The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle.

The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for maintenance of the PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.

Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PHOSBP-labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling.

Millions of hibernating bats across North America have died from white-nose syndrome (WNS), an emerging disease caused by a psychrophilic (cold-loving) fungus, Pseudogymnoascus destructans, that invades their skin. Mechanisms of P. destructans invasion of bat epidermis remain obscure. Guided by our in vivo observations, we modeled hibernation with a newly generated little brown bat (Myotis lucifugus) keratinocyte cell line. We uncovered the stealth intracellular lifestyle of P. destructans, which inhibits apoptosis of keratinocytes and spreads through the cells by two epidermal growth factor receptor (EGFR)-dependent mechanisms: active penetration during torpor and induced endocytosis during arousal. Melanin of endocytosed P. destructans blocks endolysosomal maturation, facilitating P. destructans survival and germination after return to torpor. Blockade of EGFR aborts P. destructans entry into keratinocytes.

Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

The presynapse performs an essential role in brain communication via the activity-dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity-dependent calcium influx from 3 days in vitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week in vitro. However, the most intriguing discovery was that activity-dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders.

Endocytosis is a fundamental biological process that couples exocytosis to maintain the homeostasis of the plasma membrane and sustained neurotransmission. Super-resolution microscopy enables optical imaging of exocytosis and endocytosis in live cells and makes an essential contribution to understanding molecular mechanisms of endocytosis in neuronal somata and other types of cells. However, visualization of exo-endocytic events at the single vesicular level in a synapse with optical imaging remains a great challenge to reveal mechanisms governing the synaptic exo-endocytotic coupling. In this protocol, we describe the technical details of stimulated emission depletion (STED) imaging of synaptic endocytosis at the single-vesicle level, from sample preparation and microscopy calibration to data acquisition and analysis.

Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

BACKGROUND: Enteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats.
RESULTS: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11.
CONCLUSIONS: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.