Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy

Optineurin (OPTN) plays an important role in membrane trafficking processes such as exocytosis and autophagy. The sizes and rate of formation of accumulated structures comprising OPTN, such as foci or inclusion bodies (IBs), are often disrupted by amyotrophic lateral sclerosis (ALS) and glaucoma-associated mutants of OPTN. Therefore, methods for the quantitative measurement of the size of the accumulated structure are necessary. Here, we show that, using spatial image correlation spectroscopy (ICS), the average diameter of accumulated structures of the wild-type and disease-associated mutants in living cells may be easily determined. Although OPTN was found to frequently form foci in the cytoplasm, regardless of ALS- and glaucoma-associated mutation, the diameter of OPTN foci decreased in an ALS-associated mutant and increased in a glaucoma-associated mutant. However, a portion of cells carried IBs of the ALS-associated mutant that were larger than micrometre and ellipse-like shape, suggesting that this mutant accumulates non-uniformly in the IBs. The findings suggest that changes in their accumulation, determined via quantitative comparison of the OPTN foci and IBs in the cells, are involved in pathological features of ALS. In addition, this method enables rapid comparison of the average sizes of various other intracellular structures such as granules.