A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis

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, synaptojanin2β1, 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.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

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.     

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.     

Dynamics of membrane clathrin-coated structures during cytokinesis

Remodeling of cell membranes takes place during motile processes such as cell migration and cell division. Defects of proteins involved in membrane dynamics, including clathrin and dynamin, disrupt cytokinesis. To understand the function of clathrin-containing structures (CCS) in cytokinesis, we have expressed a green fluorescent protein (GFP) fusion protein of clathrin light chain a (GFP-clathrin) in NRK epithelial cells and recorded images of dividing cells near the ventral surface with a spinning disk confocal microscope. Punctate GFP-CCS underwent dynamic appearance and disappearance throughout the ventral surface. Following anaphase onset, GFP-CCS between separated chromosomes migrated toward the equator and subsequently disappeared in the equatorial region. Movements outside separating chromosomes were mostly random, similar to what was observed in interphase cells. Directional movements toward the furrow were dependent on both actin filaments and microtubules, while the appearance/disappearance of CCS was dependent on actin filaments but not on microtubules. These results suggest that CCS are involved in remodeling the plasma membrane along the equator during cytokinesis. Clathrin-containing structures may also play a role in transporting signaling or structural components into the cleavage furrow.     

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.      

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.     

Dynamin Regulates Autophagy by Modulating Lysosomal Function

Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested w1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire(shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosomeelysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation(ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function.     

Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition

Clathrin-mediated endocytosis (CME) regulates many cell physiological processes such as the internalization of growth factors and receptors, entry of pathogens, and synaptic transmission. Within the endocytic network, clathrin functions as a central organizing platform for coated pit assembly and dissociation via its terminal domain (TD). We report the design and synthesis of two compounds named pitstops that selectively block endocytic ligand association with the clathrin TD as confirmed by X-ray crystallography. Pitstop-induced inhibition of clathrin TD function acutely interferes with receptor-mediated endocytosis, entry of HIV, and synaptic vesicle recycling. Endocytosis inhibition is caused by a dramatic increase in the lifetimes of clathrin coat components, including FCHo, clathrin, and dynamin, suggesting that the clathrin TD regulates coated pit dynamics. Pitstops provide new tools to address clathrin function in cell physiology with potential applications as inhibitors of virus and pathogen entry and as modulators of cell signaling.     

Fat-body remodeling in Drosophila melanogaster

The remodeling of the larval fat body is observed in many insects during metamorphosis, but little is known about the physiological importance or the regulation of this process. In Drosophila melanogaster, fat-body remodeling involves the dissociation of the fat body into individual fat cells, which persist throughout pupal development but are later removed by cell death in the young adult. Inhibition of fat-body dissociation is associated with pharate adult lethality and thus is likely to be an essential developmental event. As a start toward understanding the role of fat-body remodeling in the life history of insects, we carried out a detailed study of fat-body disassociation in D. melanogaster using fluorescent microscopy, and tested whether this process is mediated by hemocytes as proposed for fat-body remodeling in Sarcophaga peregrina. We identified and correlated stereotypic events in fat-body dissociation with developmental changes during metamorphosis, and have demonstrated by cell ablation studies that fat-body remodeling in D. melanogaster is a hemocyte independent process.     

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2–4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.     

Planar polarity and actin dynamics in the epidermis of Drosophila

Dorsal closure is a morphogenetic process involving the coordinated convergence of two epithelial sheets to enclose the Drosophila melanogaster embryo. Specialized populations of cells at the edges of each epithelial sheet, the dorsal-most epidermal cells, emit actin-based processes that are essential for the proper enclosure of the embryo. Here we show that actin dynamics at the leading edge is preceded by a planar polarization of the dorsal-most epidermal cells associated with a reorganization of the cytoskeleton. An important consequence of this planar polarization is the formation of actin-nucleating centres at the leading edge, which are important in the dynamics of actin. We show that Wingless (Wg) signalling and Jun amino-terminal kinase (JNK) signalling have overlapping but different roles in these events.     

Endocytosis and toxicity of clostridial binary toxins depend on a clathrin‐independent pathway regulated by Rho‐GDI

Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2II respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2‐I respectively) with ADP‐ribosyltransferase activity that disrupts the actin cytoskeleton. We show here that the endocytic pathway followed by these toxins is independent of clathrin but requires the activity of dynamin and is regulated by Rho‐GDI. This endocytic pathway is similar to a recently characterized clathrin‐independent pathway followed by the interleukin‐2 (IL2) receptor. We found indeed that Ib and C2II colocalized intracellularly with the IL2 receptor but not the transferrin receptor after different times of endocytosis. Accordingly, the intracellular effects of Iota and C2 on the cytoskeleton were inhibited by inactivation of dynamin or by Rho‐GDI whereas inhibitors of clathrin‐dependent endocytosis had no protective effect.     

Dynamin Rings: Not Just for Fission

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin‐mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin‐coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin‐binding and ‐regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell .      

Compensatory endocytosis in bladder umbrella cells occurs through an integrin‐regulated and RhoA‐ and dynamin‐dependent pathway

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin‐independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding‐stimulated CE, which depended on β₁ integrin‐associated signalling pathways, occurred by a dynamin‐, actin‐, and RhoA‐regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO‐1‐positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin‐independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA‐ and dynamin‐dependent pathway, and terminates in degradation and not recapture of membrane in DFV.     

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.     

Clathrin‐Mediated Endocytic Proteins are Involved in Regulating Mitotic Progression and Completion

A few proteins required for clathrin‐mediated endocytosis (CME) are associated with successful completion of mitosis at distinct mitotic stages. Clathrin heavy chain (CHC) and epsin are required for chromosome segregation independent of their CME function and dynamin II (dynII) functions in the abscission stage of cytokinesis. In this study we screened for mitotic roles of eight CME proteins: CHC, α‐adaptin, CALM, epsin, eps15, endophilin II (edpnII), syndapin II (sdpnII) and the GTPase dynII using a small interfering RNA targeting approach. All proteins, except for CALM, are associated with completion of the abscission stage of cytokinesis, suggesting that they function in this process in an endocytic‐dependent manner. In support of this concept, overexpression of epsin^S357D, which blocks endocytosis, induced multinucleation. Moreover, six of them have a secondary role at earlier mitotic stages that is not dependent on their endocytic function: CHC, epsin and eps15 in chromosome segregation, and sdpnII, α‐adaptin and CALM have a role in furrow ingression. Therefore, the role of endocytic proteins in mitosis is much broader than previously recognized.     

Endophilin,Lamellipodin, and Mena cooperate to regulate F‐actin‐dependent EGF‐receptor endocytosis

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin‐mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F‐actin‐dependent manner, suggesting that F‐actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin‐coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.