TITLE

Temporal resolution of protein—protein interactions in the live-cell plasma membrane

AUTHOR(S)
Weghuber, Julian; Sunzenauer, Stefan; Plochberger, Birgit; Brameshuber, Mario; Haselgrübler, Thomas; Schütz, Gerhard
PUB. DATE
August 2010
SOURCE
Analytical & Bioanalytical Chemistry;Aug2010, Vol. 397 Issue 8, p3339
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 ). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section.
ACCESSION #
52532528

 

Related Articles

  • Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis. Mamdouh, Zahra; Chen, Xia; Pierini, Lynda M.; Maxfield, Frederick R.; Muller, William A. // Nature;2/13/2003, Vol. 421 Issue 6924, p748 

    Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90?s after a leukocyte arrests on the endothelial...

  • Screening cell surface receptors using micromosaic immunoassays. Marc Wolf; Emmanuel Delamarche; Patrick Hunziker // Biomedical Microdevices;Apr2007, Vol. 9 Issue 2, p135 

    Abstract  This report presents a general method for screening cell surface receptors using so-called micromosaic immunoassays. This method employs a microfluidic chip havingn(n= 11) independent flow paths to move cells overm(m= 11) lines of surface-patterned antibodies for screening...

  • In vitro nanobody discovery for integral membrane protein targets. Doshi, Rupak; Chen, Beverly R.; Vibat, Cecile Rose T.; Huang, Norman; Chang-Wook Lee; Chang, Geoffrey // Scientific Reports;10/24/2014, p1 

    Nanobodies (Nbs) or single-domain antibodies are among the smallest and most stable binder scaffolds known. In vitro display is a powerful antibody discovery technique used worldwide. We describe the first adaptation of in vitro mRNA/cDNA display for the rapid, automatable discovery of Nbs...

  • RNA interference screens to uncover membrane protein biology. Mak, Anthony B.; Moffat, Jason // Briefings in Functional Genomics;Sep2013, Vol. 12 Issue 5, p422 

    In this review, we discuss the use of RNA interference screens to identify genes involved in the regulation and function of membrane proteins. Briefly, cells expressing the membrane protein of interest can be transduced with a pooled lentiviral short-hairpin RNA (shRNA) library containing tens...

  • Single Molecule Force Microscopy on Cells and Biological Membranes. Ebner, Andreas; Madl, Josef; Kienberger, Ferry; Chtcheglova, Lilia A.; Puntheeranurak, Theeraporn; Rong Zhu; Jilin Tang; Gruber, Hermann J.; Sch�tz, Gerhard J.; Hinterdorfer, Peter // Current Nanoscience;Feb2007, Vol. 3 Issue 1, p49 

    Atomic force microscopy (AFM) enables high resolution topographic imaging of biological samples under near-physiological conditions. Therefore, the AFM is optimally suited for investigation of biological membranes and cell surfaces, as exemplified by studies on bacterial S-layers, purple...

  • Image correlation spectroscopy of randomly distributed disks. Spendier, Kathrin; Thomas, James // Journal of Biological Physics;Sep2011, Vol. 37 Issue 4, p477 

    Image correlation spectroscopy (ICS) has been widely used to quantify spatiotemporal distributions of fluorescently labelled cell membrane proteins and receptors. When the membrane proteins are randomly distributed, ICS may be used to estimate protein densities, provided the proteins behave as...

  • Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface. Ye, K.; Shibasaki, S.; Ueda, M.; Murai, T.; Kamasawa, N.; Osumi, M.; Shimizu, K.; Tanaka, A. // Applied Microbiology & Biotechnology;Jul2000, Vol. 54 Issue 1, p90 

    An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of...

  • The Role of Membrane-Mediated Interactions in the Assembly and Architecture of Chemoreceptor Lattices. Haselwandter, Christoph A.; Wingreen, Ned S. // PLoS Computational Biology;Dec2014, Vol. 10 Issue 12, p1 

    In vivo fluorescence microscopy and electron cryo-tomography have revealed that chemoreceptors self-assemble into extended honeycomb lattices of chemoreceptor trimers with a well-defined relative orientation of trimers. The signaling response of the observed chemoreceptor lattices is remarkable...

  • Complement Fixation by Pemphigus Antibody. III. Altered Epidermal Cell Membrane Integrity Mediated by Pemphigus Antibody and Complement. Kawana, Seiji; Geoghegan, William D.; Jordon, Robert E. // Journal of Investigative Dermatology;Jan1986, Vol. 86 Issue 1, p29 

    The present study investigates the effects of pemphigus IgG and complement upon cell viability and/or membrane integrity using trypan blue exclusion, ethidium bromide (EB) staining, and fluorescein diacetate (FDA) conversion by living cells. Forty-eight-hour cultivated epidermal monolayers of...

Share

Read the Article

Courtesy of VIRGINIA BEACH PUBLIC LIBRARY AND SYSTEM

Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics