Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2015 Apr 23

PMID 25902427

Citations 2

Authors

Anna Pezzarossa

Franziska Zosel

Thomas Schmidt

Affiliations

Soon will be listed here.

Abstract

The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.

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References

Perez J, Segura J, Abankwa D, Piguet J, Martinez K, Vogel H . Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains. J Mol Biol. 2006; 363(5):918-30. DOI: 10.1016/j.jmb.2006.08.036. View

Betzig E, Patterson G, Sougrat R, Lindwasser O, Olenych S, Bonifacino J . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006; 313(5793):1642-5. DOI: 10.1126/science.1127344. View

Bates M, Huang B, Dempsey G, Zhuang X . Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science. 2007; 317(5845):1749-53. PMC: 2633025. DOI: 10.1126/science.1146598. View

Lingwood D, Ries J, Schwille P, Simons K . Plasma membranes are poised for activation of raft phase coalescence at physiological temperature. Proc Natl Acad Sci U S A. 2008; 105(29):10005-10. PMC: 2481349. DOI: 10.1073/pnas.0804374105. View

Biteen J, Thompson M, Tselentis N, Bowman G, Shapiro L, Moerner W . Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods. 2008; 5(11):947-9. PMC: 2655310. DOI: 10.1038/nmeth.1258. View

Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S . Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature. 2008; 457(7233):1159-62. DOI: 10.1038/nature07596. View

Roy S, Luetterforst R, Harding A, Apolloni A, Etheridge M, Stang E . Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains. Nat Cell Biol. 1999; 1(2):98-105. DOI: 10.1038/10067. View

Schutz G, Kada G, Pastushenko V, Schindler H . Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 2000; 19(5):892-901. PMC: 305629. DOI: 10.1093/emboj/19.5.892. View

Pralle A, Keller P, Florin E, Simons K, Horber J . Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol. 2000; 148(5):997-1008. PMC: 2174552. DOI: 10.1083/jcb.148.5.997. View

10.

Harms G, Cognet L, Lommerse P, Blab G, Kahr H, Gamsjager R . Single-molecule imaging of l-type Ca(2+) channels in live cells. Biophys J. 2001; 81(5):2639-46. PMC: 1301731. DOI: 10.1016/S0006-3495(01)75907-3. View

11.

Chiu V, Bivona T, Hach A, Sajous J, Silletti J, Wiener H . Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol. 2002; 4(5):343-50. DOI: 10.1038/ncb783. View

12.

Niv H, Gutman O, Kloog Y, Henis Y . Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells. J Cell Biol. 2002; 157(5):865-72. PMC: 2173426. DOI: 10.1083/jcb.200202009. View

13.

Prior I, Muncke C, Parton R, Hancock J . Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol. 2003; 160(2):165-70. PMC: 2172642. DOI: 10.1083/jcb.200209091. View

14.

Lommerse P, Blab G, Cognet L, Harms G, Snaar-Jagalska B, Spaink H . Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys J. 2003; 86(1 Pt 1):609-16. PMC: 1303830. DOI: 10.1016/S0006-3495(04)74139-9. View

15.

Murakoshi H, Iino R, Kobayashi T, Fujiwara T, Ohshima C, Yoshimura A . Single-molecule imaging analysis of Ras activation in living cells. Proc Natl Acad Sci U S A. 2004; 101(19):7317-22. PMC: 409916. DOI: 10.1073/pnas.0401354101. View

16.

Rotblat B, Prior I, Muncke C, Parton R, Kloog Y, Henis Y . Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane. Mol Cell Biol. 2004; 24(15):6799-810. PMC: 444858. DOI: 10.1128/MCB.24.15.6799-6810.2004. View

17.

Lommerse P, Spaink H, Schmidt T . In vivo plasma membrane organization: results of biophysical approaches. Biochim Biophys Acta. 2004; 1664(2):119-31. DOI: 10.1016/j.bbamem.2004.05.005. View

18.

Singer S, Nicolson G . The fluid mosaic model of the structure of cell membranes. Science. 1972; 175(4023):720-31. DOI: 10.1126/science.175.4023.720. View

19.

Willumsen B, Christensen A, Hubbert N, Papageorge A, Lowy D . The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984; 310(5978):583-6. DOI: 10.1038/310583a0. View

20.

Yechiel E, Edidin M . Micrometer-scale domains in fibroblast plasma membranes. J Cell Biol. 1987; 105(2):755-60. PMC: 2114748. DOI: 10.1083/jcb.105.2.755. View