Formation Of Clathrin-coated Vesicles Research Articles

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Using quantitative microscopy approaches to study the formation of clathrin-coated vesicles.

Clathrin-coated vesicles (CCVs) are endocytic carriers that mediate the internalization of ligands and their receptors from the plasma membrane. CCVs assembly requires the coordinated recruitment of many proteins that promote receptor sorting and membrane curvature formation. Clathrin triskelia together with the adaptors AP2 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are the most represented components of CCVs. CALM supports the formation of membrane curvature that is essential for the growth of CCVs. Membrane curvature can be reached only by overcoming local plasma membrane tension that, together with the availability of CCV components, determines the type of clathrin structures that forms over the plasma membrane. We hypothesized that CALM recruitment could support the formation of CCVs even under mechanically stringent conditions, such as increased membrane tension. To investigate this, we used knock-in gene editing to obtain cells expressing fluorescently tagged CALM at endogenous levels and spinning-disc and lattice light sheet live-cell microscopy calibrated to the intensity of a single molecule to quantitatively study CALM recruitment during the formation of CCVs in normal conditions, upon membrane tension increase, and/or CALM depletion. Our results demonstrate that CALM promotes CCV completion under membrane tension increase possibly by a mechanism based on protein-protein crowding at the neck of the CCV. We also observed that CALM is differentially expressed across cell types and that given the small footprint of CALM, there is sufficient room on the CCV surface to recruit CALM molecules as needed, depending on the local membrane tension and cellular availability. In conclusion, our data suggest that the competence in clathrin-mediated endocytosis differs across cells and tissues and explain the reason why CALM is essential during embryo development but dispensable in other cellular contexts.

Shear stress shifts clathrin-coated vesicle formation to favor the flat-to-curved model in endothelial cells.

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs cells. How ECs overcome such unfavorable conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to resolve how clathrin-coated vesicles (CCVs) from in fibroblast-like cells (Cos-7) and human umbilical vein endothelial cells (HUVECs). This revealed multiple productive ways of CCV formation, supporting the flexible model of CME. We next asked whether biophysical stresses generated by blood flow could favor one mechanism of CCV formation to overcome unfavorable environment present in vasculature. Addressing this question required development of an automated MATLAB-based accelerated STAR data processing pipeline (DrSTAR) to enable the processing of multiple experimental conditions and biological replicates in a robust and reproducible environment. Moreover, DrSTAR employs a dynamic local referencing algorithm, which resolves the curvature of long-lasting CCSs. We used this new platform to examine if clathrin dynamics are altered in HUVECs grown under sheer stress. We found sheer stress led to an increase in clathrin dynamics. Surprisingly, we found this was due to an increase of flat clathrin accumulations in flow-stimulated cells, while the number of curved events remained consistent between groups. The curvature-positive events had significantly delayed curvature initiation in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Moreover, CME was more resistant to increased osmotic pressure in HUVECs grown under shear stress. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local environment and represents an important regulatory mechanism.

Computational modeling of endocytic vesicle formation imaged by simultaneous two-wavelength axial ratiometry.

Clathrin-mediated endocytosis (CME) facilitates the internalization of extracellular cargoes. However, how clathrin-coated vesicles (CCVs) form remains unclear due to the limited resolution of live-cell fluorescence microscopy and the need for sample fixation in electron microscopy. To bridge this gap, our lab developed Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy that leverages the wavelength-dependent properties of Total Internal Reflection Fluorescence (TIRF) microscopy. Dual-tagging a protein of interest with two spectrally separated fluorophores allows STAR to measure the intensity ratio and retrieve the z-position of the protein. Although the exponential decay of the evanescent wave is critical for STAR, it results in an uneven excitation of fluorescently tagged proteins. This will bias STAR measurements when dual-tagged proteins are distributed on a 3D object such as CCVs. To understand the accuracy of STAR for studying vesicle formation, we used mathematical modeling. We represented the CCV as a monodisperse sphere and used the STAR equations to calculate vesicle height (∆z) and compared it to the “ground truth” center of mass (CM). We investigated the influence of vesicle formation, radius, the number and distribution of proteins, the distance of the vesicle from the plasma membrane, and Poisson noise on STAR. This mathematical model of STAR microscopy assesses how the ∆z measured by STAR compares to the ground truth. The theoretical findings will be used to relate experimental ∆z results to vesicle morphology and eventually to develop a 3D dynamic model of CCV formation from our experimental data.

Gp35/50 mucin molecules of Trypanosoma cruzi metacyclic forms that mediate host cell invasion interact with annexin A2.

Host cell invasion is a critical step for infection by Trypanosoma cruzi, the agent of Chagas disease. In natural infection, T. cruzi metacyclic trypomastigote (MT) forms establish the first interaction with host cells. The gp35/50 mucin molecules expressed in MT have been implicated in cell invasion process, but the mechanisms involved are not well understood. We performed a series of experiments to elucidate the mode of gp35/50-mediated MT internalization. Comparing two parasite strains from genetically divergent groups, G strain (TcI) and CL strain (TcVI), expressing variant forms of mucins, we demonstrated that G strain mucins participate in MT invasion. Only G strain-derived mucins bound to HeLa cells in a receptor-dependent manner and significantly inhibited G strain MT invasion. CL strain MT internalization was not affected by mucins from either strain. HeLa cell invasion by G strain MT was associated with actin recruitment and did not rely on lysosome mobilization. To examine the involvement of annexin A2, which plays a role in actin dynamic, annexin A2-depleted HeLa cells were generated. Annexin A2-deficient cell lines were significantly more resistant than wild type controls to G strain MT invasion. In a co-immunoprecipitation assay, to check whether annexin A2 might be the receptor for mucins, protein A/G magnetic beads crosslinked with monoclonal antibody to G strain mucins were incubated with detergent extracts of MT and HeLa cells. Binding of gp35/50 mucins to annexin A2 was detected. Both G strain MT and purified mucins induced focal adhesion kinase activation in HeLa cells. By confocal immunofluorescence microscopy, colocalization of invading G strain MT with clathrin was visualized. Inhibition of clathrin-coated vesicle formation reduced parasite internalization. Taken together, our data indicate that gp35/50-mediated MT invasion is accomplished through interaction with host cell annexin A2 and clathrin-dependent endocytosis.

Structure Composition and Intracellular Transport of Clathrin-Mediated Intestinal Transmembrane Tight Junction Protein.

Tight junctions (TJs) are located in the apical region of the junctions between epithelial cells and are widely found in organs such as the brain, retina, intestinal epithelium, and endothelial system. As a mechanical barrier of the intestinal mucosa, TJs can not only maintain the integrity of intestinal epithelial cells but also maintain intestinal mucosal permeability by regulating the entry of ions and molecules into paracellular channels. Therefore, the formation disorder or integrity destruction of TJs can induce damage to the intestinal epithelial barrier, ultimately leading to the occurrence of various gastrointestinal diseases, such as inflammatory bowel disease (IBD), gastroesophageal reflux disease (GERD), and irritable bowel syndrome (IBS). However, a large number of studies have shown that TJs protein transport disorder from the endoplasmic reticulum to the apical membrane can lead to TJs formation disorder, in addition to disruption of TJs integrity caused by external pathological factors and reduction of TJs protein synthesis. In this review, we focus on the structural composition of TJs, the formation of clathrin-coated vesicles containing transmembrane TJs from the Golgi apparatus, and the transport process from the Golgi apparatus to the plasma membrane via microtubules and finally fusion with the plasma membrane. At present, the mechanism of the intracellular transport of TJ proteins remains unclear. More studies are needed in the future to focus on the sorting of TJs protein vesicles, regulation of transport processes, and recycling of TJ proteins, etc.

Chlorpromazine cooperatively induces apoptosis with tyrosine kinase inhibitors in EGFR-mutated lung cancer cell lines and restores the sensitivity to gefitinib in T790M-harboring resistant cells

We previously reported that the antipsychotic drug chlorpromazine (CPZ), which inhibits the formation of clathrin-coated vesicles (CCVs) essential for endocytosis and intracellular transport of receptor tyrosine kinase (RTK), inhibits the growth/survival of acute myeloid leukemia cells with mutated RTK (KIT D816V or FLT3-ITD) by perturbing the intracellular localization of these molecules. Here, we examined whether these findings are applicable to epidermal growth factor receptor (EGFR). CPZ dose-dependently inhibited the growth/survival of the non-small cell lung cancer (NSCLC) cell line, PC9 harboring an EGFR-activating (EGFR exon 19 deletion). In addition, CPZ not only suppressed the growth/survival of gefitinib (GEF)-resistant PC9ZD cells harboring T790 M, but also restored their sensitivities to GEF. Furthermore, CPZ overcame GEF resistance caused by Met amplification in HCC827GR cells. As for the mechanism of CPZ-induced growth suppression, we found that although CPZ hardly influenced the phosphorylation of EGFR, it effectively reduced the phosphorylation of ERK and AKT. When utilized in combination with trametinib (a MEK inhibitor), dabrafenib (an RAF inhibitor), and everolimus (an mTOR inhibitor), CPZ suppressed the growth of PC9ZD cells cooperatively with everolimus but not with trametinib or dabrafenib. Immunofluorescent staining revealed that EGFR shows a perinuclear pattern and was intensely colocalized with the late endosome marker, Rab11. However, after CPZ treatment, EGFR was unevenly distributed in the cells, and colocalization with the early endosome marker Rab5 and EEA1 became more apparent, indicating that CPZ disrupted the intracellular transport of EGFR. These results suggest that CPZ has therapeutic potential for NSCLC with mutated EGFR by a novel mechanism different from conventional TKIs alone or in combination with other agents.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Advances in structural, spatial, and temporal mechanics of plant endocytosis.

Endocytic trafficking underlies processes essential for plant growth and development, including the perception of and response to abiotic and extracellular stimuli, post-Golgi and exocytic trafficking, and cytokinesis. Protein adaptors and regulatory factors of clathrin-mediated endocytosis that contribute to the formation of endocytic clathrin-coated vesicles are evolutionarily conserved. Yet, work of the last ten years has identified differences between the endocytic mechanisms of plants and Opisthokonts involving the endocytic adaptor TPLATE complex, the requirement of actin during CME, and the function of clathrin-independent endocytosis in the uptake of plant-specific plasma membrane proteins. Here, we review clathrin-mediated and -independent pathways in plants and describe recent advances enabled by new proteomic and imaging methods, and conditional perturbation of endocytosis. In addition, we summarize the formation and trafficking of clathrin-coated vesicles based on temporal and structural data garnered from high-resolution quantitative imaging studies. Finally, new information about the cross-talk between endocytosis and other endomembrane trafficking pathways and organelles will also be discussed.

Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components.

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

Imaging the dynamics of vesicle formation supports the flexible model of clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) facilitates the internalization of many cargoes. Clathrin polymerization, endocytic accessory proteins recruitment, and changes in plasma membrane architecture are necessary steps to mediate the formation of clathrin-coated vesicles (CCVs). However, simultaneous analysis of clathrin dynamics and vesicle shape in living cells is challenging due to the limited axial resolution of fluorescence microscopes, and the heterogeneity and dynamicity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies.

An online gathering about the latest on molecular membrane biology

An online gathering about the latest on molecular membrane biology

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

Regulation of Megalin membrane traffic by AMP‐activated protein kinase in kidney proximal tubule epithelial cells

Megalin is a transmembrane receptor protein that belongs to a family of proteins known as low-density lipoprotein receptors (LDLRs) that functions in reabsorption of various molecules from the urine in the proximal tubule epithelial cells (PTECs) of the kidney. Disruption of the regulation of Megalin is associated with various stages of renal disease. Megalin mediates reabsorption by functioning as a scavenger receptor: binding of molecules (cargo) within the kidney proximal tubule lumen to the ectodomain of Megalin followed by internalization of cargo-bound Megalin via clathrin-mediated endocytosis, the first stage in transcytosis of cargo molecules across the kidney epithelium. Kidney proximal tubule cells experience metabolic stress due to low oxygen availability. We have recently shown that metabolic stress resulting in activation of AMP-activated protein kinase (AMPK) regulates the endocytosis of a large number of cell surface proteins, including that of beta1-integrin, that has a similar requirement for dab2 for internalization as does Megalin. How AMPK may regulate Megalin internalization in kidney PTECs is not well understood. Megalin internalization occurs via recruitment of the receptor to clathrin-coated pits (CCPs), which are ~100 nm structures comprised of clathrin and other proteins at the cell surface. Upon assembly and receptor recruitment, CCPs mediate membrane budding and eventually scission, leading to an internalized clathrin-coated vesicle (CCV). Megalin recruitment to CCPs is mediated by the clathrin adaptor protein dab2 that binds to NPXY motif that is present on Megalin and other LDLR family proteins. Here, we examine how metabolic stress and AMPK regulate the formation of CCPs and the dab2-dependent recruitment of Megalin and related receptors for internalization. Using cells engineered to express fluorescently tagged clathrin and other proteins, together with total internal reflection fluorescence microscopy and automated image analysis, we uncovered that AMPK activation triggers a reduction in the size and rate of initiation of CCPs, but an increased efficiency of formation of clathrin-coated vesicles. In addition, AMPK activation resulted in a robust increase in the recruitment of dab2 to CCPs. We have complemented this approach with assays that monitor the cell surface membrane traffic of Megalin and related receptors such as integrins. Collectively, these studies reveal a novel signaling pathway that mediates the regulation of the clathrin internalization of several receptors by AMPK and metabolic stress. A better understanding of signals responsive to cell metabolism and metabolic stress in kidney PTECs and how these lead to remodeling of endocytic membrane traffic may reveal novel ways to treat certain forms of kidney diseases.

Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome.

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.

Evolving models for assembling and shaping clathrin-coated pits.

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

Wbox2: A clathrin terminal domain-derived peptide inhibitor of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane-permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.

Epsin but not AP-2 supports reconstitution of endocytic clathrin-coated vesicles.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based invitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia.

Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.