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Related Concept Videos

Clathrin Coated Vesicles01:12

Clathrin Coated Vesicles

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Clathrin-coated vesicles use endocytosis to transport receptors and lysosomal hydrolases from the Golgi to the lysosome in the late secretory pathway. Clathrin-mediated endocytosis was the first described endocytic process, and Clathrin-coated vesicles remain one of the most well-studied transport vesicles. The molecular machinery that generates clathrin-coated vesicles comprises over 50 proteins that precisely coordinate vesicle formation. Cell surface receptors concentrated in indented sites...
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Fusion of Secretory Vesicles with the Plasma Membrane01:26

Fusion of Secretory Vesicles with the Plasma Membrane

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Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. Vesicles are docked and primed in preparation for the quick exocytosis of their contents in response to a stimulus. The fusion process is mainly carried out by a SNAP Receptor or SNARE complex, consisting of synaptobrevin, syntaxin-1, and SNAP-25.
In 1993, Jim Rothman proposed that the antiparallel pairing of vesicular and transmembrane SNAREs, or...
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Pinching-off of Coated Vesicles01:32

Pinching-off of Coated Vesicles

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Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming...
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Mechanisms of Membrane Domain Formation00:59

Mechanisms of Membrane Domain Formation

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Different physical properties of lipids and proteins allow them to localize and form distinct islands or domains in the membrane. Some membrane domains are formed due to protein-protein interactions, whereas others are formed due to the presence of specific lipids such as sphingolipids and sterols—for example, large proteins, such as bacteriorhodopsin, aggregate and create distinct domains.
Another mechanism for membrane domain formation involves membrane proteins interacting with...
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SNAREs and Membrane Fusion01:43

SNAREs and Membrane Fusion

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Once a transport vesicle has recognized its target organelle, the vesicular membrane needs to fuse with the target membrane to unload the cargo. Transmembrane proteins called SNAREs present on organelle membranes and their vesicles, mediate vesicle fusion.
SNAREs exist in pairs that symmetrically interact and catalyze the fusion of the lipid bilayers in vesicle and target organelle. v-SNARE in the vesicle membrane are single polypeptide chains that bind to a complementary t-SNARE, composed of 2...
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Protein Translocation Machinery on the ER Membrane01:28

Protein Translocation Machinery on the ER Membrane

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The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the...
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Related Experiment Video

Updated: Jul 17, 2025

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds
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Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

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Membrane binding and pore formation is Ca 2+ -dependent for the Clostridioides difficile binary toxin.

Dinendra L Abeyawardhane, Spiridon E Sevdalis, Kaylin A Adipietro

    Biorxiv : the Preprint Server for Biology
    |August 30, 2023
    PubMed
    Summary

    Clostridioides difficile toxin (CDT) uses calcium (Ca2+) depletion to trigger pore formation in host cells. This mechanism, distinct from pH changes, involves calcium dissociation from CDTb

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    Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
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    Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
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    Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

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    Area of Science:

    • Microbiology
    • Molecular Biology
    • Biochemistry

    Background:

    • Clostridioides difficile binary toxin (CDT) is an AB-type toxin that enters host cells via endosomal pathways.
    • Many binary toxins utilize pH changes within endosomes to activate their cell-binding components.
    • The precise mechanism by which CDT interacts with and permeabilizes endosomal membranes remains incompletely understood.

    Purpose of the Study:

    • To elucidate the molecular mechanism by which the cell-binding component of CDT, CDTb, mediates pore formation in lipid bilayers.
    • To determine the role of calcium ions (Ca2+) and pH in the activation of CDTb.
    • To identify the structural basis for CDTb-induced membrane permeabilization.

    Main Methods:

    • Cryoelectron microscopy (Cryo-EM) for structural analysis.
    • Nuclear magnetic resonance (NMR) spectroscopy to study protein dynamics and interactions.
    • Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) to assess binding and pore formation kinetics.
    • Site-directed mutagenesis to probe the function of specific residues.
    • Toxicity studies to evaluate the biological activity of CDT.

    Main Results:

    • CDTb binds to lipid bilayers and forms pores specifically upon depletion of free Ca2+ ions, not due to a decrease in pH.
    • Ca2+ dissociation from a single site in receptor binding domain 1 (RBD1) of CDTb acts as a trigger.
    • This Ca2+ dissociation induces a conformational change in CDTb, enabling membrane binding and pore formation.
    • Structural and biophysical data support a model where decreasing Ca2+ concentrations during endosomal delivery activate CDTb.

    Conclusions:

    • The mechanism of CDT-mediated cell entry relies on Ca2+ depletion-induced conformational changes in CDTb, leading to pore formation.
    • This Ca2+ -dependent mechanism differs from the pH-dependent activation observed in other binary toxins.
    • Understanding this unique activation pathway provides insights into C. difficile pathogenesis and potential therapeutic targets.