Construction Materials Are Carried in the Vesicles and Are Continually Deposited

Coated Vesicle

These coated vesicles rapidly lose their clathrin coats through the action of a chaperone from the Hsp70 family, an event that facilitates local fusion with early endosomes (EEs).

From: Basic Neurochemistry (Eighth Edition) , 2012

Complementary Strategies to Understand Virus Structure and Function

Martin Obr , Florian K.M. Schur , in Advances in Virus Research, 2019

5.3 Structural studies of intracellular transport vesicles by cryo-ET and subtomogram averaging

Coated vesicles and enveloped viruses share certain similar mechanisms of structural assembly and can be considered to perform homologous processes. Both are higher-order pleomorphic structures, assembled to transport their selectively recruited cargo to distinct target locations. Hence, results obtained via cryo-ET and subtomogram averaging for coated vesicles demonstrate the possibilities that these methods also have for pleomorphic viruses.

Cargo-selective trafficking vesicles can derive from the plasma membrane, Golgi, ER or endosomal membranes and their formation is driven by assembly of different classes of coat proteins, i.e., Clathrin, COPI, COPII, or retromer complex, respectively. While clathrin coats can more routinely be determined via single-particle approaches due to their more regular structure (Fotin et al., 2006), other vesicular coats display significant structural variability, which requires cryo-ET and subtomogram averaging for visualizing interactions within the coat assembly. Recent work was successful in describing in vitro the intricate coat architecture of part of the COPI and retromer coat at subnanometer resolution (Dodonova et al., 2017; Kovtun et al., 2018) and the COPII coat at resolutions beyond 5   Å (Hutchings et al., 2018). In situ experiments visualizing COPI and retromer coated vesicles also confirmed the in vitro observations (Bykov et al., 2017; Kovtun et al., 2018). These results underscore the potential and versatility offered by cryo-ET and subtomogram averaging to elucidate important protein-protein interactions of relevant large macromolecular assemblies.

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Exocytosis

Merri Lynn Casem BA, PhD , in Case Studies in Cell Biology, 2016

Introduction

The pathway of exocytosis starts with the synthesis of a protein on the rough endoplasmic reticulum (RER). From there, the protein must travel through each of the compartments of the Golgi complex, finally arriving at the trans Golgi network (TGN) before being transported to the plasma membrane, lysosome, or vacuole. The collections of membrane-bound organelles that participate in exocytosis are part of the endomembrane system . It is generally accepted that proteins move along the exocytic pathway inside small transport vesicles. Transport vesicles are formed through a process of "budding" from the membrane of the RER or Golgi, capturing protein cargo in the process. Fusion of the vesicles with the membrane of the target organelle releases the protein cargo into that compartment. These vesicles carry a collection of peripheral proteins on the cytoplasmic side of their membranes, forming a "coat." COPI and COPII are the two distinct types of coated vesicles associated with transport between the RER and the Golgi complex.

Three models were developed to explain the role of coated vesicles in exocytosis. The first model proposed that COPII vesicles were responsible for the anterograde (forward) movement of materials in the cell. The second model proposed that both COPI and COPII vesicles moved material in parallel, independent of anterograde pathways. The third model proposed a sequential role for coated vesicles. In this model the COPII vesicles carry material from the RER to an intermediate compartment. COPI vesicles are then formed from the intermediate compartment to carry the material to the cis Golgi. The work presented in this case study seeks to determine which of these models is correct.

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Research/review the types of proteins that are synthesized on the RER.

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Summarize how a newly synthesized protein is modified from the time it enters the lumen of the RER to the time it reaches the trans Golgi network.

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Describe another example of a coated vesicle found in cells.

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G Protein Pathways

Jun Kuai , Richard A. Kahn , in Methods in Enzymology, 2002

Coat Protein Recruitment Assay

ARF is found to copurify with coatomer-coated vesicles that are generated in vitro by the incubation of Golgi-enriched membranes with cytosol and GTPγS. ⁸ The ARF dependence of coatomer recruitment can be assayed in vitro with purified myrARF, ARF-free cytosol, and Golgi-enriched membranes. The binding of coatomer to the Golgi membrane is detected by immunoblot using β-COP antibody (EAGE ²⁷ ). Golgi membranes from Chinese hamster ovary (CHO) cells gives a lower background than membranes from other sources, for example, rat liver. The most variable aspect of this assay is the enriched Golgi membranes, as some preparations are less active than others. Controls for electrophoretic transfer to nitrocellulose and an internal control are required for quantification or comparison between experiments.

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Organizational Cell Biology

J.A. Swanson , S. Yoshida , in Encyclopedia of Cell Biology, 2016

Other Necessary Molecules

Dynamin

Dynamin facilitates scission of clathrin-coated pits into coated vesicles ( Schmid and Frolov, 2011). Its contribution to macropinocytosis is unsettled. Inhibition of dynamin 2 function inhibits macropinosome formation (Liu et al., 2008). However, Cao et al., found that dynamin was not required for macropinocytosis (Cao et al., 2007). Dynamin 2 interacts with cortactin and Arp2/3 in the formation of CDR (Krueger et al., 2003).

SNX proteins

The nascent macropinosome transforms from an irregular profile to a more rounded morphology with thin tubulovesicular extensions radiating along cytoplasmic microtubules. These extensions are formed by SNX-family proteins, which are regulated by PtdIns3P or PtdIns(3,4)P₂. The morphological changes may increase hydrostatic pressure inside the macropinosome (Kerr et al., 2006). SNX1, 5, 9, 18, and 33 affect macropinosome formation or maturation (Lim et al., 2012; Wang et al., 2010).

Microtubules

Microtubule-depolymerizing drugs inhibit macropinocytosis by unknown mechanisms (Racoosin and Swanson, 1992; Swanson et al., 1987). Conversely, microtubule destabilization promotes macropinosome formation. Histone deacetylase-6 (HDAC6) removes acetyl groups from tubulin in microtubules, leading to microtubule destabilization. HDAC6 localizes to ruffles and macropinosomes and stimulates macropinocytosis (Gao et al., 2007). Moreover, a Shigella effector protein stimulates macropinocytosis for bacteria entry into host cells using an effector protein which destabilizes microtubules (Yoshida et al., 2002). This suggests that dynamic microtubules are required for macropinosome formation.

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Volume 2

Bernardo Ortega , Paul A. Welling , in Physiology of the Gastrointestinal Tract (Sixth Edition), 2018

45.6.2 Vesicle Formation and Budding

Sorting signal recognition is often coupled to vesicle formation. Coated vesicles have a critical role in concentrating, packaging, and shuttling cargo between different intracellular compartments and plasma membrane domains (reviewed in Refs. 186,187). Three different types of coated vesicles have been described based on their compositions and intracellular compartments where they originate. The CCVs mediate transport between TGN, endosomes, and the plasma membrane. Coatomer complex I (COPI) and coatomer complex II (COPII) coated vesicles mediate intra-Golgi or Golgi to ER retrograde transport, and ER to Golgi transport, respectively. Formation of all the three types of coated vesicles shares a common sequential mechanism, whereby coat subunits assemble on the membrane and recognize cargo. As cargo and coat proteins concentrate, the underlying membrane deforms, buds from the parent membrane and is cut off, forming a coated vesicle.

The three types of coated vesicles recognize specific sets of cargo and have different protein and lipid compositions. The CCVs are composed of two cytosolic protein complexes: clathrin triskelia and heterotetrameric adaptor complexes (AP-1 and AP-2). The COPII vesicles have a similar structure, but the two protein complexes are Sec13-31 and Sec23-24. The COPI coat is somewhat more simple and contains a single multisubunit coatomer complex. Despite their differences, all these protein complexes have a common tendency to self-assemble into empty spherical cages, ^188–190 similar to the outer cage found in vesicles in vivo. Cage subunits cannot bind directly to the membrane, and require an inner adaptor layer capable of simultaneous interaction with cage protein complexes, sorting motifs in cargo proteins, and the compartment membrane via negatively charged lipids. ^187,191

Sculpting the membrane into the curved shape of a vesicle is an energy-intensive process that requires recruitment of additional proteins. Two related GTPases, Arf1 in COPI vesicles, and Sar1 in COPII vesicles, appear to manage both coat recruitment and curvature generation. ¹⁸⁶ A similar role is played by the GTPase dynamin in CCV, although additional proteins such as amphiphysin, epsin, endophilin, and members of the sorting nexin family are also recruited and play a role in binding to members of the core component, or recognizing, generating, and stabilizing the membrane curvature. ^186,192 The GDP-bound Arf1 associates with the membrane, and upon GTP binding it undergoes a conformational change. This change exposes a short amphipathic N-terminal helix that gets inserted into the lipid bilayer, causing an asymmetric expansion of the outer versus the inner leaflet, allowing Arf1 to remodel the membrane into highly curved structures. ^193,194

Similar to Arf1, Sar1 action also follow a cycle of GTP binding, followed by association to the membrane, recruitment of the adaptor complex Sec23-24, and a conformational change that induces curvature of the membrane. ^195–197 Formation of CCVs appears to be more complex, with multiple accessory proteins interacting with the membrane through structural elements such as BAR (Bin, amphiphysin, Rvs) or ENTH (epsin N-terminal homology) domains. ¹⁹⁸ In addition, dynamin contains a PH domain that provides docking sites for negatively charged lipid headgroups. The GTP-bound dynamin readily associates with the membrane and inserts its PH domain in the lipid bilayer, inducing curvature of the membrane. The scaffolding role of dynamin allows this protein to interact with multiple SH3-domain containing accessory proteins, which in turn contribute to stabilize the membrane curvature. ^199,200 Finally, dynamin has also been shown to play an important role in vesicle scission and release from the donor membrane, a process also known to require GTP hydrolysis. ¹⁹⁷

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Intracellular trafficking of bacterial and plant protein toxins

Christophe Lamaze , Ludger Johannes , in The Comprehensive Sourcebook of Bacterial Protein Toxins (Third Edition), 2006

MULTIPLE ENDOCYTIC PATHWAYS OPERATE IN MAMMALIAN CELLS

It was long assumed that endocytosis involving the formation of clathrin-coated pits and vesicles was the only efficient process for the internalization of exogenous molecules bound to their specific transmembrane receptors. Several recent reviews have described the enormous progress that has been made in the last 10 years in the molecular understanding of the clathrin-dependent pathway (Brodsky et al., 2001; Owen et al., 2004; Rappoport et al., 2004). Nevertheless, it has been known for almost 20 years that some toxins can enter cells by means other than the well-described clathrin-dependent endocytosis (Montesano et al., 1982; Moya et al., 1985; Tran et al., 1987). Since these pioneering studies, our understanding of the molecular mechanisms involved in these so-called clathrin-independent endocytic pathways has not progressed much. Paradoxically, it is the recent progress in the understanding of clathrin-dependent endocytosis that has made the largest contribution to our understanding of clathrin-independent endocytosis (see Figure 8.1) (Johannes and Lamaze, 2002). There is no longer any doubt about the existence and the importance of these alternative pathways, which constitute a major portal of entry for many toxins. After a brief summary of the main characteristics of each of the known endocytic systems operating in mammalian cells, we will examine how different toxins use these routes to enter cells. It should be noted, however, that most toxins can use several entry pathways and that endocytic plasticity is a trait common to many known toxins.

FIGURE 8.1. Multiple endocytic pathways operate in mammalian cells. Besides the best-characterized clathrin-dependent endocytosis (II), several clathrin-independent endocytic pathways have been identified by molecular and morphological means and represent potential carriers for toxins delivery in the cell. Macropinocytosis (I) is a transient response to growth factors and mitogenic agents that contributes to fluid phase uptake through the formation of large vesicles of heterogeneous size. Non-coated invaginations (III) that pinch off to form smooth vesicles are commonly observed by conventional microscopy in different cell types and participate to fluid phase uptake. Certain lipid-based membrane microdomains including rafts (IV) and caveolae (V) are enriched in cholesterol and glycosphingolipids. Caveolin is an integral caveolar protein that gives a striated aspect to caveolae and allows us to distinguish them from other smooth invaginations. Caveolar endocytosis is mostly induced and can lead to the formation of "caveosomes" from which endocytosed molecules (SV40, CTx) could reach the endoplasmic reticulum and/or the nucleus. While it is not known whether lipid rafts tubulate or vesiculate to form smooth vesicles similar to (III), the vesicle budding GTPase dynamin is involved in clathrin-dependent endocytosis and some, but not all clathin-independent endocytosis. Black arrows represent established intracellular tracks whereas pathways with question marks have been less investigated.

(adapted from Johannes and Lamaze, 2002) Copyright © 2002

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Antigen Uptake by M Cells for Effective Mucosal Vaccines

Marian R. Neutra , Jean-Pierre Kraehenbuhl , in Mucosal Vaccines, 1996

B Endocytosis and Transcytosis

M cells take up macromolecules, particles and microorganisms by adsorptive endocytosis via clathrin-coated pits and vesicles (Neutra et al., 1987; Sicinski et al., 1990), fluid-phase endocytosis (Bockman and Cooper, 1973; Owen, 1977), and phagocytosis involving extension of cellular processes and reorganization of submembrane actin assemblies (Jones et al., 1994; Neutra et al., 1994a). All of these uptake mechanisms result in transport of foreign material into endosomal tubules and vesicles and large multivesicular bodies that lie apically, between the apical membrane and the intraepithelial pocket (Neutra and Kraehenbuhl, 1992). The large vesicles contain the late endosome/lysosome membrane marker lgp120 and generate an acidic internal milieu (Allan et al., 1993). Immunocytochemical analysis revealed the presense of an endosomal protease, cat-hepsin E in rabbit M cells (Finzi et al., 1993) but the possible presence of other endosomal hydrolases in M-cell transport vesicles has not yet been examined. MHC class II antigens on M-cell membranes have been documented in subpopulations of M cells of some species (Allan et al., 1993). Because of the complexity of these tissues and the lack of an in vitro M-cell system, however, it is not yet known to what extent endocytosed materials are degraded during transepithelial transport, or whether M cells participate in the processing and presentation of antigens.

Adherent macromolecules or particles bound to the apical plasma membranes of M cells are efficiently endocytosed or phagocytosed (Neutra et al., 1987). It follows that pathogens or vaccines that can bind selectively to M cells would be most effective in mucosal invasion and induction of mucosal immune responses; this assumption underlies many of the current approaches to vaccine design discussed below. Macro-molecules and particles that are endocytosed by M cells can be released at the pocket membrane as rapidly as 10–15   min later. Rapid binding and uptake of polystyrene or latex beads that adhere to M cells (Pappo and Ermak, 1989), along with the lectin binding studies described above, suggest that particles or microorganisms with hydrophobic surfaces as well as those with appropriate lectin-like adhesins could interact with M-cell surfaces. Lectins that fail to recognize mucins and other cells could be highly M-cell-selective, whereas hydrophobic particles would also interact with mucus and the glycocalyx of enterocytes on villi, and this would tend to reduce the efficiency of M-cell uptake.

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Imaging and Spectroscopic Analysis of Living Cells

Comert Kural , Tom Kirchhausen , in Methods in Enzymology, 2012

2 Dynamics of Clathrin Assembly

Our current picture of coated pit formation derives primarily from analysis by live-cell imaging of coated pits and vesicles at the plasma membrane (Fig. 4.1). The most important structural components of the assembly are clathrin and the AP-2 (α-β2-μ2-σ2) heterotetrameric adaptor complex. A number of accessory proteins associate with coated pits at specific stages of assembly/disassembly (Henne et al., 2010; Reider et al., 2009; Toshima et al., 2006; Traub, 2009). Eps15, epsin, FCHo1/2, and intersectin form an interacting complex that appears to be localized at the rim of a coated pit (Henne et al., 2010; Reider et al., 2009; Saffarian et al., 2009; Tebar et al., 1996; Traub and Wendland, 2010 ). This "rim complex" accumulates during early stages of coated pit assembly, but its components are excluded from a budded coated vesicle. Dynamin, the GTPase that drives membrane scission, accumulates both gradually during pit assembly and in a burst following clathrin lattice completion ( Ehrlich et al., 2004; Loerke et al., 2009; Macia et al., 2006; Rappoport and Simon, 2003). Auxilin and Hsc70 arrive following scission, to direct uncoating (Lee et al., 2006; Massol et al., 2006). Hip1R, which binds clathrin light chains, recruits actin, required in some instances for coated vesicle maturation and budding (Ferguson et al., 2009; Merrifield et al., 2002, 2004; Saffarian et al., 2009).

Figure 4.1. Coated pit formation proceeds by sequential addition of clathrin triskelions to an initial nucleus, generating a sharply curved coat; adaptor-mediated interactions with membrane-bound proteins (and lipids) deform the underlying membrane; dynamin mediates scission when the deformation has created a suitably narrow neck; auxilin, which arrives immediately following scission, recruits the uncoating ATPase, Hsc70. Under conditions of membrane tension (hyposmolarity, cell stretching, apical membranes of polarized cells, elongated cargo), coated pit maturation requires the formation of short-branched actin filaments; by contrast, actin polymerization does not generally accompany the assembly or budding of coated pits in membranes without tension. Continuous line, plasma membrane; dashed stripe, a clathrin coat (clathrin plus AP-2 adaptor); red rods, rim proteins (Eps15, epsin, FHCo1/2, SIG1a); gray lines, short-branched actin polymers plus the Arp2/3 complex, cortactin, N-Wasp; green bar dynamin; blue bar, F-BAR containing proteins; green dots, uncoating ATP Hsc70; red lines, auxilin. We use stable and transient expression of recombinant, fluorescently-tagged constructs to follow the dynamic behavior of plasma membrane structures containing different combinations of clathrin, AP-2, auxilin, Arp2/3 complex, cortactin, dynamin, etc. We mostly use TIRF and spinning disc confocal microscopy to obtain live-cell imaging data.

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Lipids

Marnix Wieffer , ... Michael Krauß , in Methods in Cell Biology, 2012

Abstract

Clathrin plays key roles in endocytic and endo-lysosomal membrane dynamics by facilitating the formation of coated vesicles at the plasma membrane and at the trans-Golgi network (TGN)/endosomal boundary. Assembly of the clathrin lattice critically depends on adaptor proteins and accessory proteins, which connect the clathrin scaffold to the membrane and to transmembrane cargo including receptors, transporters, channels, and SNARE proteins. The recruitment of adaptor proteins to membrane surfaces is triggered by coincidence-detection mechanisms involving phosphoinositides (PIs), cargo proteins, and in many cases small GTPases. To tightly regulate coat formation, there is extensive cross-talk between PI-metabolizing enzymes and adaptor proteins. One of the best studied examples is the endocytic clathrin adaptor complex AP-2, which binds plasma membrane-enriched PI(4,5)P ₂. In neurons, PI(4,5)P₂ is synthesized from PI(4)P primarily by the γ-isoform of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIPKIγ), whose enzymatic activity is regulated by direct binding to, amongst others, the small GTPase Arf6 and AP-2. Cargo-bound AP-2 potently stimulates PIPK1γ activity and thereby drives AP-2-membrane interactions. This feed-forward loop is thought to facilitate membrane translocation of additional AP-2 molecules and concomitantly clathrin, but also of endocytic accessory proteins, many of which directly associate with PI(4,5)P₂. It is likely that similar mechanisms support the formation of coated vesicles at the trans-Golgi network (TGN) and on endosomes, involving PI(4)P and PI(3)P respectively, but detailed knowledge is lacking to date. To explore how coat proteins regulate kinase activity, assays are needed to sensitively detect subtle changes in PI synthesis and to discriminate between the various PI species. Here we describe a sensitive and specific radioactivity-based assay to measure PI kinase activity.

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Clathrin and Clathrin-Adaptors

R.M. Twyman , in Encyclopedia of Neuroscience, 2009

Regulation of Clathrin Adaptors: Interactions and Functions

It is clear that clathrin adaptors play the most significant role in the formation of clathrin-coated pits and vesicles, which suggests they are the prime target for upstream regulation. There is evidence that posttranslational modification is used to regulate the activity and potential interactions of these proteins, allowing them to be controlled by upstream signaling pathways.

One of the most abundant forms of regulatory modification in eukaryotic cells is the phosphorylation of tyrosine, serine, and threonine residues. A relevant example is the phosphorylation of AP2 subunit μ2 by protein kinase AP2-associated kinase (AAK1). The phosphorylation of a single threonine residue within the lipid-binding domain of subunit μ2 has a profound effect on its ability to interact with PtdIns(_4,5)P₂ head groups, increasing its affinity over 100-fold. The additional negative charge introduced by phosphorylation is thought to cause a conformation change that alters the relative spacing of the cargo-binding and lipid-binding domains, allowing both sites to bind their respective ligands simultaneously, and leading to a stable complex at the membrane. Other adaptor proteins are negatively regulated by phosphorylation. For example, epsin phosphorylation prevents endocytosis, presumably because the additional negatively charged residue interferes with crucial interactions with the endocytic machinery.

As already stated, the ubiquitylation of cargo molecules can be important for the initiation of vesicle formation by epsin, but an additional complication is that such adaptors can also undergo ubiquitylation at a site distinct from their ubiquitin-binding domain (UBD). In vitro, it has been shown that the presence of a single ubiquitin moiety on epsin inhibits its ability to bind to accessory proteins and ubiquitinylated cargo, therefore reducing the efficiency of vesicle formation. It is possible that the ubiquitin moiety interacts with the UBD on the same protein and that this intramolecular binding outcompetes such interactions with other molecules, whereas the UBD on the nonubiquitinylated form of the protein is more likely to partake in intermolecular interactions.

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