Using multi-channel level sets to measure the cytoplasmic localization of HCMV pUL97 in GFP-B-gal fusion constructs.
Human cytomegalovirus UL97-encoded protein kinase (pUL97) phosphorylates cellular and viral proteins and is critical for viral replication. To quantify the efficiency of nuclear translocation and to elucidate the role of putative nuclear localization signal (NLS) elements, immunofluorescence analysis of different pUL97 expression constructs was performed. Since manual quantitation of respective expression levels lacks objectivity and reproducibility, and is time-consuming as well, a computer-based model is established. This model enables objective quantitation of the degree of cytoplasmic localization λ. To determine the degree of cytoplasmic localization of different pUL97-GFP-β-gal fusion proteins automatically, a multi-channel segmentation of the nucleus and cytoplasm of transfected HeLa cells is performed in DAPI and GFP micrographs. A watershed transform-based segmentation scheme is used for the segmentation of the cell nuclei. Subsequently, the cytoplasm is segmented using a fast marching level set method. Based on the segmentation of cell nuclei and cytoplasm, λ can be determined for each HeLa cell by quantitation of the ratio of average signal intensity outside and inside the nucleus. The degree of cytoplasmic localization of an individual construct is then determined by evaluating the average and standard deviation of λ for the corresponding HeLa cells. Evaluation demonstrates that nuclear transport of pUL97 is a multilayered mechanism resulting in different efficiencies of nuclear translocation between a small and a large isoform and objective quantitation of the cytoplasmic localization is possible with a high accuracy (96.7% and 94.3%).
Department of Image Processing and Biomedical Engineering, Fraunhofer Institute for Integrated Circuits IIS, Erlangen, Germany. Electronic address: christian.held@iis.fraunhofer.de.
Christian
C
Held
Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany. Electronic address: rewebel@viro.med.uni-erlangen.de.
Rike
R
Webel
Optical Imaging Center Erlangen, OICE, Erlangen, Germany. Electronic address: Ralf.Palmisano@fau.de.
Ralf
R
Palmisano
Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany. Electronic address: corina.hutterer@viro.med.uni-erlangen.de.
Corina
C
Hutterer
Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany. Electronic address: manfred.marschall@viro.med.uni-erlangen.de.
Manfred
M
Marschall
Department of Image Processing and Biomedical Engineering, Fraunhofer Institute for Integrated Circuits IIS, Erlangen, Germany. Electronic address: thomas.wittenberg@iis.fraunhofer.de.
Thomas
T
Wittenberg
D019677:Artificial Gene Fusion; D003584:Cytological Techniques / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014779:Virus Replication; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D001616:beta-Galactosidase
Cell segmentation; Fluorescence imaging; Image analysis; Level set; Optimization