Unraveling Protein-protein Interactions in Clathrin Assemblies Via Atomic Force Spectroscopy

Overview

Journal Methods

Specialty Biochemistry

Date 2012 Dec 29

PMID 23270814

Citations 1

Authors

Albert J Jin

Eileen M Lafer

Jennifer Q Peng

Paul D Smith

Ralph Nossal

Affiliations

Soon will be listed here.

Abstract

Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate.

Citing Articles

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Cordella N, Lampo T, Mehraeen S, Spakowitz A Biophys J. 2014; 106(7):1476-88.

PMID: 24703309 PMC: 3976529. DOI: 10.1016/j.bpj.2013.11.4505.

References

Bocking T, Aguet F, Harrison S, Kirchhausen T . Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol. 2011; 18(3):295-301. PMC: 3056279. DOI: 10.1038/nsmb.1985. View

Zhuo Y, Ilangovan U, Schirf V, Demeler B, Sousa R, Hinck A . Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly. J Mol Biol. 2010; 404(2):274-90. PMC: 2981644. DOI: 10.1016/j.jmb.2010.09.044. View

Hearty S, Conroy P, Ayyar B, Byrne B, OKennedy R . Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends. Expert Rev Vaccines. 2010; 9(6):645-64. DOI: 10.1586/erv.10.52. View

Ybe J, Brodsky F, Hofmann K, Lin K, Liu S, Chen L . Clathrin self-assembly is mediated by a tandemly repeated superhelix. Nature. 1999; 399(6734):371-5. DOI: 10.1038/20708. View

Hao W, Luo Z, Zheng L, Prasad K, Lafer E . AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin. J Biol Chem. 1999; 274(32):22785-94. DOI: 10.1074/jbc.274.32.22785. View

Kirchhausen T . Imaging endocytic clathrin structures in living cells. Trends Cell Biol. 2009; 19(11):596-605. PMC: 2796618. DOI: 10.1016/j.tcb.2009.09.002. View

Xing Y, Bocking T, Wolf M, Grigorieff N, Kirchhausen T, Harrison S . Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly. EMBO J. 2009; 29(3):655-65. PMC: 2830701. DOI: 10.1038/emboj.2009.383. View

Jin A, Prasad K, Smith P, Lafer E, Nossal R . Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy. Biophys J. 2006; 90(9):3333-44. PMC: 1432129. DOI: 10.1529/biophysj.105.068742. View

Wakeham D, Chen C, Greene B, Hwang P, Brodsky F . Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation. EMBO J. 2003; 22(19):4980-90. PMC: 204494. DOI: 10.1093/emboj/cdg511. View

10.

Ungewickell E, Ungewickell H, Holstein S, Lindner R, Prasad K, Barouch W . Role of auxilin in uncoating clathrin-coated vesicles. Nature. 1995; 378(6557):632-5. DOI: 10.1038/378632a0. View

11.

McMahon H, Boucrot E . Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2011; 12(8):517-33. DOI: 10.1038/nrm3151. View

12.

Svoboda K, Block S . Biological applications of optical forces. Annu Rev Biophys Biomol Struct. 1994; 23:247-85. DOI: 10.1146/annurev.bb.23.060194.001335. View

13.

Dudko O, Hummer G, Szabo A . Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proc Natl Acad Sci U S A. 2008; 105(41):15755-60. PMC: 2572921. DOI: 10.1073/pnas.0806085105. View

14.

den Otter W, Renes M, Briels W . Asymmetry as the key to clathrin cage assembly. Biophys J. 2010; 99(4):1231-8. PMC: 2920741. DOI: 10.1016/j.bpj.2010.06.011. View

15.

Qian H, Shapiro B . Graphical method for force analysis: macromolecular mechanics with atomic force microscopy. Proteins. 2000; 37(4):576-81. DOI: 10.1002/(sici)1097-0134(19991201)37:4<576::aid-prot8>3.0.co;2-o. View

16.

Shapiro B, Qian H . A quantitative analysis of single protein-ligand complex separation with the atomic force microscope. Biophys Chem. 2006; 67(1-3):211-9. DOI: 10.1016/s0301-4622(97)00045-8. View

17.

Gauglitz G . Direct optical detection in bioanalysis: an update. Anal Bioanal Chem. 2010; 398(6):2363-72. DOI: 10.1007/s00216-010-3904-4. View

18.

Dannhauser P, Ungewickell E . Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat Cell Biol. 2012; 14(6):634-9. DOI: 10.1038/ncb2478. View

19.

Anker J, Hall W, Lyandres O, Shah N, Zhao J, Van Duyne R . Biosensing with plasmonic nanosensors. Nat Mater. 2008; 7(6):442-53. DOI: 10.1038/nmat2162. View

20.

Rappoport J, Kemal S, Benmerah A, Simon S . Dynamics of clathrin and adaptor proteins during endocytosis. Am J Physiol Cell Physiol. 2006; 291(5):C1072-81. DOI: 10.1152/ajpcell.00160.2006. View