title: Simultaneous measurements of cytoplasmic viscosity and intracellular vesicle sizes for live human brain cancer cells.
abstract: Intracellular vesicles, comprised of protein clusters, were individually tracked inside human brain cancer cells and characterized to simultaneously determine the average vesicle size and effective cytoplasmic viscosity. The cells were transfected with a TGF-β superfamily gene, non-steroidal anti-inflammatory drug-Activated Gene-1 (NAG-1) tagged with green fluorescent proteins (GFPs). Using total internal reflection fluorescent microscopy (TIRFM) the individual movements of the vesicles were categorized into either Brownian, caged, or directional type motion. In the near-field region confined by the evanescent wave field of TIRFM, the hindrance of these vesicles was created by interactions with the glass coverslip and/or sub-cellular structures. Measured particle motions were compared with theoretical predictions of hindered motion to estimate the unknown size and viscosity parameters using a nonlinear regression technique. For the tested human brain cancer cells, the average vesicle size and effective intracellular fluid viscosity were calculated to be 496 nm and 0.068 Pa s, respectively. This finding suggests that most of the hindrance experienced by vesicles can be due to non-hydrodynamic interactions with microtubules and other intracellular structures. It should be also noted that this method provides a way to examine changes in vesicle size due to outside stimulus such as drug interaction, cytotoxicity, etc., unlike standard measurement techniques which require fixing the cells themselves.
authors.affiliation: Department of Mechanical, Aerospace, and Biomedical Engineering University of Tennessee, Knoxville, Tennessee 37996; telephone: +906-487-1463, fax: +906-487-2822.
authors.forename: Charles
authors.initials: C
authors.lastname: Margraves
authors.forename: Kenneth
authors.initials: K
authors.lastname: Kihm
authors.forename: Sang Youl
authors.initials: SY
authors.lastname: Yoon
authors.forename: Chang Kyoung
authors.initials: CK
authors.lastname: Choi
authors.forename: Seong-ho
authors.initials: SH
authors.lastname: Lee
authors.forename: Jason
authors.initials: J
authors.lastname: Liggett
authors.forename: Seung Joon
authors.initials: SJ
authors.lastname: Baek
mesh_terms: D001932:Brain Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D055436:Growth Differentiation Factor 15 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014783:Viscosity
chemical_list: C526140:GDF15 protein, human; D055436:Growth Differentiation Factor 15; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins