Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells
Open accessAbstract
During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.
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Endocytosis typically requires force generation by active actin polymerization at the maturing endocytic pit.
“Endocytosis typically requires force generation by active actin polymerization at the maturing endocytic pit52–57.”
From Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis (2023) · cites this paper as [56]
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Cortactin: the gray eminence of the cytoskeleton.
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“(69) studied the dynamics of individual CCPs using evanescent field microscopy and a witty technique, the pH-sensitive labeling of transferrin receptors, with which the closure and scission of CCPs can be resolved in real time.”
BackgroundCureton, D., Massol, R. H., Whelan, S. P. J., & Kirchhausen, T. (2010) · PLoS Pathogens · doi:10.1371/journal.ppat.1001127 · Cited by 184 · see citing works
Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains.
Co, C., Wong, D. T., Gierke, S., Chang, V., & Taunton, J. (2007) · Cell · doi:10.1016/j.cell.2006.12.049 · Cited by 182 · see citing works
Jain, K. (2008) · Humana Press · doi:10.1007/978-1-61779-983-9 · Cited by 179
Soykan, T., Kaempf, N., Sakaba, T., Vollweiter, D., Goerdeler, F., Puchkov, D., Kononenko, N. L., & Haucke, V. (2017) · Neuron · doi:10.1016/j.neuron.2017.02.011 · Cited by 178 · see citing works
Dynamics of endocytic vesicle creation.
Perrais, D., & Merrifield, C. J. (2005) · Developmental Cell · doi:10.1016/j.devcel.2005.10.002 · Cited by 177 · see citing works
Self-citationTwo synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages
Perera, R., Zoncu, R., Lucast, L. J., Camilli, P. D., & Toomre, D. (2006) · Proceedings of the National Academy of Sciences of the United States of America · doi:10.1073/pnas.0609795104 · Cited by 173 · see citing works
Focusing on clathrin-mediated endocytosis.
Rappoport, J. Z. (2008) · Biochemical Journal · doi:10.1042/bj20080474 · Cited by 172 · see citing works
A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis
Taylor, M. J., Lampe, M., & Merrifield, C. J. (2012) · PLoS Biology · doi:10.1371/journal.pbio.1001302 · Cited by 166 · see citing works
“This finding is consistent with previous studies that have observed latrunculinresistant clathrin endocytic events [7,33,47,48].”
MethodInfluentialSelf-citationSirotkin, V., Berro, J., Macmillan, K., Zhao, L., & Pollard, T. D. (2010) · Molecular Biology of the Cell · doi:10.1091/mbc.e10-02-0157 · Cited by 166 · see citing works
Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis
Mettlen, M., Loerke, D., Yarar, D., Danuser, G., & Schmid, S. (2010) · Journal of Cell Biology · doi:10.1083/jcb.200908078 · Cited by 166 · see citing works
“…as sites at which multiple rounds of CCV formation were observed to occur from an apparently single CCP. Nonterminal events are frequently observed in HeLa cells (Merrifield et al., 2005; Loerke et al., 2009), but rarely detected in control BSC1 cells (Ehrlich et al., 2004; Loerke et al., 2009).”
BackgroundInfluentialSubcellular membrane curvature mediated by the BAR domain superfamily proteins.
Suetsugu, S., Toyooka, K., & Senju, Y. (2010) · Seminars in Cell and Developmental Biology · doi:10.1016/j.semcdb.2009.12.002 · Cited by 165 · see citing works
Kirkbride, K. C., Sung, B., Sinha, S., & Weaver, A. M. (2011) · Cell Adhesion & Migration · doi:10.4161/cam.5.2.14773 · Cited by 164 · see citing works
Membrane deformation by protein coats.
Antonny, B. (2006) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2006.06.003 · Cited by 163 · see citing works
Antonescu, C., Aguet, F., Danuser, G., & Schmid, S. (2011) · Molecular Biology of the Cell · doi:10.1091/mbc.e11-04-0362 · Cited by 157 · see citing works
“…we and others have begun to establish a temporal hierarchy of functional requirements of EAPs in CCP initiation, stabilization, and maturation (Merrifield et al., 2005; Rappoport et al., 2006; Saffarian and Kirchhausen, 2008; Mettlen et al., 2009; Loerke et al., 2011; Taylor et al., 2011).”
BackgroundDynamin recruitment and membrane scission at the neck of a clathrin-coated pit
Cocucci, E., Gaudin, R., & Kirchhausen, T. (2014) · Molecular Biology of the Cell · doi:10.1091/mbc.e14-07-1240 · Cited by 156 · see citing works
“…neck of a coated pit and release of a coated vesicle from the plasma membrane coincide with a peak in dynamin recruitment (Ehrlich et al., 2004; Merrifield et al., 2005; Taylor et al., 2011, 2012; Aguet et al., 2013), but direct information concerning the number of dynamin molecules that…”
BackgroundRole of lipids and actin in the formation of clathrin-coated pits.
Boucrot, E., Saffarian, S., Massol, R. H., Kirchhausen, T., & Ehrlich, M. (2006) · Experimental Cell Research · doi:10.1016/j.yexcr.2006.09.025 · Cited by 153 · see citing works
Molecular basis for SH3 domain regulation of F-BAR–mediated membrane deformation
Rao, Y., Ma, Q., Vahedi-Faridi, A., Sundborger, A., Pechstein, A., Puchkov, D., Luo, L., Shupliakov, O., Saenger, W., & Haucke, V. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1003478107 · Cited by 149 · see citing works
Wu, M., Huang, B., Graham, M. M., Raimondi, A., Heuser, J., Zhuang, X., & Camilli, P. D. (2010) · Nature Cell Biology · doi:10.1038/ncb2094 · Cited by 149 · see citing works
Keyel, P. A., Mishra, S., Roth, R., Heuser, J., Watkins, S. C., & Traub, L. (2006) · Molecular Biology of the Cell · doi:10.1091/mbc.e06-05-0421 · Cited by 149 · see citing works
“Protein levels were standardized using a Coomassie blue–based filter paper binding assay (Minamide and Bamburg, 1990); 25 g was loaded per lane.”
MethodThe cellular functions of clathrin
Royle, S. J. (2006) · Cellular and Molecular Life Sciences (CMLS) · doi:10.1007/s00018-005-5587-0 · Cited by 148 · see citing works
Cortactin has an essential and specific role in osteoclast actin assembly.
Tehrani, S., Faccio, R., Chandrasekar, I., Ross, F., & Cooper, J. A. (2006) · Molecular Biology of the Cell · doi:10.1091/mbc.e06-03-0187 · Cited by 146 · see citing works
“This viral titer had no cytotoxic effects on target cells.”
BackgroundEndocytic adaptors: recruiters, coordinators and regulators.
Maldonado-Báez, L., & Wendland, B. (2006) · Trends in Cell Biology · doi:10.1016/j.tcb.2006.08.001 · Cited by 141 · see citing works
Clathrin‐Coated Pits: Vive La Différence?
Benmerah, A., & Lamaze, C. (2007) · Traffic : the International Journal of Intracellular Transport · doi:10.1111/j.1600-0854.2007.00585.x · Cited by 138 · see citing works
Imaging single events at the cell membrane.
Jaiswal, J., & Simon, S. (2007) · Nature Chemical Biology · doi:10.1038/nchembio855 · Cited by 135 · see citing works
Alto, N. M., Weflen, A. W., Rardin, M. J., Yarar, D., Lazar, C., Tonikian, R., Koller, A., Taylor, S. S., Boone, C., Sidhu, S., et al. (2007) · Journal of Cell Biology · doi:10.1083/jcb.200705021 · Cited by 134 · see citing works
<i>Candida albicans</i>internalization by host cells is mediated by a clathrin-dependent mechanism
Moreno-Ruiz, E., Galán-Díez, M., Zhu, W., Fernández-Ruiz, E., d'Enfert, C., Filler, S. G., Cossart, P., & Veiga, E. (2009) · Cellular Microbiology · doi:10.1111/j.1462-5822.2009.01319.x · Cited by 133 · see citing works
“…C. albicans internalization The GTPase dynamin is required for clathrin-coated pits to pinch off the plasma membrane and form endocytic vesicles (Hinshaw, 2000; Kirchhausen, 2000; Conner and Schmid, 2003; Orth and McNiven, 2003; Ehrlich et al., 2004; Merrifield et al., 2005; Macia et al., 2006).”
BackgroundRecruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis
Lee, D.-W., Wu, X.-F., Eisenberg, E., & Greene, L. (2006) · Journal of Cell Science · doi:10.1242/jcs.03092 · Cited by 133 · see citing works
“…field about 30 seconds after it disappeared from the evanescent field (Merrifield et al., 2002); also during the period of decreasing dynamin and clathrin intensity, actin, Arp2/3 and cortactin were recruited to the CCPs (Merrifield et al., 2002; Merrifield et al., 2004; Merrifield et al., 2005).”
BackgroundResultInfluential
- Authors
- Merrifield, C. J., Perrais, D., & Zenisek, D.
- Affiliations
- MRC Laboratory of Molecular Biology (GB)
- Centre National de la Recherche Scientifique (FR)
- Yale University (US)
- Corresponding author
- Merrifield, Christien J.
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2005-05
- Volume
- 121
- Issue
- 4
- Pages
- 593-606
- ISSN
- 0092-8674 (ISSN-L)
- OpenAlex
- W2001532503
- Cited by
- 476
- References
- 0
- Citations by year
- 2026: 3 · 2025: 6 · 2024: 6 · 2023: 14 · 2022: 15 · 2021: 10 · 2020: 9 · 2019: 11 · 2018: 18 · 2017: 17 · 2016: 13 · 2015: 21 · 2014: 36 · 2013: 12 · 2012: 34
- FWCI
- 16.53
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Caveolin-1 and cellular processes
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
- Keywords
- clathrin-mediated endocytosis, membrane fission, clathrin-coated vesicles, cortactin, actin polymerization, latrunculin B, vesicle formation, dynamin, F-actin
- MeSH terms
- Actins, Animals, Cell Membrane, Coated Pits, Cell-Membrane, Endocytosis, Microfilament Proteins, Microscopy, Fluorescence, Polymers, Thiazoles, Microscopy, Video, Bridged Bicyclo Compounds, Heterocyclic, Clathrin-Coated Vesicles, Swiss 3T3 Cells, Cortactin, Mice, Thiazolidines
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