ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Regulation of cargo‐selective endocytosis by dynamin 2 GTPase‐activating protein girdin

In clathrin‐mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo‐specific adaptors for distinct cellular functions. Here, we show that the actin‐binding protein girdin is a regulator of cargo‐selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase‐activating protein. Interestingly, girdin depletion leads to the defect in clathrin‐coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E‐cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo‐specific adaptor.     

Endocytic activity of HIV‐1 Vpu: Phosphoserine‐dependent interactions with clathrin adaptors

HIV‐1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune‐evasion, triggering ubiquitin‐mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP‐1 and AP‐2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α‐chain. CD8‐VpuCD was rapidly endocytosed in a clathrin‐ and AP‐2‐dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β‐TrCP binding site and a leucine‐based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV‐dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP‐2 and showed that this is enhanced by serine‐phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP‐2 and AP‐1; this interaction was dependent on serine‐phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.      

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin‐mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin‐coated pits and bulk endocytosis‐like structures increase on the plasma membrane in Syt11‐knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.     

Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.     

Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis

Endocytosis is a vital cellular process maintaining the cell surface, modulating signal transduction and facilitating nutrient acquisition. In metazoa, multiple endocytic modes are recognized, but for many unicellular organisms the process is likely dominated by the ancient clathrin‐mediated pathway. The endocytic system of the highly divergent trypanosomatid Trypanosoma brucei exhibits many unusual features, including a restricted site of internalization, dominance of the plasma membrane by GPI‐anchored proteins, absence of the AP2 complex and an exceptionally high rate. Here we asked if the proteins subtending clathrin trafficking in trypanosomes are exclusively related to those of higher eukaryotes or if novel, potentially taxon‐specific proteins operate. Co‐immunoprecipitation identified twelve T. brucei clathrin‐associating proteins (TbCAPs), which partially colocalized with clathrin. Critically, eight TbCAPs are restricted to trypanosomatid genomes and all of these are required for robust cell proliferation. A subset, TbCAP100, TbCAP116, TbCAP161 and TbCAP334, were implicated in distinct endocytic steps by detailed analysis of knockdown cells. Coupled with the absence of orthologs for many metazoan and fungal endocytic factors, these data suggest that clathrin interactions in trypanosomes are highly lineage‐specific, and indicate substantial evolutionary diversity within clathrin‐mediated endocytosis mechanisms across the eukaryotes.     

An internally eGFP‐tagged α‐adaptin is a fully functional and improved fiduciary marker for clathrin‐coated pit dynamics

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby‐CLCa and/or μ2‐eGFP, σ2‐eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α‐eGFP). Using biochemical and TIRFM‐based assays, we compared the functionality of the AP2 markers. All of the eGFP‐tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image‐based CCP analyses than mRuby‐CLCa. However, overexpression of either μ2‐eGFP or σ2‐eGFP impaired transferrin receptor uptake. In addition, μ2‐eGFP reduced the rates of CCP initiation and σ2‐eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α‐eGFP. Moreover, α‐eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby‐CLCa. Thus, our work establishes α‐eGFP as a robust, fully functional marker for CME.     

The Surface Density of the Glutamate Transporter EAAC1 is Controlled by Interactions with PDZK1 and AP2 Adaptor Complexes

The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell‐surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post‐synaptic density‐95/Discs large/Zonula occludens (PDZ) proteins (‐SQF) and a tyrosine‐based internalization signal (‐YVNG‐) in the C‐terminus of EAAC1, and investigated their role in the transporter localization. We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+‐exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin‐Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ‐target motif promoted the EAAC1 binding to α‐adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant‐negative µ2‐W421A‐subunit of AP‐2 clathrin‐adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis. We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in Mammalian cells

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated beta(2)-adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM-Ub interactions to overcome the low affinity binding of mono-Ub to UIM.     

Disruption of adaptor protein 2μ (AP‐2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP‐2μ) is required for release site replenishment and hearing. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation.     

Regulators of yeast endocytosis identified by systematic quantitative analysis

Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.     

Yeast Endocytic Adaptor AP‐2 Binds the Stress Sensor Mid2 and Functions in Polarized Cell Responses

The AP‐2 complex is a heterotetrameric endocytic cargo‐binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin‐mediated endocytosis. While budding yeast has clear homologues of all four AP‐2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP‐2 has remained enigmatic. Here, we demonstrate that AP‐2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo‐binding mu subunit of AP‐2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP‐2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP‐2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth.      

Conformational regulation of AP1 and AP2 clathrin adaptor complexes

Heterotetrameric clathrin adaptor protein complexes (APs) orchestrate the formation of coated vesicles for transport among organelles of the cell periphery. AP1 binds membranes enriched for phosphatidylinositol 4‐phosphate, such as the trans Golgi network, while AP2 associates with phosphatidylinositol 4,5‐bisphosphate of the plasma membrane. At their respective membranes, AP1 and AP2 bind the cytoplasmic tails of transmembrane protein cargo and clathrin triskelions, thereby coupling cargo recruitment to coat polymerization. Structural, biochemical and genetic studies have revealed that APs undergo conformational rearrangements and reversible phosphorylation to cycle between different activity states. While membrane, cargo and clathrin have been demonstrated to promote AP activation, growing evidence supports that membrane‐associated proteins such as Arf1 and FCHo also stimulate this transition. APs may be returned to the inactive state via a regulated process involving phosphorylation and a protein called NECAP. Finally, because antiviral mechanisms often rely on appropriate trafficking of membrane proteins, viruses have evolved novel strategies to evade host defenses by influencing the conformation of APs. This review will cover recent advances in our understanding of the molecular inputs that stimulate AP1 and AP2 to adopt structurally and functionally distinct configurations.     

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, β2, μ2 and σ2 subunits, facilitates clathrin‐mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl‐terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand‐mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand‐mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2‐μ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, μ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2‐μ2, is replaced with μ2 mutated in Patch 1 and/or Patch 2 domains, ligand‐mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2‐μ2 Patch 1, but not Patch 2. AP2‐σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2‐σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2‐mediated receptor internalization can be dissociated from AP2‐mediated chemotaxis.      

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.     

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.     

Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis

Chlorpromazine is a phenothiazine‐derived antipsychotic drug (APD) that inhibits clathrin‐mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine‐derived APDs inhibited dynamin I (dynI) in the 2–12 µm range, the most potent being trifluoperazine (IC₅₀ 2.6 ± 0.7 µm ). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8‐ to 10‐fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine‐derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine‐derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin‐active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure–activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP‐2 recruitment in the formation of clathrin‐coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP‐2 interaction with amphiphysin or clathrin. Super‐resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP‐2, nor AP‐2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine‐derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in‐cell target in endocytosis.      

The Machado–Joseph disease‐associated form of ataxin‐3 impacts dynamics of clathrin‐coated pits

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin‐3 is the main cause of neurodegeneration in Machado–Joseph disease. Ataxin‐3 contains an N‐terminal catalytic domain, called Josephin domain, and a highly aggregation‐prone C‐terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin‐mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin‐3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin‐3 could impact CME. Here, we report that both the wild‐type and the expanded ataxin‐3 reduce transferrin internalization and expanded ataxin‐3 impacts dynamics of clathrin‐coated pits (CCPs) by reducing CCP nucleation and increasing short‐lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.