PMID- 21806277
OWN - NLM
STAT- MEDLINE
DCOM- 20120105
LR  - 20191210
IS  - 1560-2281 (Electronic)
IS  - 1083-3668 (Linking)
VI  - 16
IP  - 7
DP  - 2011 Jul
TI  - Robust assessment of protein complex formation in vivo via single-molecule 
      intensity distributions of autofluorescent proteins.
PG  - 076016
LID - 10.1117/1.3600002 [doi]
AB  - The formation of protein complexes or clusters in the plasma membrane is 
      essential for many biological processes, such as signaling. We develop a tool, 
      based on single-molecule microscopy, for following cluster formation in vivo. 
      Detection and tracing of single autofluorescent proteins have become standard 
      biophysical techniques. The determination of the number of proteins in a cluster, 
      however, remains challenging. The reasons are (i) the poor photophysical 
      stability and complex photophysics of fluorescent proteins and (ii) noise and 
      autofluorescent background in live cell recordings. We show that, despite those 
      obstacles, the accurate fraction of signals in which a certain (or set) number of 
      labeled proteins reside, can be determined in an accurate an robust way in vivo. 
      We define experimental conditions under which fluorescent proteins exhibit 
      predictable distributions of intensity and quantify the influence of noise. 
      Finally, we confirm our theoretical predictions by measurements of the 
      intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in 
      living cells. Quantification of the average number of EYFP-C10HRAS chimeras in 
      diffraction-limited spots finally confirm that the membrane anchor of human 
      Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of 
      living Chinese hamster ovary cells.
FAU - Meckel, Tobias
AU  - Meckel T
AD  - Technische Universitat Darmstadt, Membrane Dynamics, Department of Biology, 
      Darmstadt, Germany.
FAU - Semrau, Stefan
AU  - Semrau S
FAU - Schaaf, Marcel J M
AU  - Schaaf MJ
FAU - Schmidt, Thomas
AU  - Schmidt T
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Validation Study
PL  - United States
TA  - J Biomed Opt
JT  - Journal of biomedical optics
JID - 9605853
RN  - 0 (Bacterial Proteins)
RN  - 0 (Luminescent Proteins)
RN  - 0 (Multiprotein Complexes)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (yellow fluorescent protein, Bacteria)
RN  - EC 3.6.5.2 (HRAS protein, human)
RN  - EC 3.6.5.2 (Proto-Oncogene Proteins p21(ras))
SB  - IM
MH  - Animals
MH  - Bacterial Proteins/chemistry/metabolism
MH  - Biophysical Phenomena
MH  - CHO Cells
MH  - Cell Line, Tumor
MH  - Cell Membrane/metabolism
MH  - Cricetinae
MH  - Cricetulus
MH  - Fluorescence
MH  - Humans
MH  - Luminescent Proteins/chemistry/*metabolism
MH  - Microscopy, Fluorescence
MH  - Models, Biological
MH  - Multiprotein Complexes/chemistry/metabolism
MH  - Optical Phenomena
MH  - Proto-Oncogene Proteins p21(ras)/chemistry/metabolism
MH  - Rats
MH  - Recombinant Fusion Proteins/chemistry/metabolism
MH  - Spectrometry, Fluorescence
EDAT- 2011/08/03 06:00
MHDA- 2012/01/06 06:00
CRDT- 2011/08/03 06:00
PHST- 2011/08/03 06:00 [entrez]
PHST- 2011/08/03 06:00 [pubmed]
PHST- 2012/01/06 06:00 [medline]
AID - 10.1117/1.3600002 [doi]
PST - ppublish
SO  - J Biomed Opt. 2011 Jul;16(7):076016. doi: 10.1117/1.3600002.