in our crystal structure. The mutational study of C632 revealed that this residue is important for the function of SGIP1 during cellular endocytosis. Our study revealed a new dimerization and/or oligomerization manner in theses adaptor proteins, which is a critical prerequisite for their proper function.

Targeted pancreatic cancer drug-delivery utilizing sigma-2 ligand/receptor internalization is energy-dependent.

331 Background: Pancreatic cancer is a devastating disease that is poorly responsive to traditional systemic therapies. Cancer-selective drug delivery can improve survival and reduce systemic toxicities. We have developed a pancreatic cancer drug delivery platform based on sigma-2 receptor ligands conjugated to small molecule drug cargos, which improves delivery and efficacy through efficient drug internalization. However, the mechanism of drug internalization via the sigma-2 receptor/ligand interaction remains unclear. Methods: Uptake of fluorescently conjugated sigma-2 ligand SW120 was studied in ASPC-1 human pancreatic cancer cells. Uptake was measured at 37C, 4C, after competitive inhibition with sigma-2 ligand SW43, and after pretreatment with Pitstop 2 (Abcam), an inhibiter of clathrin-mediated endocytosis. Uptake was visualized using live-cell imaging using A1Rsi confocal laser scanning microscope (Nikon). Image analysis was performed using FIJI (NIH) and Matlab (Mathworks). Results: SW120 (10 nM) was rapidly internalized into ASPC-1 cells at 37C with maximal fluorescence at 12 minutes. As a negative control, incubation of ASPC-1 cells with the unconjugated flourophore NBD Cl demonstrated no uptake after 15 minutes, indicating that uptake of SW120 depends on the specific sigma-2 receptor/ligand interaction. Fluorescence at 15 minutes was reduced by 85% in ASPC-1 cells incubated at 4C, indicating uptake of SW120 into cells is an energy dependent process. Pretreatment of cells with Pitstop 2 decreased total flourescence at 15 minutes by 76%, suggesting an important role of clathrin-mediated endocytosis in sigma-2 receptor uptake, while pretreatment with competitive inhibitor SW43 reduced uptake by 84%, suggesting that sigma-2 ligands are internalized via a specific receptor capable of saturation. Conclusions: Sigma-2 receptor ligands are rapidly internalized into pancreatic cancer cells via a specific, targetable, energy-dependent pathway that appears to rely on clathrin-mediated endocytosis. Further understanding of sigma-2 mediated drug internalization can help optimize targeted drug development and delivery for pancreatic cancer patients.

Dynamin 1- and 3-Mediated Endocytosis Is Essential for the Development of a Large Central Synapse In Vivo.

Dynamin is a large GTPase crucial for endocytosis and sustained neurotransmission, but its role in synapse development in the mammalian brain has received little attention. We addressed this question using the calyx of Held (CH), a large nerve terminal in the auditory brainstem in mice. Tissue-specific ablation of different dynamin isoforms bypasses the early lethality of conventional knock-outs and allows us to examine CH development in a native brain circuit. Individual gene deletion of dynamin 1, a primary dynamin isoform in neurons, as well as dynamin 2 and 3, did not affect CH development. However, combined tissue-specific knock-out of both dynamin 1 and 3 (cDKO) severely impaired CH formation and growth during the first postnatal week, and the phenotypes were exacerbated by further additive conditional knock-out of dynamin 2. The developmental defect of CH in cDKO first became evident on postnatal day 3 (P3), a time point when CH forms and grows abruptly. This is followed by a progressive loss of postsynaptic neurons and increased glial infiltration late in development. However, early CH synaptogenesis before protocalyx formation was not altered in cDKO. Functional maturation of synaptic transmission in the medial nucleus of the trapezoid body in cDKO was impeded during development and accompanied by an increase in the membrane excitability of medial nucleus of the trapezoid body neurons. This study provides compelling genetic evidence that CH formation requires dynamin 1- and 3-mediated endocytosis in vivo, indicating a critical role of dynamin in synaptic development, maturation, and subsequent maintenance in the mammalian brain. Synaptic development has been increasingly implicated in numerous brain disorders. Dynamin plays a crucial role in clathrin-mediated endocytosis and synaptic transmission at nerve terminals, but its potential role in synaptic development in the native brain circuitry is unclear. Using the calyx of Held, a giant nerve terminal in the mouse brainstem, we evaluated the role of dynamin in this process by using tissue-specific knock-out (KO) of three different dynamin isoforms (dynamin 1, 2, and 3) individually and in combination. Our data demonstrated that dynamin is required for the formation, functional maturation, and subsequent survival of the calyx of Held. This study highlights the important role of dynamin-mediated endocytosis in the development of central synapses in the mammalian brain.