Clathrin-Independent Pathways of Endocytosis

Purpose: Clathrin-mediated endocytosis (CME) has long been the dominant paradigm for cellular uptake, but many endocytic pathways operate without clathrin. These clathrin-independent endocytic (CIE) pathways are increasingly recognized as distinct routes with different cargo selectivity, molecular machinery, kinetics, and intracellular destinations. The review addresses the need to organize and understand this diversity, especially for small-scale CIE processes that operate without clathrin coats and, in some cases, without dynamin. ---

Hypothesis: The central thesis is: CIE pathways are not merely alternative or redundant uptake mechanisms; they represent distinct, mechanistically diverse, and physiologically important endocytic systems. If their cargo selection, vesicle formation, trafficking itineraries, and regulatory logic are systematically mapped, then their roles in cell physiology, membrane tension control, cell migration, signaling, and disease can be understood and potentially exploited. ---

Aims: - Explore pathways of endocytosis that occur in the absence of clathrin, with emphasis on small-scale CIE processes. - Classify CIE pathways based on dynamin dependence, presence/absence of coats, and involvement of actin machinery. - Review cargo selection, vesicle budding, and post-endocytic trafficking itineraries of CIE cargo. - Discuss functional roles of CIE pathways in cell and tissue physiology, including membrane area/tension regulation and cell migration. - Provide a perspective on future challenges, including the need for a molecular “parts list” and in vivo validation. ---

Delivery system: This review does not describe an engineered drug delivery system. Its “platform” is the set of endogenous cellular endocytic pathways: CIE pathways discussed: - Caveolar endocytosis: dynamin-dependent, caveolin/cavin-coated, 50–80 nm flask-shaped invaginations. - IL-2 receptor endocytic route: dynamin-dependent, RhoA/Rac1-dependent, cholesterol-sensitive, small noncoated invaginations (50–100 nm). - EGFR endocytosis at high EGF: switches from clathrin-dependent to CIE; cholesterol/raft-associated; dynamin role controversial. - CLIC/GEEC pathway: dynamin-independent, uncoated tubulovesicular carriers; high-capacity fluid-phase and GPI-AP uptake; Cdc42/Arf1/GRAF1-dependent. - Arf6-associated pathway: dynamin-independent; involved in recycling of MHC-I, integrins, and other cargo. - Flotillin-dependent pathways: dynamin-independent or possibly dynamin-dependent depending on context. - Bungarotoxin/nicotinic acetylcholine receptor pathway: dynamin-independent, Rac1/actin-dependent. - Yeast actin-mediated endocytosis: clathrin-facilitated but clathrin-dispensable; actin and myosin-driven membrane invagination and scission. Representative cargo: GPI-anchored proteins, MHC class I, CD44, CD98, CD147, folate receptor, dysferlin, IL-2 receptor subunits, EGFR, cholera toxin, Shiga toxin, SV40, ricin, and various pathogens. Key molecular machinery: dynamin, caveolin-1/2/3, cavins, EHD2, pacsin2, actin, Arp2/3, Cdc42, RhoA, Rac1, N-WASP, cortactin, Arf1, GBF1, ARHGAP10/21, GRAF1, flotillins, Rab5, Rab11, Rab22, Rab35, Arf6, Hook1, ESCRT, and ubiquitin ligases. ---

Approach: Narrative review of published literature. No primary experimental groups. Model systems discussed include: - Mammalian cell lines: HeLa, CHO, BHK, mouse embryonic fibroblasts, endothelial cells, and others. - Primary cells: Drosophila hemocytes, neurons, lymphocytes. - Organisms: yeast (Saccharomyces cerevisiae), Drosophila shibire mutants, caveolin-deficient mice, zebrafish. - In vivo contexts: lung endothelium, muscle, immune cells, migrating cells. - Disease/physiology context: cell proliferation, immune signaling, cell migration, membrane tension, muscle disease, pathogen entry. ---

Key methods: Techniques highlighted across cited studies: - Live-cell fluorescence imaging, including TIRF microscopy and photobleaching. - Electron microscopy (EM), including correlative light and electron microscopy and EM tomography. - Genetic mutants and genome-edited cells (e.g., caveolin-deficient, dynamin mutants, shibire). - Dominant-negative constructs and small-molecule inhibitors. - siRNA knockdown of CIE regulators (e.g., EHD2, cavin1, GRAF1, Cdc42). - Antibody labeling of endogenous surface markers (e.g., GPI-AP aminopeptidase P). - Biochemical and ultrastructural assays for caveolar budding and endocytic carrier formation. - Tracking of cargo trafficking to early endosomes, GEECs, recycling endosomes, and lysosomes. ---

Key results: - Caveolae are dynamic endocytic carriers: In interphase cells, ~85% of caveolae were internalized within a 10-min observation period, with lifetimes ranging from <2 s (30% of caveolae) to >7 min. Budded structures were cavin-positive, and dynamin dependence was confirmed. - EHD2 is a negative regulator of caveolar endocytosis: Knockdown of EHD2 increases caveolar budding. EHD2 links caveolae to actin and associates with pacsin2. - CLIC/GEEC pathway is high-capacity and constitutive: In mouse embryonic fibroblasts, the entire cell surface can be recycled in <15 min. In some cell lines, ~50% of newly endocytosed material is recycled early; the rest matures into GEECs. - IL-2 receptor route: Small noncoated invaginations (50–100 nm) concentrate IL-2 receptors; endocytosis is dynamin-dependent, cholesterol-sensitive, and requires RhoA/Rac1, cortactin, N-WASP, and Arp2/3. - EGFR switches to CIE at high EGF: Low EGF favors clathrin-dependent uptake; high EGF and enhanced receptor ubiquitination shunt EGFR to a CIE route. - Actin polymerization is a unifying mechanism: In the absence of clathrin and dynamin, specialized actin-based platforms drive membrane remodeling and scission. - Caveolin-deficient mice show relatively mild phenotypes: This suggests specialized rather than universal essential functions for caveolae, or significant compensation. ---

Interpretation: The authors conclude that CIE pathways are diverse, mechanistically distinct, and physiologically important. They argue that these pathways likely regulate plasma membrane area and tension, control membrane availability during cell migration, and influence signaling, immune responses, and pathogen entry. The field needs a systematic “parts list” for each CIE pathway, better in vivo studies, and specific small-molecule inhibitors to dissect their roles. The authors also emphasize that overexpression artifacts and the prior “caveosome” model need reassessment. ---

Limitations: - Lack of a complete molecular parts list for most CIE pathways. - In vivo relevance remains underexplored, especially for different tissues and physiological states. - Overexpression artifacts can confound interpretation; the caveosome is now largely considered an artifact of caveolin overexpression. - Redundancy vs. distinct roles of CIE pathways are unresolved. - Dynamin dependence is controversial for some pathways, notably EGFR endocytosis at high EGF. - Specific inhibitors are lacking, making acute functional dissection difficult. - Mild phenotypes in caveolin-deficient mice complicate assignment of essential functions. - As a review, this article does not provide primary data or systematic meta-analysis.