HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis

Herpes simplex virus‐1 (HSV‐1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin‐dependent endocytosis plays a major role in this process. Dominant‐negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin‐dependent and ‐independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non‐infectious HSV‐1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein‐sorting event during HSV‐1 envelopment.      

μ2‐Dependent endocytosis of N‐cadherin is regulated by β‐catenin to facilitate neurite outgrowth

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N‐cadherin, a calcium‐dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N‐cadherin internalizes through clathrin‐mediated endocytosis (CME). Two tyrosine‐based motifs in the cytoplasmic domain of N‐cadherin recognized by the μ2 subunit of the AP‐2 adaptor complex are responsible for CME of N‐cadherin. Moreover, β‐catenin, a core component of the N‐cadherin adhesion complex, inhibits N‐cadherin endocytosis by masking the 2 tyrosine‐based motifs. Removal of β‐catenin facilitates μ2 binding to N‐cadherin, thereby increasing clathrin‐mediated N‐cadherin endocytosis and neurite outgrowth without affecting the steady‐state level of surface N‐cadherin. These results identify and characterize the mechanism controlling N‐cadherin endocytosis through β‐catenin‐regulated μ2 binding to modulate neurite outgrowth.      

Microtubule Motors Power Plasma Membrane Tubulation in Clathrin‐Independent Endocytosis

How the plasma membrane is bent to accommodate clathrin‐independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin‐independent carriers by binding and cross‐linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin‐induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B‐subunit into surface‐derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin‐independent endocytosis.      

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin‐independent endocytosis, and recently, flotillin‐1 was implicated in the protein kinase C (PKC)‐triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin‐mediated, we re‐examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin‐1, flotillin‐2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC‐dependent internalization and degradation of HA‐DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP‐2 as well as a dynamin inhibitor Dyngo‐4A significantly decreased PKC‐dependent endocytosis of HA‐DAT. Similarly, endocytosis and degradation of DAT that is not epitope‐tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co‐localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA‐DAT in the plasma membrane, suggesting that flotillin‐organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin‐mediated endocytosis is the major pathway of PKC‐dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis .     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Sphingomyelin synthase 1-generated sphingomyelin plays an important role in transferrin trafficking and cell proliferation

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.     

Stable Cell Surface Expression of GPI‐Anchored Proteins,but not Intracellular Transport,Depends on their Fatty Acid Structure

Glycosylphosphatidylinositol‐anchored proteins (GPI‐APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI‐APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI‐APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI‐AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi‐to‐plasma membrane transport or selective endocytosis via GPI‐enriched early endosomal compartment (GEEC)/ clathrin‐independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI‐APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI‐APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI‐APs particularly under potential membrane lipid perturbation.      

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR4^GFP, confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.

     

Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers

Lipid metabolites, free fatty acids and lysophospholipids, modify the function of membrane proteins including ion channels. Such alterations can occur through signal transduction pathways, but may also result from "direct" effects of the metabolite on the protein. To investigate possible mechanisms for such direct effects, we examined the alterations of gramicidin channel function by lysophospholipids (LPLs): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The experiments were done on planar bilayers formed by diphytanoylphosphatidylcholine in n-decane a system where receptor- mediated effects can be excluded. At aqueous concentrations below the critical micelle concentration (CMC), LPLs can increase the dimerization constant for membrane-bound gramicidin up to 500-fold (at 2 microM). The relative potency increases as a function of the size of the polar head group, but does not seem to vary as a function of head group charge. The increased dimerization constant results primarily from an increase in the rate constant for channel formation, which can increase more than 100-fold (in the presence of LPC and LPI), whereas the channel dissociation rate constant decreases only about fivefold. The LPL effect cannot be ascribed to an increased membrane fluidity, which would give rise to an increased channel dissociation rate constant. The ability of LPC to decrease the channel dissociation rate constant varies as a function of channel length (which is always less than the membrane's equilibrium thickness): as the channel length is decreased, the potency of LPC is increased. LPC has no effect on membrane thickness or the surface tension of monolayers at the air/electrolyte interface. The bilayer-forming glycerolmonooleate does not decrease the channel dissociation rate constant. These results show that LPLs alter gramicidin channel function by altering the membrane deformation energy, and that the changes in deformation energy can be related to the molecular "shape" of the membrane-modifying compounds. Similar alterations in the mechanical properties of biological membranes may form a general mechanism by which one can alter membrane protein function.     

Regulation of G protein‐coupled receptor signaling by plasma membrane organization and endocytosis

The trafficking of G protein coupled‐receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine‐tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor‐receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin‐coated pit dynamics, and through the scaffolding adaptor protein β‐arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.      

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

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.     

Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics

The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.     

Endocytosis of Ubiquitylation‐Deficient EGFR Mutants via Clathrin‐Coated Pits is Mediated by Ubiquitylation

Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP‐2‐binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin‐dependent pathway in human duodenal adenocarcinoma HuTu‐80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin‐conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin‐binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu‐80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation‐deficient EGFR mutant was endocytosed through a limited population of epsin‐enriched clathrin‐coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP‐2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA‐MD‐231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP‐2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.      

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono‐ and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl‐MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl‐MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12‐oxo‐phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA‐containing galactolipids in the plant kingdom. While acyl‐MGDG was found to be ubiquitous in green tissue of plants ranging from non‐vascular plants to angiosperms, OPDA‐containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non‐oxidized and OPDA‐containing acyl‐MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl‐MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.     

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.      

Nonmuscle Myosin II Is a Critical Regulator of Clathrin‐Mediated Endocytosis

Variable requirements for actin during clathrin‐mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.      

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

Dynamics of clathrin and adaptor proteins during endocytosis

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for 30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis. adaptor complex; epsin; total internal reflection fluorescence microscopy     

Glycosylation and glycan interactions can serve as extracellular machinery facilitating clathrin‐independent endocytosis

In contrast to clathrin‐mediated endocytosis (CME) which is well characterized and understood, little is known about the regulation and machinery underlying clathrin‐independent endocytosis (CIE). There is also a wide variation in the requirements each individual CIE cargo has for its internalization. Recent studies have shown that CIE is affected by glycosylation and glycan interactions. We briefly review these studies and explore how these studies mesh with one another. We then discuss what this sensitivity to glycan interactions could indicate for the regulation of CIE. We address the spectrum of responses CIE has been shown to have with respect to changes in glycan interactions and attempt to reconcile disparate observations onto a shared conceptual landscape. We focus on the mechanisms by which cells can alter the glycan interactions at the plasma membrane and propose that glycosylation and glycan interactions could provide cells with a tool box with which cells can manipulate CIE. Altered glycosylation is often associated with a number of diseases and we discuss how under different disease settings, glycosylation‐based modulation of CIE could play a role in disease progression.