Analysis of Protein Degradation Rates in Live Cells

 

Michael Halter, Alex Tona, Kiran Bhadriraju, Anne Plant, John Elliott

Biochemical Science Division, N.I.S.T., Gaithersburg, MD 20899, USA

 

ABSTRACT

 

Fluorescent proteins provide the potential for probing the dynamics of gene activity within single cells. Measurements of the differences in gene expression between cells and the temporal variability of gene expression within a single cell may broadly impact our understanding cell signaling networks and the processes that give rise to cell behavior. When a promoter driven GFP reporter is stabily transfected into cells, the genetically homogeneous cell population exhibits a wide range of the amount of green fluorescence protein in different cells. Understanding the processes that result in the distribution of responses from cell populations requires techniques for characterizing large numbers of living cells. Automated microscopy and image analysis is used to measure the protein degradation rates from large numbers of live, single cells confined on micropatterned arrays. The distribution of degradation rates may be a measure of the heterogeneity of proteasome activity across the population. Observed single cell fluorescence intensities and the GFP degradation rates do not appear correlated, suggesting the heterogeneity in protein abundances is not due to differences in degradation rates.  These measurements of GFP half-lives in statistically significant numbers of cells provide assurance that higher GFP abundance in some cells is due to higher rates of gene expression and not due to lower rates of protein degradation.

 

Name: Michael Halter

Mentor: John Elliott

Building 227

Room 261

Mail Stop 8310

Phone: (301) 975-4195

Fax: (301) 330-3447

Email: michael.halter@nist.gov

Not a Sigma Xi member

Category: Biotechnology