GO:0072583 clathrin-dependent endocytosis: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0072583 clathrin-dependent endocytosis (CME) is the biological process in which extracellular material is internalized through clathrin-coated pits that pinch off to form clathrin-coated endocytic vesicles.
• CME is the major route for uptake of nutrients, signaling receptors, adhesion molecules, and pathogens, and it controls receptor-ligand signaling.
• Core machinery includes clathrin triskelia, AP-2 adaptor complexes, dynamin, and accessory factors such as amphiphysin, synaptojanin, and auxilin that drive coat assembly, membrane scission, and uncoating.
• CME regulates G protein-coupled receptor (GPCR) signaling, integrin turnover, synaptic vesicle recycling, and HER2 internalization in cancer.
• Dysregulated CME is linked to cancer progression, bacterial pathogenesis, and altered receptor signaling, making it a target for mechanistic and therapeutic studies.
• CRISPR knockout, point-mutation, knock-in, and overexpression models enable causal dissection of CME genes and their disease relevance.
Description
Clathrin-dependent endocytosis (GO:0072583) is a fundamental cellular process by which cells internalize extracellular cargo and plasma membrane proteins through clathrin-coated pits that invaginate and pinch off to form clathrin-coated vesicles. This pathway is essential for nutrient uptake, receptor downregulation, synaptic vesicle recycling, and pathogen entry, and it is one of the best-characterized membrane trafficking routes in eukaryotic cells. Because CME controls the surface availability of signaling receptors, it directly influences cell signaling, adhesion, and proliferation. Researchers study CME to understand how cells communicate with their environment, how pathogens exploit host machinery, and how defects in endocytic trafficking contribute to cancer and other diseases. The pathway is highly conserved and involves a defined set of core proteins, making it amenable to genetic and biochemical dissection. Advances in CRISPR genome editing now allow precise manipulation of CME genes to test their causal roles in health and disease.
clathrin-dependent endocytosis At A Glance
| GO ID | GO:0072583 |
|---|---|
| GO term | clathrin-dependent endocytosis |
| Ontology | biological_process |
| Synonym | clathrin-mediated endocytosis; CME; clathrin coated pit-dependent endocytosis |
| Major function | Internalization of extracellular material and plasma membrane proteins via clathrin-coated pits and vesicles |
| Key machinery | Clathrin triskelia, AP-2 adaptor complex, dynamin, accessory factors (amphiphysin, synaptojanin, auxilin) |
| Cellular location | Plasma membrane, clathrin-coated pits, clathrin-coated vesicles, endosomes |
| Biological context | Nutrient uptake, receptor signaling, synaptic vesicle recycling, pathogen entry |
| Disease relevance | Cancer, bacterial infection, neurological disorders |
What Is GO:0072583?
According to the Gene Ontology, clathrin-dependent endocytosis (GO:0072583) is an endocytosis process that begins when material is taken up into clathrin-coated pits, which then pinch off to form clathrin-coated endocytic vesicles. In other words, it is a receptor-mediated uptake mechanism in which cargo is concentrated into small membrane invaginations coated with the protein clathrin, followed by scission of the vesicle from the plasma membrane and subsequent uncoating. This process is synonymous with clathrin-mediated endocytosis (CME) and clathrin coated pit-dependent endocytosis.
Why Is clathrin-dependent endocytosis Important in Cell Biology?
Clathrin-dependent endocytosis is a central hub for cellular communication and homeostasis, controlling the internalization of nutrients, signaling receptors, adhesion molecules, and pathogens. It is the primary mechanism by which cells downregulate activated receptors, thereby shaping the duration and intensity of signaling cascades. In the nervous system, CME is essential for synaptic vesicle recycling and neurotransmitter release. Pathogens such as bacteria exploit CME to gain entry into host cells, making it a key interface in infection biology. In cancer, altered CME can affect receptor tyrosine kinase trafficking, as shown for HER2 internalization in non-small cell lung cancer. Consequently, understanding CME is critical for basic cell biology, neurobiology, infectious disease, and oncology research.
• Controls uptake of nutrients and signaling molecules, influencing cell growth and survival.
• Regulates G protein-coupled receptor (GPCR) signaling by mediating receptor internalization and recycling.
• Modulates integrin turnover and cell adhesion, affecting migration and tissue architecture.
• Essential for synaptic vesicle endocytosis and neurotransmission.
• Mediates internalization of bacterial pathogens, contributing to infection.
• Regulates receptor-ligand signaling through RLIP and other accessory proteins.
• Influences HER2 internalization and downstream signaling in cancer cells.
• Provides a target for therapeutic intervention in cancer and infectious diseases.
• Serves as a paradigm for studying membrane trafficking and protein-lipid interactions.
• Enables precise genetic dissection using CRISPR-based models.
What Happens During clathrin-dependent endocytosis?
Initiation and cargo selection
In simple terms: The cell decides what to bring in by marking cargo with tags that attract the clathrin coat.
Clathrin-dependent endocytosis begins when cargo molecules, such as receptors or nutrients, are recognized at the plasma membrane by adaptor proteins, most notably the AP-2 complex. These adaptors link cargo to the forming clathrin coat and help concentrate cargo into nascent pits. The process is highly selective and can be regulated by accessory factors that control receptor-ligand signaling.
Clathrin coat assembly and membrane invagination
In simple terms: Clathrin molecules assemble into a basket-like coat that bends the membrane inward.
Clathrin triskelia, composed of three heavy and three light chains, assemble into a polyhedral lattice on the cytoplasmic face of the plasma membrane. This assembly, aided by adaptors and accessory proteins, drives membrane invagination to form a clathrin-coated pit. The mechanical forces and energy requirements of this step have been studied in detail, revealing constraints and frustration in the pathway.
Membrane scission and vesicle formation
In simple terms: The neck of the pit is cut to release a coated vesicle inside the cell.
Once the coated pit is deeply invaginated, the GTPase dynamin assembles at the neck and mediates membrane scission, releasing a clathrin-coated vesicle into the cytoplasm. This step requires GTP hydrolysis and is regulated by accessory factors such as amphiphysin and endophilin. The newly formed vesicle is still coated with clathrin and associated proteins.
Uncoating and vesicle trafficking
In simple terms: The clathrin coat is removed so the vesicle can fuse with its target compartment.
After scission, the clathrin coat is disassembled through the action of accessory factors including auxilin and synaptojanin, which recruit the ATPase Hsc70 to remove clathrin. The uncoated vesicle then fuses with early endosomes, delivering its cargo for sorting, recycling, or degradation. This uncoating step is essential for subsequent membrane fusion events.
Regulation by accessory and signaling proteins
In simple terms: Many helper proteins fine-tune the speed and specificity of the whole process.
Accessory factors such as RLIP control receptor-ligand signaling by regulating clathrin-dependent endocytosis. In synaptic terminals, a specialized set of accessory proteins ensures rapid and efficient vesicle recycling. Additionally, mechanical forces and integrin endocytosis can tune CME through force-dependent regulation. These regulatory layers allow CME to adapt to different cellular contexts and cargoes.
Key Genes Involved in GO:0072583 clathrin-dependent endocytosis
The following genes and proteins are core components or regulators of clathrin-dependent endocytosis, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CLTC | Clathrin heavy chain; major structural component of the coat | Essential for coat assembly and vesicle formation |
| CLTA | Clathrin light chain A; regulates coat assembly and disassembly | Modulates clathrin lattice dynamics |
| CLTB | Clathrin light chain B; regulates coat assembly and disassembly | Modulates clathrin lattice dynamics |
| AP2A1 | AP-2 adaptor complex subunit alpha 1; links cargo to clathrin | Cargo selection and coat recruitment |
| AP2B1 | AP-2 adaptor complex subunit beta 1; links cargo to clathrin | Cargo selection and coat recruitment |
| AP2M1 | AP-2 adaptor complex subunit mu 1; binds cargo sorting signals | Cargo recognition |
| DNM1 | Dynamin 1; GTPase mediating membrane scission | Vesicle scission |
| DNM2 | Dynamin 2; GTPase mediating membrane scission | Vesicle scission |
| SH3GL2 | Endophilin A1; accessory factor in membrane curvature and scission | Synaptic vesicle endocytosis |
| BIN1 | Amphiphysin 1; accessory factor in membrane curvature and dynamin recruitment | Synaptic vesicle endocytosis |
| SYNJ1 | Synaptojanin 1; phosphoinositide phosphatase involved in uncoating | Uncoating and synaptic vesicle recycling |
| DNAJC6 | Auxilin; recruits Hsc70 for clathrin uncoating | Uncoating |
| HSPA8 | Hsc70; ATPase that disassembles clathrin coats | Uncoating |
| RLIP | RalBP1; regulates receptor-ligand signaling and CME | Signaling regulation |
| ITGB1 | Integrin beta 1; cargo for CME, regulates adhesion | Force tuning and adhesion |
| ERBB2 | HER2 receptor tyrosine kinase; cargo for CME | Cancer signaling and internalization |
| PIP5K1C | Phosphatidylinositol-4-phosphate 5-kinase; generates PI(4,5)P2 for CME | Coat assembly and cargo recruitment |
How Is clathrin-dependent endocytosis Regulated?
Clathrin-dependent endocytosis is regulated at multiple levels, including by accessory proteins that control receptor-ligand signaling. RLIP (RalBP1) has been shown to regulate CME and thereby modulate signaling downstream of receptor-ligand interactions. Mechanical forces also tune CME, as demonstrated for integrin endocytosis, where force-dependent regulation affects adhesion dynamics. In synaptic terminals, a specialized set of accessory factors ensures rapid and efficient vesicle recycling. Additionally, the pathway is influenced by the availability of phosphoinositides and the activity of kinases and phosphatases that control coat assembly and uncoating.
clathrin-dependent endocytosis and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ERBB2 | Non-small cell lung cancer with HER2 mutations | Knock-in of HER2 mutations in lung cancer cell lines; CME assays |
| RLIP | Cancer signaling and receptor-ligand regulation | Knockout or knockdown in cancer cell lines; signaling assays |
| SYNJ1 | Neurological disorders, synaptic dysfunction | Knockout in neurons; synaptic vesicle recycling assays |
| DNAJC6 | Neurological disorders, uncoating defects | Knockout in neuronal cells; clathrin uncoating assays |
| ITGB1 | Cell adhesion and migration | Knockout or point mutation; force-dependent endocytosis assays |
Cancer
Altered clathrin-dependent endocytosis can affect the internalization and signaling of receptor tyrosine kinases such as HER2. In non-small cell lung cancer positive for HER2 mutations, enhanced HER2 internalization by CME has been observed, suggesting that CME modulates oncogenic signaling. RLIP, a regulator of CME, controls receptor-ligand signaling and may influence cancer progression.
Bacterial infection
Several bacterial pathogens exploit clathrin-dependent endocytosis to enter host cells. The role of CME in bacterial internalization has been documented, highlighting it as a target for anti-infective strategies.
Neurological disorders
CME is essential for synaptic vesicle endocytosis, and defects in accessory factors such as synaptojanin and auxilin have been linked to neurological dysfunction. Proper regulation of CME is critical for neurotransmission and neuronal survival.
From clathrin-dependent endocytosis-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is a candidate gene required for CME? | CRISPR knockout cell line followed by transferrin uptake assay |
| Does a specific mutation alter CME efficiency? | Point-mutation knock-in cell line |
| How does a tagged CME protein localize dynamically? | Knock-in of fluorescent tag (e.g., GFP) |
| Does overexpression of a CME regulator affect signaling? | Overexpression cell line |
| What is the role of a CME gene in synaptic vesicle recycling? | Neuronal knockout or knock-in models |
| Does a cancer-associated mutation affect receptor internalization? | Knock-in of the mutation in cancer cells |
How to Study the clathrin-dependent endocytosis Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Transferrin uptake assay | Rate of CME | Quantifying endocytic activity in cells |
| Surface biotinylation | Internalization of surface proteins | Measuring receptor endocytosis |
| Live-cell fluorescence microscopy | Dynamics of clathrin-coated pits and vesicles | Visualizing CME in real time |
| Immunofluorescence | Localization of CME proteins | Assessing coat assembly and uncoating |
| Co-immunoprecipitation | Protein-protein interactions | Identifying CME complexes |
| Mass spectrometry | Proteome of clathrin-coated vesicles | Discovering new CME components |
| CRISPR knockout | Loss-of-function phenotype | Testing gene requirement for CME |
| CRISPR knock-in | Effect of specific mutations or tags | Dissecting domain functions |
Fluorescence microscopy and live-cell imaging
Fluorescence microscopy using fluorescently labeled cargo (e.g., transferrin) or tagged CME proteins allows visualization of clathrin-coated pits and vesicles in real time. Live-cell imaging can track the dynamics of coat assembly, scission, and uncoating.
Biochemical assays for endocytosis
Biochemical assays such as surface biotinylation and receptor internalization assays measure the rate and extent of CME. These assays are often used to quantify the effects of genetic perturbations.
Proteomics and interactomics
Mass spectrometry-based proteomics can identify proteins associated with clathrin-coated vesicles and their post-translational modifications. Interactomics approaches reveal the network of accessory factors and cargo adaptors.
Genetic perturbation with CRISPR
CRISPR knockout, point mutation, knock-in, and overexpression models enable causal testing of CME gene function. These models can be combined with imaging and biochemical assays to dissect the pathway.
How CRISPR Can Be Used to Study GO:0072583 clathrin-dependent endocytosis
Knockout
CRISPR knockout of core CME genes such as CLTC, AP2M1, or DNM1 abolishes or severely impairs clathrin-dependent endocytosis, providing a clean loss-of-function background to test cargo internalization and downstream signaling. Knockout of regulatory genes like RLIP can reveal their specific contributions to receptor-ligand signaling.
Point Mutation
Point mutations can be introduced into CME genes to mimic disease-associated variants or to disrupt specific protein domains, such as the GTPase domain of dynamin or the cargo-binding site of AP-2. These models allow precise structure-function analysis without confounding effects of complete protein loss.
Knock-in
Knock-in of fluorescent tags (e.g., GFP or mCherry) into endogenous CME genes enables real-time visualization of protein dynamics at physiological expression levels. Knock-in of disease-relevant mutations, such as those in HER2, can model altered receptor internalization in cancer.
Overexpression
Overexpression of CME components or regulators can amplify the pathway and reveal dominant effects on receptor internalization and signaling. This approach is useful for testing whether increased levels of a protein enhance or perturb CME.
How EDITGENE Supports clathrin-dependent endocytosis Research
Researchers studying clathrin-dependent endocytosis-related genes often need to determine whether a candidate gene is causally involved in the pathway or merely correlated with it. CRISPR-based genome editing provides the gold-standard approach to establish causality by creating isogenic cell lines with defined genetic alterations. EDITGENE offers a comprehensive suite of services to support such studies, from knockout to knock-in and overexpression models, as well as library screening and bioinformatics.
Contact EDITGENE today to design your custom CRISPR model for clathrin-dependent endocytosis research.
Frequently Asked Questions About clathrin-dependent endocytosis
What is clathrin-dependent endocytosis?
Clathrin-dependent endocytosis (GO:0072583) is a biological process in which cells internalize extracellular material and membrane proteins through clathrin-coated pits that pinch off to form clathrin-coated vesicles.
What genes are involved in clathrin-dependent endocytosis?
Key genes include CLTC, CLTA, CLTB, AP2A1, AP2B1, AP2M1, DNM1, DNM2, SH3GL2, BIN1, SYNJ1, DNAJC6, HSPA8, RLIP, ITGB1, ERBB2, and PIP5K1C.
What is the function of clathrin-dependent endocytosis?
It mediates the uptake of nutrients, signaling receptors, adhesion molecules, and pathogens, and regulates receptor-ligand signaling and synaptic vesicle recycling.
How is clathrin-dependent endocytosis regulated?
It is regulated by accessory proteins such as RLIP, mechanical forces, and phosphoinositide metabolism, which control coat assembly, scission, and uncoating.
What diseases are associated with clathrin-dependent endocytosis?
Dysregulation is linked to cancer (e.g., HER2 internalization), bacterial infections, and neurological disorders affecting synaptic vesicle recycling.
What is the role of clathrin in endocytosis?
Clathrin triskelia assemble into a polyhedral lattice that deforms the plasma membrane and forms the coat of endocytic vesicles.
How do you study clathrin-dependent endocytosis?
Common methods include transferrin uptake assays, surface biotinylation, live-cell fluorescence microscopy, proteomics, and CRISPR-based genetic perturbation.
What is the difference between clathrin-dependent and clathrin-independent endocytosis?
Clathrin-dependent endocytosis requires the clathrin coat and dynamin for vesicle formation, whereas clathrin-independent pathways use different mechanisms and machinery.
Which proteins are involved in clathrin-coated vesicle scission?
Dynamin GTPases, together with accessory factors such as amphiphysin and endophilin, mediate membrane scission at the neck of the coated pit.
How can CRISPR help study clathrin-dependent endocytosis?
CRISPR knockout, point mutation, knock-in, and overexpression models allow precise manipulation of CME genes to test their causal roles in the pathway and disease.
Conclusion
Clathrin-dependent endocytosis (GO:0072583) is a cornerstone of cellular uptake and signaling, with essential roles in nutrient acquisition, receptor regulation, synaptic function, and host-pathogen interactions. Its dysfunction is implicated in cancer, infection, and neurological disorders, making it a compelling area of research. Advances in CRISPR genome editing provide powerful tools to dissect the molecular mechanisms and disease relevance of this pathway. EDITGENE offers comprehensive CRISPR services to support mechanistic and translational studies of clathrin-dependent endocytosis.
References
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- 3. Wolfe BL et al.. 2007. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis.. Traffic 8(5):462-70 PMID: 17376169
- 4. Kyumurkov A et al.. 2023. Force tuning through regulation of clathrin-dependent integrin endocytosis.. J Cell Biol 222(1) PMID: 36250940
- 5. Bruna-Gauchoux J et al.. 2022. Constraints and frustration in the clathrin-dependent endocytosis pathway.. C R Biol 345(2):43-56 PMID: 36847464
- 6. Shimauchi A et al.. 2025. Enhanced HER2 internalization by clathrin-dependent endocytosis in non-small cell lung cancer positive for HER2 mutations.. Br J Cancer 133(7):976-985 PMID: 40721523
- 7. Slepnev VI et al.. 2000. Accessory factors in clathrin-dependent synaptic vesicle endocytosis.. Nat Rev Neurosci 1(3):161-72 PMID: 11257904
- 8. Veiga E et al.. 2006. The role of clathrin-dependent endocytosis in bacterial internalization.. Trends Cell Biol 16(10):499-504 PMID: 16962776
