Qusous, Ala, Parker, Eleanor, Geewan, Corinne, Kapasi, Arva, Getting, Stephen J., Hucklebridge, Frank, Keshavarz, Tajalli and Kerrigan, Mark J.P. (2012) Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling. Microscopy Research & Technique, 75 (7). pp. 991-999. ISSN 1059-910X (doi:10.1002/jemt.22055)

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Abstract

We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/μm3). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm3 (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10−5). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling.

Item Type:	Article
Additional Information:	[1] Article first published online: 19 APR 2012. [2] This article was published online on 19 April 2012. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected on 4 May 2012. [3] Additional Supporting Information may be found in the online version of the article. [4] ISSN 1059-910X (Print), 1097-0029 (Electronic).
Uncontrolled Keywords:	confocal, phalloidin, volume, microscopy, fluorescence
Subjects:	R Medicine > RM Therapeutics. Pharmacology
Pre-2014 Departments:	School of Science
Related URLs:	Publisher Organisation
Last Modified:	26 Oct 2016 10:54
URI:	http://gala.gre.ac.uk/id/eprint/8215

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