Dynamics of Dynamin during Clathrin Mediated Endocytosis in PC12 Cells

Rappoport, Joshua Z.; Heyman, Katherine P.; Kemal, Shahrnaz; Simon, Sanford M.

doi:10.1371/journal.pone.0002416

Cited by 30 publications

(24 citation statements)

References 34 publications

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“…Our findings suggest that dynamin could be recruited to the neck of closing clathrin-coated pits when their curvature is sufficiently high, and that its polymerization further constricts them to 10 nm. In vivo, dynamin appears at the clathrin-coated pits at the end of their formation (11)(12)(13), when the neck has a radius of ∼20 nm (35), close to the value of the critical radius (19 nm) we found for dynamin at 440 nM. Giant Unilamellar Vesicles.…”

Section: Discussionsupporting

confidence: 79%

“…The recruitment of dynamin to endocytic buds is thought to depend on the local synthesis of phosphatidylinositol (4,5)bisphosphate (PIP 2 ), as dynamin has a PIP 2 binding pleckstrin homology (PH) domain (10). Dynamin is recruited late in clathrin-coated vesicle formation, as seen by total internal reflection fluorescence (TIRF) microscopy (11)(12)(13). Because PIP 2 is also responsible for the binding of clathrin coats, it is expected to be present at the clathrin bud from the beginning of its formation.…”

mentioning

confidence: 99%

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Membrane curvature controls dynamin polymerization

Roux

Koster

Lenz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

279

270

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The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds' necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations (∼0.1-10 μM), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration.force | nucleation | fission | endocytosis | dynamin-like proteins M embrane remodeling is an essential task of proteins involved in membrane traffic (1, 2). Dynamin is a large GTPase that has been shown to polymerize into a helical collar at the neck of endocytic buds (3), where it subsequently plays a key role in the formation of endocytic vesicles through fission (4-8). This function is fundamental, as the knockout of the dynamin neuronal isoform leads to striking defects in synapse organization and results in a strong dysfunction of neuronal activity (9). The recruitment of dynamin to endocytic buds is thought to depend on the local synthesis of phosphatidylinositol(4,5)bisphosphate (PIP 2 ), as dynamin has a PIP 2 binding pleckstrin homology (PH) domain (10). Dynamin is recruited late in clathrin-coated vesicle formation, as seen by total internal reflection fluorescence (TIRF) microscopy (11-13). Because PIP 2 is also responsible for the binding of clathrin coats, it is expected to be present at the clathrin bud from the beginning of its formation. Thus, another explanation was suggested: Proteins that interact with dynamin and possess a curvature-sensing Bin-Amphiphysin-Rvs (BAR) domain (such as endophilin and amphiphysin) were proposed to sense the high curvature of the neck and recruit dynamin (14). Because the curvature of the neck is increasing during clathrin-coated vesicle formation to finally reach the range needed for BAR recruitment, this could explain the arrival of dynamin at the very late stage of clathrin-coated vesicle formation. However, in solution, dynamin spontaneously associates into helices and rings (15) with an internal radius of ≈10 nm. Dynamin can also polymerize around pre...

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Section: Discussionsupporting

confidence: 79%

mentioning

confidence: 99%

Membrane curvature controls dynamin polymerization

Roux

Koster

Lenz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

279

270

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“…However, the upstream event of nanoparticle entry to the cellular compartment and the molecular machinery that drives the dynamics of the internalization to cancer cells is not known. Evidence suggests an important role of dyn-2 in many cellular processes such as EGFR and other receptor endocytosis (28,29,31). EGF stimulated EGFRs have been found to involve both clathrin-dependent and independent pathways (32).…”

Section: Discussionmentioning

confidence: 99%

Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis

Bhattacharyya¹,

Bhattacharya²,

Curley

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

124

123

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Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs such as cetuximab, herceptin, etc., have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells. However, mechanisms of nanoparticles-mAbs interactions with the target cells and its effect on intracellular trafficking and mechanism are currently unknown. In this paper, we demonstrate that the distinct patterning and dynamics of anti-EGFR (epidermal growth factor receptor) antibody cetuximab (C225)-induced EGFR internalization in pancreatic cancer cells with variable receptor expression is altered upon nanoconjugation. Nanoconjugation uniformly enhanced C225-induced EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influenced its compartmentalization and regulated the involvement of dynamin-2 in the endocytic processes. Receptor endocytosis and its intracellular trafficking were monitored by confocal microscopy and transmission electron microscopy. The role of dynamin-2 in EGFR endocytosis was determined after overexpressing either wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer cells followed by tracking the receptor-antibody complex internalization by confocal microscopy. Significantly, these findings demonstrate that the nanoconjugation cannot be construed as an innocuous reaction involved in attaching the targeting agent to the nanoparticle, instead it may distinctly alter the cellular processes at the molecular level, at least antibody induced receptor endocytosis. This information is critical for successful design of a nanoparticle-based targeted drug delivery system for future clinical translation.EGFR | nanoparticle | dynamin | pancreatic cancer N anotechnology provides opportunities for biomedical applications that may improve human health care in the future (1-13). Engineered nanoparticles are evolving as promising candidates for various biological applications due to their tunable biophysical and biochemical properties (14-16). However, fundamental studies to understand the mechanism of cell-nanomaterial interactions are still lacking. To advance the successful development of biomedical nanotechnology for clinical use, vivid understanding of such mechanisms is essential.Epidermal growth factor receptor (EGFR) represents a unique target as over expression of EGFR has been implicated in the pathogenesis of many cancers (17, 18). Cetuximab (C225), a monoclonal anti-EGFR antibody, has been approved by the FDA for treatment of colon and head and neck cancers (19). Cetuximab is also in different phases of clinical trials for other cancers (20,21). However, the mechanism and pattern of C225 induced endocytosis of EGFR in pancreatic cancer cells of variable EGFR expression is not fully understood (20,21). Furthermore, the effect of nanoconjugation on the mechanism of C225 induced endocytosis remains to be investigated. Here, we demonstrate the mechanism and endocytic pattern of C225 and gold conjugated-C225 induced internalization of EGFR in pancreatic ...

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“…clathrin and epsin) (Rappoport et al, 2006;, whereas the localization of other proteins to the coated vesicle either increased over time (e.g. dynamin) (Merrifield et al, 2002;Rappoport et al, 2008; or decreased (e.g. AP-2) Rappoport et al, 2005).…”

Section: Endocytosismentioning

confidence: 99%

Imaging with total internal reflection fluorescence microscopy for the cell biologist

Mattheyses

Simon

Rappoport

2010

Journal of Cell Science

Self Cite

318

278

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SummaryTotal internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

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Dynamics of Dynamin during Clathrin Mediated Endocytosis in PC12 Cells

Cited by 30 publications

References 34 publications

Membrane curvature controls dynamin polymerization

Membrane curvature controls dynamin polymerization

Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis

Imaging with total internal reflection fluorescence microscopy for the cell biologist

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