Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy

Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy Abstract 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. foci, image correlation spectroscopy, inclusion body, optineurin, vesicular trafficking Vesicular trafficking, which involves endocytosis, exocytosis and autophagy, is a well-conserved and highly regulated cellular process in eukaryotic cells; this process enables targeted delivery of specific components to selective compartments such as the extracellular space, endosome and lysosome. Adapter or regulatory proteins play an important role in the efficient delivery of these components. Regulatory proteins temporarily accumulate on vesicular membranes to regulate functions (1, 2). When diffusing proteins temporarily attach to the membrane, their localization on vesicles may be observed as foci in the cytoplasm in the autophagosomes, endosomes and endoplasmic reticulum exit sites. However, misfolded or denatured proteins in the cells often form aggregates that accumulate in the inclusion bodies (IBs) (3, 4). The foci and IBs may be observed by optical microscopy as well as electron microscopy; accordingly, the characterization of the foci and IBs via size quantification is a straightforward procedure. However, the measurement of foci diameter individually is complex; therefore, a facile method for the estimation of the diameter of foci and IBs is important. Fluorescence fluctuation-based methods, such as fluorescence correlation spectroscopy (FCS), image correlation spectroscopy (ICS) and number and brightness (N&B) procedure, are used for various analyses for accumulated proteins in cell biology. In FCS and ICS, temporal and/or spatial correlation functions reflecting molecular size or dynamics are calculated from the recorded fluorescence fluctuation. Specifically, FCS is suitable for measuring the diffusion rate of molecules in living cells and solution (3, 5). The N&B procedure enables detection of oligomeric units of aggregates or oligomers (6). Importantly, ICS includes two kinds of flexibility: the spatial and temporal persistence of the fluorescence intensity, which correspond to size and residence time of molecules or particles, respectively (7). Therefore, ICS is an attractive method for the quantitative determination of the size of the foci or IBs. Optineurin (OPTN), which is also called as FIP2 or NRP, is a multidomain protein encoded by the OPTN gene; this protein comprises several coiled-coil, leucine zipper, zinc finger and ubiquitin (Ub)-associating region, and mediates various functions by interacting with numerous different proteins. OPTN was initially identified as a regulator of NF-κB and interferon signalling; however, recently, its role in post-Golgi vesicular trafficking and selective autophagy, including mitophagy and xenophagy, has been reported (8, 9). Thus, the majority of OPTN foci are autophagosome- or trafficking vesicle-associated (10–12). Mutations in OPTN that lead to amino acid substitutions have been implicated in several genetic diseases: amyotrophic lateral sclerosis (ALS) and primary open-angle glaucoma (POAG) (9, 13, 14). From familial ALS patients, Q398X nonsense or E478G missense mutation in OPTN gene have been identified (10). In motor neurons from patients with sporadic and familial ALS, Ub- and TDP-43-positive OPTN IBs have been observed (10). The ALS-linked Q398X and E478G mutants of OPTN cause defects of vesicular trafficking (15). Since the Q398X mutant of OPTN does not efficiently expressed probably because of nonsense-mediated mRNA decay (10), aggregation property of the E478G missense mutation lacking binding ability to poly-Ub chain has been investigated. On the other hand, overexpression of the most common E50K mutant of OPTN in POAG leads to progressive retinal degeneration in mice (16). In the POAG-linked E50K mutant of OPTN, enlarged foci are often observed (11, 12, 16). However, the quantitative size comparison of OPTN foci and IBs has not been reported to date. Therefore, we established an ICS-based procedure to quantitatively compare the diameter of the foci and IBs of the wild-type (WT) and disease-associated mutants of OPTN (E50K and E478G). Materials and Methods Preparation of plasmids coding for GFP-tagged OPTN The plasmid coding for the complementary DNA sequence of OPTN was obtained from the MGC collection (BC032762; Thermo Fisher Scientific, Waltham, MA). The cDNA lacks 628–645 bp relative to the splicing variant type 1 of the OPTN transcript (NM_001008211); thus, in order to complement the lacking sequence, double-stranded synthetic oligo (5′-gggcccacgagaacagtctccactggcacggcattgtctaaatataggagcagatct-3′) was inserted into the original plasmid via ApaI and BglII restriction sites. A clone carrying the correct sequence was selected using a genetic analyser (Applied Biosystems, Waltham, MA) and used for PCR as a template, with forward (5′-agcagaattctatgtcccatcaacctctcagctgcctc-3′) and reverse (5′-tgctggatcccgaatgatgcaatccatcacgtgaatc-3′) primers. The amplified fragments were cut and inserted into the pBluescript II KS(+) vector. The plasmids coding for OPTN with E50K and E478G amino acid substitutions were constructed using the PCR-based mutagenesis method with the following primers: E50K, 5′-gagctcctgaccAagaaccaccagc-3′ and 5′-gctggtggttctTggtcaggagctc-3′; E478G, 5′-gattttcatgctgGaagagcagcgag-3′ and 5′-ctcgctgctcttCcagcatgaaaatc-3′. In order to construct plasmids coding for green fluorescent protein (GFP)-tagged OPTN, fragments encoding OPTN-WT, -E50K and -E478G were cut using EcoRI and BamHI, and inserted into the pmeGFP-C1 vector (5) (GFP-OPTN-WT, -E50K, and -E478G, respectively). Cell culture and transfection Mouse neuroblastoma Neuro2A cells were maintained as reported previously (5), and 2.0 × 105 cells were grown in a glass-based dish (#3910-035, Asahi-Techno-glass, Shizuoka, Japan) for 16 h prior to transfection. The plasmid coding for GFP-OPTN (1.0 μg) was transfected into the cells with 2.0 μl of Lipofectamine 2000 (Thermo Fisher Scientific). After incubation of the cells for 24 h, subsequent experiments were performed. Quantification of efficiency of foci or IB formation To determine the proportion of the cells containing OPTN foci or IBs, confocal fluorescence images were acquired using an LSM 510 META (Carl Zeiss, Jena, Germany) through a C-Apochromat 40×/1.2 NA Korr UV-VIS-IR water-immersion objective (Carl Zeiss). A confocal pinhole diameter was adjusted to 71 μm. GFPs were excited at 488 nm and emission signals were detected using a 505-nm long-pass filter. The zoom factor was 1. The number of cells and cells containing foci or IBs was counted manually. Spatial ICS (SICS) Confocal fluorescence images were acquired using an LSM 510 META (Carl Zeiss) through a C-Apochromat 40×/1.2 NA Korr UV-VIS-IR water-immersion objective (Carl Zeiss). A confocal pinhole diameter was adjusted to 71 μm. GFPs were excited at 488 nm and emission signals were detected using a 505-nm long-pass filter. The zoom factor was 8 (the pixel size was 55 nm). After the measurement of cytoplasmic background fluorescence intensities (not included foci or IBs), the background was subtracted using ImageJ 1.51j8 (NIH, Bethesda, MD, USA). The two-dimensional (2D) spatial autocorrelation function (ACF) was defined as Eq. 1: gξ,η=δix,yδix+ξ,y+η (1) with the fluctuation in fluorescence, δi(x, y), given by Eq. 2: δix,y=ix,y-i(x,y) (2) where i(x, y) is the intensity at pixel (x, y) in the image; ξ and η are the lag pixels for x- and y-directions, respectively; and i is the average intensity of the image. These functions are typically calculated using Fourier transform methods with GNU Octave software version 4.2.1 platform (17) (Eq. 3): gξ,η=F-1Fix,y·F*ix,y (3) where F denotes the 2D spatial Fourier transform, F* is the complex conjugate of this transform and F-1 is the inverse 2D spatial Fourier transform. To obtain the distribution of the spatial ACF, modified 2D Gaussian function (Eq. 4) was used: g(ξ,η)=g0+A·exp[-12ξcosθ+ηsinθ-ξccosθ-ηcsinθwl2-12-ξsinθ+ηcosθ+ξcsinθ-ηccosθws]2 (4) where g0 is the base line; A is the maximum amplitude; ξc and ηc are the centre coordinates of the 2D distribution for ξ and η-direction, respectively; wl and ws are the major and minor standard deviations of the distribution, respectively; θ is the orientation factor. Non-linear fitting analysis and data visualization were performed using Origin Pro 2017 software (Origin Lab., Northampton, MA, USA). Major and minor radii of the foci or IBs were calibrated from wl and ws using Eq. 5 modified from linear regression equation (represented in Fig. 2D): ri=wi+0.06470.829 (5) where ri is the calibrated radii (i = l or s). Average major and minor diameters (dl and ds) were doubled rl and rs value, respectively. For demonstration of size determination using SICS, images including dot(s) were generated using ImageJ 1.50i and then modified with Photoshop CC platform (Adobe Systems Inc., San Jose, CA, USA). Images including random noise were created using a plugin for ImageJ (Salt and Pepper tool). Data analysis and statistics Representative images were processed using ImageJ 1.50i (NIH). Student’s t-tests were performed using MS-Excel in Office 365 ProPlus Ver. 1708 (Microsoft Corp., Redmond, WA, USA). Plots were created using Origin Pro 2017, and their appearance such as colour and axis labels was modified using Illustrator CC (Adobe Systems Inc.). Results Cytoplasmic foci or inclusion body formation of GFP-OPTN Confocal microscopy was used to evaluate the subcellular localization of GFP-OPTN. GFP-OPTN-WT, -E50K and -E478G were mainly distributed in the cytoplasm, and their abundance in the nucleus was low (Fig. 1A). Since various functional analysis using GFP-tagged OPTN has been performed (18, 19), there is little influence of GFP tag in the subcellular localization of OPTN. Although almost all cells expressing GFP-OPTN-WT and -E50K carried cytoplasmic foci, the proportion of cells carrying foci of GFP-OPTN-E50K was slightly higher than that carrying WT (Fig. 1B). The fluorescence intensity of the foci of the E50K mutant was brighter than that of WT (Fig. 1A); this may be attributed to the accumulation of high levels of the E50K mutant of OPTN in the foci. The proportion of cells carrying foci of GFP-OPTN-E478G was decreased, which agreed with the findings of previous reports (10, 12) (Fig. 1B); however, a proportion of cells expressing GFP-OPTN-E478G (5.8%) exhibited enlarged cytoplasmic structure (> 2 μm as a diameter) that shows extremely high fluorescence intensity compared with the WT and E50K mutant (Fig. 1A, white arrow; Fig. 1C). As intracytoplasmic IBs have been identified in motor neurons form patients with ALS (10), the enlarged structure of E478G mutant was defined as an IB. These results suggest that the disease-associated mutant of OPTN may exhibit altered accumulation in the cytoplasm. Therefore, we subsequently attempted to quantitatively measure the size of foci or IBs. Fig. 1 View largeDownload slide Formation of cytoplasmic foci or IBs in GFP-tagged WT and glaucoma- or ALS-associated mutant of OPTN. (A) Confocal fluorescence image of typical Neuro2A cells expressing GFP-OPTN-WT, -E50K and E478G. Cells expressing E478G mutant exhibit two distinct patterns: foci and IB (white arrow); bar = 5 μm. (B) The proportion of cells carrying cytoplasmic foci of GFP-OPTN-WT, -E50K and -E478G. (C) The proportion of cells carrying cytoplasmic IBs of GFP-OPTN-WT, -E50K and -E478G. (B and C) In three trials, the total number of analyzed cells: 59, 74 and 82 for WT; 94, 87 and 117 for E50K; and 125, 44 and 100 for E478G. Bars: mean ± SEM (n = 3). Inset numerical value indicates mean. Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001. Fig. 1 View largeDownload slide Formation of cytoplasmic foci or IBs in GFP-tagged WT and glaucoma- or ALS-associated mutant of OPTN. (A) Confocal fluorescence image of typical Neuro2A cells expressing GFP-OPTN-WT, -E50K and E478G. Cells expressing E478G mutant exhibit two distinct patterns: foci and IB (white arrow); bar = 5 μm. (B) The proportion of cells carrying cytoplasmic foci of GFP-OPTN-WT, -E50K and -E478G. (C) The proportion of cells carrying cytoplasmic IBs of GFP-OPTN-WT, -E50K and -E478G. (B and C) In three trials, the total number of analyzed cells: 59, 74 and 82 for WT; 94, 87 and 117 for E50K; and 125, 44 and 100 for E478G. Bars: mean ± SEM (n = 3). Inset numerical value indicates mean. Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001. Fig. 2 View largeDownload slide Demonstration of circle size measurement using SICS. (A) Original images including dot(s) (a–g) and 2D spatial autocorrelation function (ACF) images using SICS calculation (h–n) are shown. Line and cross indicate images including numerous circles with a radius of 5 pixels, which are drawn with 30 or −30 pixels in the x-direction around the centre and additionally in the y-direction at the same distance, respectively; bar = 10 pixels. The colour scale for the ACF image is shown in the right side of the ACF images. (B) Scheme of SICS analysis: (a) The ACF image is the same as in (m) in (A). (b) An image of the cropped centre region (c) and (d). Before and after non-linear fitting analysis using 2D Gaussian function (Eq. 4, see Materials and Methods section) on Origin 2017, respectively. Colour scales are shown at the right side of the ACF images. Contour lines in (d) indicate the height of fitted 2D Gaussian function from 0 to 0.85 in 0.05 increments. (C) Overlap of 1D-dropped intensity profile of a circle with 5-pixel radius (green) and Gaussian function when A is 1.0, ξc, ηc, θ, η, and g0 are 0, and standard deviation (w) is 4.02 in Eq. 4 (magenta). The intensity of the original circle was normalized to 0.5. Double arrow indicates the width of w and e−0.5. (D) Linear regression analysis between real radii of the circle and fitted w values; inset function indicates the calibration formula (blue line). (E) Fitted w value and calibrated radii of the circles using the equation in (D) were shown (light and dark grey, respectively). The inset graph indicates residuals between fitted w values and calibrated radii. Fig. 2 View largeDownload slide Demonstration of circle size measurement using SICS. (A) Original images including dot(s) (a–g) and 2D spatial autocorrelation function (ACF) images using SICS calculation (h–n) are shown. Line and cross indicate images including numerous circles with a radius of 5 pixels, which are drawn with 30 or −30 pixels in the x-direction around the centre and additionally in the y-direction at the same distance, respectively; bar = 10 pixels. The colour scale for the ACF image is shown in the right side of the ACF images. (B) Scheme of SICS analysis: (a) The ACF image is the same as in (m) in (A). (b) An image of the cropped centre region (c) and (d). Before and after non-linear fitting analysis using 2D Gaussian function (Eq. 4, see Materials and Methods section) on Origin 2017, respectively. Colour scales are shown at the right side of the ACF images. Contour lines in (d) indicate the height of fitted 2D Gaussian function from 0 to 0.85 in 0.05 increments. (C) Overlap of 1D-dropped intensity profile of a circle with 5-pixel radius (green) and Gaussian function when A is 1.0, ξc, ηc, θ, η, and g0 are 0, and standard deviation (w) is 4.02 in Eq. 4 (magenta). The intensity of the original circle was normalized to 0.5. Double arrow indicates the width of w and e−0.5. (D) Linear regression analysis between real radii of the circle and fitted w values; inset function indicates the calibration formula (blue line). (E) Fitted w value and calibrated radii of the circles using the equation in (D) were shown (light and dark grey, respectively). The inset graph indicates residuals between fitted w values and calibrated radii. Circle size measurement using SICS In order to determine the dot size using SICS, we constructed images containing circle(s) or dots with known radii (0.5, 2.5, 5, 7.5, 10 pixels) by image processing (Fig. 2A, a–g). The SICS images obtained from single-circle image showed strong amplitude at the centre of the image (Fig. 2A, h–k). The SICS images calculated from images in which circles were regularly arranged showed not only strong amplitude at the centre of the image but also ordered spread patterns according to the original circle arrangement (Fig. 2A, l–m). As the SICS image was calculated from the image containing randomly arranged dots (Fig. 2A, n), the ordered spread pattern in SICS image reflected the ordered arrangement of structures in the original image. To analyse average size of the circles in the original images, the centre region in the SICS image was cropped (Fig. 2B, a and b); then, non-linear fitting was performed using the 2D Gaussian function (Eq. 4; Fig. 2B, c and d). The fitted function successfully corresponded to the intensity distribution of the original circle; however, the obtained w value when using Eq. 4 (also known as the standard deviation of the Gaussian distribution) represents the distance from the distribution centre at the position at which the maximum peak of the Gaussian function decreases e−0.5 times, and therefore does not represent the radius of the circle (Fig. 2C). Therefore, in order to determine the radius of the circle, we obtained a calibration formula between real radii of the circles and fitted w values using linear regression analysis (Fig. 2D). Using the calibration formula, the radii of the circles were successfully obtained with an error of less than 0.2 pixels (Fig. 2E). Therefore, using SICS, the mean radius of foci in the cells may be determined. Measurement of OPTN foci or IBs using SICS in living cells To quantitatively determine the shape of the foci or IBs of OPTN, we adopted SICS analysis. As shown in the previous section and Fig. 2, SICS analysis is able to determine the radius of the structure in the image. However, the centre peak of spatial ACF was dramatically affected by the fluorescence intensity distribution of GFP-OPTN throughout the cytoplasm (Supplementary Fig. S1); thus, fluorescence intensity of the cytoplasmic region that did not form foci or IBs was subtracted from the original confocal images; then, SICS analysis was performed (Fig. 3A). The spatial ACFs of cells containing OPTN foci showed the distribution of low amplitude in addition to high amplitude at the centre (Fig. 3A, i–k). The two types of distribution of amplitude may be attributable to the average size of each foci, as well as to the foci being distributed and localized in the cytoplasm with a certain order. In contrast, the spatial ACF of IB of the E478G mutant of OPTN showed only a high peak at the centre (Fig. 3A, l). This may be attributed to the relatively high fluorescence intensity of the IBs. Moreover, the directionality in which the foci were distributed in the fluorescence image is also reflected in the directionality of the intensity distribution in the spatial ACF (Fig. 3A, i–k), suggesting that it would be possible to determine the average space in which foci are distributed in the cytoplasm. Fig. 3 View largeDownload slide Comparison of the size of foci or IBs of ALS- and POAG-linked mutant of OPTN using SICS. (A) Confocal fluorescence, cytoplasmic background intensity-subtracted, spatial ACF images are shown. Colour scales are indicated at the right side of the images; bar = 5 μm. (B) Top view of 2D Gaussian function (Eq. 4) for the ξ and η coordinates when ξc and ηc is 0. wl and ws are major and minor standard deviation, respectively. θ is the orientation factor. (C) Major and minor diameter (dl and ds) are plotted (magenta and cyan, respectively). The diameters are twice the calibrated radii from wl and ws using the Eq. 5; bars: means ± SEM (n = 10). Inset numerical values indicate means. Student’s t-test: ***P < 0.001. The number sign on E478G (IB) are significance when compared with the others (P < 0.001). (D) Ratios of the diameter in each cell are plotted; ns, no significant difference. Fig. 3 View largeDownload slide Comparison of the size of foci or IBs of ALS- and POAG-linked mutant of OPTN using SICS. (A) Confocal fluorescence, cytoplasmic background intensity-subtracted, spatial ACF images are shown. Colour scales are indicated at the right side of the images; bar = 5 μm. (B) Top view of 2D Gaussian function (Eq. 4) for the ξ and η coordinates when ξc and ηc is 0. wl and ws are major and minor standard deviation, respectively. θ is the orientation factor. (C) Major and minor diameter (dl and ds) are plotted (magenta and cyan, respectively). The diameters are twice the calibrated radii from wl and ws using the Eq. 5; bars: means ± SEM (n = 10). Inset numerical values indicate means. Student’s t-test: ***P < 0.001. The number sign on E478G (IB) are significance when compared with the others (P < 0.001). (D) Ratios of the diameter in each cell are plotted; ns, no significant difference. Next, to quantify the distribution of the amplitude at the centre in spatial ACF, 2D Gaussian function, including the orientation factor, was employed (Eq. 4; Fig. 3B). As the orientation of the cells was not unified during image acquisition, the orientation factor served to increase the accuracy of fitting the Gaussian function to the distribution of the peaks in spatial ACF. Using calculated major and minor standard deviations from the fitting and calibrated function obtained in Fig. 2D, the average major and minor diameters (dl and ds, respectively) of the foci and IBs of GFP-OPTN in living cells were determined. Both the dl and ds of foci of GFP-OPTN-E50K were longer than that of the WT and E478G mutant (Fig. 3C). The dl and ds of foci of the E478G mutant showed a tendency to be short (Fig. 3C), suggesting that this mutant may be difficult to assemble compared with WT. The dl and ds of IBs of the E478G mutant were dramatically longer than the foci (Fig. 3C), suggesting that the IBs may contain large amounts of OPTN compared with the foci. The ratio between the dl and ds of the foci showed an increasing tendency (Fig. 3D); this was considered to occur because a portion of the foci was adjoined, even though the shape of the foci was almost uniform. Moreover, a proportion of the IBs of the E478G mutant showed a large ratio between dl and ds, and there were few cases where the ratio showed values close to 1 (Fig. 3D), suggesting that the shape of the IBs of the E478G mutant may not be a uniform sphere. Discussion In the present work, we established a spatial fluorescence fluctuation-based procedure to determine the average size of intracytoplasmic foci or IBs of OPTN in cells. The procedure may be adapted to other proteins that form granules, foci or IBs, in fixed or living cells. The method additionally enables the quantitative comparison of the sizes of foci and IBs in a facile and rapid manner. In particular, it is effective for rapid determination of average size of multiple foci; thus, SICS analysis may be applied to high-throughput detection of the structure and shape of cells. Using images with dots of known sizes, the accuracy of the radii of the dots determined from SICS analysis was determined to be less than 0.2 pixel (Fig. 2). This corresponds to 11 nm in terms of pixel size, as shown in Fig. 3. Therefore, the accuracy is sufficient to determine the size at the sub-diffraction limit when using a confocal fluorescence microscope. OPTN is associated with vesicular trafficking processes such as the maintenance of Golgi apparatus, exocytosis and autophagy (9, 13). Almost all cells expressing OPTN-WT possessed cytoplasmic foci (Fig. 1); thus, foci may be involved in vesicular trafficking including autophagy. TBK1, a kinase for OPTN regulation, is known to colocalize with OPTN foci (11); thus, the foci are thought to provide an environment suited to effective enzymatic activity in the cytoplasm by facilitating concentration of the proteins. Almost all cells expressing WT and the E50K mutant of OPTN carried cytoplasmic foci; however, the proportion of cells was decreased in the E478G mutant; in addition, the size of E478G foci tended to be slightly smaller than that of the WT. As the E478G mutant binds more weakly to linear (M1)-linked poly-Ub than does the WT (20), the decrease in the proportion of the cells carrying the E478G foci may be attributed to the low binding efficiency between OPTN-E478G and the M1-linked Ub chain. Moreover, the E478G foci were not entirely lost, and the E478G mutant of OPTN retained the ability to bind the Lys63 (K63)-linked Ub chain (20). K63 chains act as proteasome independent signals for endocytosis, DNA damage responses and immune responses (21). Therefore, the E478G foci may represent the position of the scaffold associated with signal transduction via the K63-linked Ub chain, and the scaffold size would be smaller than that associated with the M1-linked chain. A proportion of cells expressing E478G mutant harboured structures with extremely large diameter (2.7–3.4 μm). The IBs of OPTN in motor neurons from patients with ALS colocalize with Ub and TDP-43 aggregates, even though the E478G mutant does not bind the M1-linked Ub chain. Hence, OPTN in the IBs may be misfolded, and the IBs are considered to sequestrate several protein aggregates. The diameter of the E50K foci was enlarged (1.2–1.3 μm); however, these foci were dramatically smaller than the IBs of the E478G mutant. The E50K mutation altered the oligomeric state of OPTN and induced its tetramerization (11). The enlargement of the E50K foci may occur because the oligomeric OPTN efficiently sequestrates several OPTN-binding proteins such as LC3, TBC1D17 and TBK1 (11, 15, 22). The increase in the ratio between the dl and ds of the foci (Fig. 3D) enables the proximity of the OPTN-WT foci to be determined. As OPTN is involved in vesicular trafficking and mitophagy in the cytoplasm (19, 23), the intermediate fusion and fission of the membrane vesicle containing OPTN may be visualized. However, individual E478G IBs were observed to possess an ellipse-like shape (Fig. 3C and D), suggesting that misfolded OPTN may accumulate non-uniformly in the IBs. The ellipse-like shape would be involved in the physical properties of aggregates in the IBs such as solid or gel phase. Accordingly, the ALS-associated E478G mutant and glaucoma-associated E50K mutant of OPTN may possess distinct functions, especially with regard to vesicular trafficking and/or signal transduction. The present method using SICS enables facile determination of the average size of foci such as granules, autophagosomes, endosomes and other physiological structures in the cell. In future, the function of the foci of OPTN should be clarified in detail. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We are grateful to Dr J. Yamamoto and Mr R. Fukushima for their assistance with SICS analysis. Funding A.K. was supported by the Japan Society for Promotion of Science (JSPS) Grant-in-Aid for the Promotion of Joint International Research (Fostering Joint International Research) (16KK0156); the JSPS Grant-in-Aid for Scientific Research (C) (#26440090); by the Grant-in-Aid of The Nakabayashi Trust for ALS Research (Tokyo, Japan); by the Grant-in-Aid of the Japan Amyotrophic Lateral Sclerosis Association (JALSA, Tokyo, Japan) for ALS research and by a grant from the Akiyama Life Science Foundation (Sapporo, Japan). Conflict of Interest None declared. References 1 Kaksonen M. , Roux A. ( 2018 ) Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19 , 313 – 326 2 Witkos T.M. , Lowe M. ( 2017 ) Recognition and tethering of transport vesicles at the Golgi apparatus . Curr. Opin. Cell Biol. 47 , 16 – 23 Google Scholar CrossRef Search ADS PubMed 3 Kitamura A. , Nagata K. , Kinjo M. 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Cells 3 , 1027 – 1088 Google Scholar CrossRef Search ADS PubMed 22 Chalasani M.L. , Kumari A. , Radha V. , Swarup G. ( 2014 ) E50K-OPTN-induced retinal cell death involves the Rab GTPase-activating protein, TBC1D17 mediated block in autophagy . PLoS One 9 , e95758 Google Scholar CrossRef Search ADS PubMed 23 Chen K. , Dai H. , Yuan J. , Chen J. , Lin L. , Zhang W. , Wang L. , Zhang J. , Li K. , He Y. ( 2018 ) Optineurin-mediated mitophagy protects renal tubular epithelial cells against accelerated senescence in diabetic nephropathy . Cell death Dis . 9 , 105 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ACF autocorrelation function ALS amyotrophic lateral sclerosis GFP green fluorescent protein IB inclusion body ICS image correlation spectroscopy OPTN optineurin POAG primary open-angle glaucoma SICS spatial ICS © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy044
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Abstract

Abstract 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. foci, image correlation spectroscopy, inclusion body, optineurin, vesicular trafficking Vesicular trafficking, which involves endocytosis, exocytosis and autophagy, is a well-conserved and highly regulated cellular process in eukaryotic cells; this process enables targeted delivery of specific components to selective compartments such as the extracellular space, endosome and lysosome. Adapter or regulatory proteins play an important role in the efficient delivery of these components. Regulatory proteins temporarily accumulate on vesicular membranes to regulate functions (1, 2). When diffusing proteins temporarily attach to the membrane, their localization on vesicles may be observed as foci in the cytoplasm in the autophagosomes, endosomes and endoplasmic reticulum exit sites. However, misfolded or denatured proteins in the cells often form aggregates that accumulate in the inclusion bodies (IBs) (3, 4). The foci and IBs may be observed by optical microscopy as well as electron microscopy; accordingly, the characterization of the foci and IBs via size quantification is a straightforward procedure. However, the measurement of foci diameter individually is complex; therefore, a facile method for the estimation of the diameter of foci and IBs is important. Fluorescence fluctuation-based methods, such as fluorescence correlation spectroscopy (FCS), image correlation spectroscopy (ICS) and number and brightness (N&B) procedure, are used for various analyses for accumulated proteins in cell biology. In FCS and ICS, temporal and/or spatial correlation functions reflecting molecular size or dynamics are calculated from the recorded fluorescence fluctuation. Specifically, FCS is suitable for measuring the diffusion rate of molecules in living cells and solution (3, 5). The N&B procedure enables detection of oligomeric units of aggregates or oligomers (6). Importantly, ICS includes two kinds of flexibility: the spatial and temporal persistence of the fluorescence intensity, which correspond to size and residence time of molecules or particles, respectively (7). Therefore, ICS is an attractive method for the quantitative determination of the size of the foci or IBs. Optineurin (OPTN), which is also called as FIP2 or NRP, is a multidomain protein encoded by the OPTN gene; this protein comprises several coiled-coil, leucine zipper, zinc finger and ubiquitin (Ub)-associating region, and mediates various functions by interacting with numerous different proteins. OPTN was initially identified as a regulator of NF-κB and interferon signalling; however, recently, its role in post-Golgi vesicular trafficking and selective autophagy, including mitophagy and xenophagy, has been reported (8, 9). Thus, the majority of OPTN foci are autophagosome- or trafficking vesicle-associated (10–12). Mutations in OPTN that lead to amino acid substitutions have been implicated in several genetic diseases: amyotrophic lateral sclerosis (ALS) and primary open-angle glaucoma (POAG) (9, 13, 14). From familial ALS patients, Q398X nonsense or E478G missense mutation in OPTN gene have been identified (10). In motor neurons from patients with sporadic and familial ALS, Ub- and TDP-43-positive OPTN IBs have been observed (10). The ALS-linked Q398X and E478G mutants of OPTN cause defects of vesicular trafficking (15). Since the Q398X mutant of OPTN does not efficiently expressed probably because of nonsense-mediated mRNA decay (10), aggregation property of the E478G missense mutation lacking binding ability to poly-Ub chain has been investigated. On the other hand, overexpression of the most common E50K mutant of OPTN in POAG leads to progressive retinal degeneration in mice (16). In the POAG-linked E50K mutant of OPTN, enlarged foci are often observed (11, 12, 16). However, the quantitative size comparison of OPTN foci and IBs has not been reported to date. Therefore, we established an ICS-based procedure to quantitatively compare the diameter of the foci and IBs of the wild-type (WT) and disease-associated mutants of OPTN (E50K and E478G). Materials and Methods Preparation of plasmids coding for GFP-tagged OPTN The plasmid coding for the complementary DNA sequence of OPTN was obtained from the MGC collection (BC032762; Thermo Fisher Scientific, Waltham, MA). The cDNA lacks 628–645 bp relative to the splicing variant type 1 of the OPTN transcript (NM_001008211); thus, in order to complement the lacking sequence, double-stranded synthetic oligo (5′-gggcccacgagaacagtctccactggcacggcattgtctaaatataggagcagatct-3′) was inserted into the original plasmid via ApaI and BglII restriction sites. A clone carrying the correct sequence was selected using a genetic analyser (Applied Biosystems, Waltham, MA) and used for PCR as a template, with forward (5′-agcagaattctatgtcccatcaacctctcagctgcctc-3′) and reverse (5′-tgctggatcccgaatgatgcaatccatcacgtgaatc-3′) primers. The amplified fragments were cut and inserted into the pBluescript II KS(+) vector. The plasmids coding for OPTN with E50K and E478G amino acid substitutions were constructed using the PCR-based mutagenesis method with the following primers: E50K, 5′-gagctcctgaccAagaaccaccagc-3′ and 5′-gctggtggttctTggtcaggagctc-3′; E478G, 5′-gattttcatgctgGaagagcagcgag-3′ and 5′-ctcgctgctcttCcagcatgaaaatc-3′. In order to construct plasmids coding for green fluorescent protein (GFP)-tagged OPTN, fragments encoding OPTN-WT, -E50K and -E478G were cut using EcoRI and BamHI, and inserted into the pmeGFP-C1 vector (5) (GFP-OPTN-WT, -E50K, and -E478G, respectively). Cell culture and transfection Mouse neuroblastoma Neuro2A cells were maintained as reported previously (5), and 2.0 × 105 cells were grown in a glass-based dish (#3910-035, Asahi-Techno-glass, Shizuoka, Japan) for 16 h prior to transfection. The plasmid coding for GFP-OPTN (1.0 μg) was transfected into the cells with 2.0 μl of Lipofectamine 2000 (Thermo Fisher Scientific). After incubation of the cells for 24 h, subsequent experiments were performed. Quantification of efficiency of foci or IB formation To determine the proportion of the cells containing OPTN foci or IBs, confocal fluorescence images were acquired using an LSM 510 META (Carl Zeiss, Jena, Germany) through a C-Apochromat 40×/1.2 NA Korr UV-VIS-IR water-immersion objective (Carl Zeiss). A confocal pinhole diameter was adjusted to 71 μm. GFPs were excited at 488 nm and emission signals were detected using a 505-nm long-pass filter. The zoom factor was 1. The number of cells and cells containing foci or IBs was counted manually. Spatial ICS (SICS) Confocal fluorescence images were acquired using an LSM 510 META (Carl Zeiss) through a C-Apochromat 40×/1.2 NA Korr UV-VIS-IR water-immersion objective (Carl Zeiss). A confocal pinhole diameter was adjusted to 71 μm. GFPs were excited at 488 nm and emission signals were detected using a 505-nm long-pass filter. The zoom factor was 8 (the pixel size was 55 nm). After the measurement of cytoplasmic background fluorescence intensities (not included foci or IBs), the background was subtracted using ImageJ 1.51j8 (NIH, Bethesda, MD, USA). The two-dimensional (2D) spatial autocorrelation function (ACF) was defined as Eq. 1: gξ,η=δix,yδix+ξ,y+η (1) with the fluctuation in fluorescence, δi(x, y), given by Eq. 2: δix,y=ix,y-i(x,y) (2) where i(x, y) is the intensity at pixel (x, y) in the image; ξ and η are the lag pixels for x- and y-directions, respectively; and i is the average intensity of the image. These functions are typically calculated using Fourier transform methods with GNU Octave software version 4.2.1 platform (17) (Eq. 3): gξ,η=F-1Fix,y·F*ix,y (3) where F denotes the 2D spatial Fourier transform, F* is the complex conjugate of this transform and F-1 is the inverse 2D spatial Fourier transform. To obtain the distribution of the spatial ACF, modified 2D Gaussian function (Eq. 4) was used: g(ξ,η)=g0+A·exp[-12ξcosθ+ηsinθ-ξccosθ-ηcsinθwl2-12-ξsinθ+ηcosθ+ξcsinθ-ηccosθws]2 (4) where g0 is the base line; A is the maximum amplitude; ξc and ηc are the centre coordinates of the 2D distribution for ξ and η-direction, respectively; wl and ws are the major and minor standard deviations of the distribution, respectively; θ is the orientation factor. Non-linear fitting analysis and data visualization were performed using Origin Pro 2017 software (Origin Lab., Northampton, MA, USA). Major and minor radii of the foci or IBs were calibrated from wl and ws using Eq. 5 modified from linear regression equation (represented in Fig. 2D): ri=wi+0.06470.829 (5) where ri is the calibrated radii (i = l or s). Average major and minor diameters (dl and ds) were doubled rl and rs value, respectively. For demonstration of size determination using SICS, images including dot(s) were generated using ImageJ 1.50i and then modified with Photoshop CC platform (Adobe Systems Inc., San Jose, CA, USA). Images including random noise were created using a plugin for ImageJ (Salt and Pepper tool). Data analysis and statistics Representative images were processed using ImageJ 1.50i (NIH). Student’s t-tests were performed using MS-Excel in Office 365 ProPlus Ver. 1708 (Microsoft Corp., Redmond, WA, USA). Plots were created using Origin Pro 2017, and their appearance such as colour and axis labels was modified using Illustrator CC (Adobe Systems Inc.). Results Cytoplasmic foci or inclusion body formation of GFP-OPTN Confocal microscopy was used to evaluate the subcellular localization of GFP-OPTN. GFP-OPTN-WT, -E50K and -E478G were mainly distributed in the cytoplasm, and their abundance in the nucleus was low (Fig. 1A). Since various functional analysis using GFP-tagged OPTN has been performed (18, 19), there is little influence of GFP tag in the subcellular localization of OPTN. Although almost all cells expressing GFP-OPTN-WT and -E50K carried cytoplasmic foci, the proportion of cells carrying foci of GFP-OPTN-E50K was slightly higher than that carrying WT (Fig. 1B). The fluorescence intensity of the foci of the E50K mutant was brighter than that of WT (Fig. 1A); this may be attributed to the accumulation of high levels of the E50K mutant of OPTN in the foci. The proportion of cells carrying foci of GFP-OPTN-E478G was decreased, which agreed with the findings of previous reports (10, 12) (Fig. 1B); however, a proportion of cells expressing GFP-OPTN-E478G (5.8%) exhibited enlarged cytoplasmic structure (> 2 μm as a diameter) that shows extremely high fluorescence intensity compared with the WT and E50K mutant (Fig. 1A, white arrow; Fig. 1C). As intracytoplasmic IBs have been identified in motor neurons form patients with ALS (10), the enlarged structure of E478G mutant was defined as an IB. These results suggest that the disease-associated mutant of OPTN may exhibit altered accumulation in the cytoplasm. Therefore, we subsequently attempted to quantitatively measure the size of foci or IBs. Fig. 1 View largeDownload slide Formation of cytoplasmic foci or IBs in GFP-tagged WT and glaucoma- or ALS-associated mutant of OPTN. (A) Confocal fluorescence image of typical Neuro2A cells expressing GFP-OPTN-WT, -E50K and E478G. Cells expressing E478G mutant exhibit two distinct patterns: foci and IB (white arrow); bar = 5 μm. (B) The proportion of cells carrying cytoplasmic foci of GFP-OPTN-WT, -E50K and -E478G. (C) The proportion of cells carrying cytoplasmic IBs of GFP-OPTN-WT, -E50K and -E478G. (B and C) In three trials, the total number of analyzed cells: 59, 74 and 82 for WT; 94, 87 and 117 for E50K; and 125, 44 and 100 for E478G. Bars: mean ± SEM (n = 3). Inset numerical value indicates mean. Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001. Fig. 1 View largeDownload slide Formation of cytoplasmic foci or IBs in GFP-tagged WT and glaucoma- or ALS-associated mutant of OPTN. (A) Confocal fluorescence image of typical Neuro2A cells expressing GFP-OPTN-WT, -E50K and E478G. Cells expressing E478G mutant exhibit two distinct patterns: foci and IB (white arrow); bar = 5 μm. (B) The proportion of cells carrying cytoplasmic foci of GFP-OPTN-WT, -E50K and -E478G. (C) The proportion of cells carrying cytoplasmic IBs of GFP-OPTN-WT, -E50K and -E478G. (B and C) In three trials, the total number of analyzed cells: 59, 74 and 82 for WT; 94, 87 and 117 for E50K; and 125, 44 and 100 for E478G. Bars: mean ± SEM (n = 3). Inset numerical value indicates mean. Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001. Fig. 2 View largeDownload slide Demonstration of circle size measurement using SICS. (A) Original images including dot(s) (a–g) and 2D spatial autocorrelation function (ACF) images using SICS calculation (h–n) are shown. Line and cross indicate images including numerous circles with a radius of 5 pixels, which are drawn with 30 or −30 pixels in the x-direction around the centre and additionally in the y-direction at the same distance, respectively; bar = 10 pixels. The colour scale for the ACF image is shown in the right side of the ACF images. (B) Scheme of SICS analysis: (a) The ACF image is the same as in (m) in (A). (b) An image of the cropped centre region (c) and (d). Before and after non-linear fitting analysis using 2D Gaussian function (Eq. 4, see Materials and Methods section) on Origin 2017, respectively. Colour scales are shown at the right side of the ACF images. Contour lines in (d) indicate the height of fitted 2D Gaussian function from 0 to 0.85 in 0.05 increments. (C) Overlap of 1D-dropped intensity profile of a circle with 5-pixel radius (green) and Gaussian function when A is 1.0, ξc, ηc, θ, η, and g0 are 0, and standard deviation (w) is 4.02 in Eq. 4 (magenta). The intensity of the original circle was normalized to 0.5. Double arrow indicates the width of w and e−0.5. (D) Linear regression analysis between real radii of the circle and fitted w values; inset function indicates the calibration formula (blue line). (E) Fitted w value and calibrated radii of the circles using the equation in (D) were shown (light and dark grey, respectively). The inset graph indicates residuals between fitted w values and calibrated radii. Fig. 2 View largeDownload slide Demonstration of circle size measurement using SICS. (A) Original images including dot(s) (a–g) and 2D spatial autocorrelation function (ACF) images using SICS calculation (h–n) are shown. Line and cross indicate images including numerous circles with a radius of 5 pixels, which are drawn with 30 or −30 pixels in the x-direction around the centre and additionally in the y-direction at the same distance, respectively; bar = 10 pixels. The colour scale for the ACF image is shown in the right side of the ACF images. (B) Scheme of SICS analysis: (a) The ACF image is the same as in (m) in (A). (b) An image of the cropped centre region (c) and (d). Before and after non-linear fitting analysis using 2D Gaussian function (Eq. 4, see Materials and Methods section) on Origin 2017, respectively. Colour scales are shown at the right side of the ACF images. Contour lines in (d) indicate the height of fitted 2D Gaussian function from 0 to 0.85 in 0.05 increments. (C) Overlap of 1D-dropped intensity profile of a circle with 5-pixel radius (green) and Gaussian function when A is 1.0, ξc, ηc, θ, η, and g0 are 0, and standard deviation (w) is 4.02 in Eq. 4 (magenta). The intensity of the original circle was normalized to 0.5. Double arrow indicates the width of w and e−0.5. (D) Linear regression analysis between real radii of the circle and fitted w values; inset function indicates the calibration formula (blue line). (E) Fitted w value and calibrated radii of the circles using the equation in (D) were shown (light and dark grey, respectively). The inset graph indicates residuals between fitted w values and calibrated radii. Circle size measurement using SICS In order to determine the dot size using SICS, we constructed images containing circle(s) or dots with known radii (0.5, 2.5, 5, 7.5, 10 pixels) by image processing (Fig. 2A, a–g). The SICS images obtained from single-circle image showed strong amplitude at the centre of the image (Fig. 2A, h–k). The SICS images calculated from images in which circles were regularly arranged showed not only strong amplitude at the centre of the image but also ordered spread patterns according to the original circle arrangement (Fig. 2A, l–m). As the SICS image was calculated from the image containing randomly arranged dots (Fig. 2A, n), the ordered spread pattern in SICS image reflected the ordered arrangement of structures in the original image. To analyse average size of the circles in the original images, the centre region in the SICS image was cropped (Fig. 2B, a and b); then, non-linear fitting was performed using the 2D Gaussian function (Eq. 4; Fig. 2B, c and d). The fitted function successfully corresponded to the intensity distribution of the original circle; however, the obtained w value when using Eq. 4 (also known as the standard deviation of the Gaussian distribution) represents the distance from the distribution centre at the position at which the maximum peak of the Gaussian function decreases e−0.5 times, and therefore does not represent the radius of the circle (Fig. 2C). Therefore, in order to determine the radius of the circle, we obtained a calibration formula between real radii of the circles and fitted w values using linear regression analysis (Fig. 2D). Using the calibration formula, the radii of the circles were successfully obtained with an error of less than 0.2 pixels (Fig. 2E). Therefore, using SICS, the mean radius of foci in the cells may be determined. Measurement of OPTN foci or IBs using SICS in living cells To quantitatively determine the shape of the foci or IBs of OPTN, we adopted SICS analysis. As shown in the previous section and Fig. 2, SICS analysis is able to determine the radius of the structure in the image. However, the centre peak of spatial ACF was dramatically affected by the fluorescence intensity distribution of GFP-OPTN throughout the cytoplasm (Supplementary Fig. S1); thus, fluorescence intensity of the cytoplasmic region that did not form foci or IBs was subtracted from the original confocal images; then, SICS analysis was performed (Fig. 3A). The spatial ACFs of cells containing OPTN foci showed the distribution of low amplitude in addition to high amplitude at the centre (Fig. 3A, i–k). The two types of distribution of amplitude may be attributable to the average size of each foci, as well as to the foci being distributed and localized in the cytoplasm with a certain order. In contrast, the spatial ACF of IB of the E478G mutant of OPTN showed only a high peak at the centre (Fig. 3A, l). This may be attributed to the relatively high fluorescence intensity of the IBs. Moreover, the directionality in which the foci were distributed in the fluorescence image is also reflected in the directionality of the intensity distribution in the spatial ACF (Fig. 3A, i–k), suggesting that it would be possible to determine the average space in which foci are distributed in the cytoplasm. Fig. 3 View largeDownload slide Comparison of the size of foci or IBs of ALS- and POAG-linked mutant of OPTN using SICS. (A) Confocal fluorescence, cytoplasmic background intensity-subtracted, spatial ACF images are shown. Colour scales are indicated at the right side of the images; bar = 5 μm. (B) Top view of 2D Gaussian function (Eq. 4) for the ξ and η coordinates when ξc and ηc is 0. wl and ws are major and minor standard deviation, respectively. θ is the orientation factor. (C) Major and minor diameter (dl and ds) are plotted (magenta and cyan, respectively). The diameters are twice the calibrated radii from wl and ws using the Eq. 5; bars: means ± SEM (n = 10). Inset numerical values indicate means. Student’s t-test: ***P < 0.001. The number sign on E478G (IB) are significance when compared with the others (P < 0.001). (D) Ratios of the diameter in each cell are plotted; ns, no significant difference. Fig. 3 View largeDownload slide Comparison of the size of foci or IBs of ALS- and POAG-linked mutant of OPTN using SICS. (A) Confocal fluorescence, cytoplasmic background intensity-subtracted, spatial ACF images are shown. Colour scales are indicated at the right side of the images; bar = 5 μm. (B) Top view of 2D Gaussian function (Eq. 4) for the ξ and η coordinates when ξc and ηc is 0. wl and ws are major and minor standard deviation, respectively. θ is the orientation factor. (C) Major and minor diameter (dl and ds) are plotted (magenta and cyan, respectively). The diameters are twice the calibrated radii from wl and ws using the Eq. 5; bars: means ± SEM (n = 10). Inset numerical values indicate means. Student’s t-test: ***P < 0.001. The number sign on E478G (IB) are significance when compared with the others (P < 0.001). (D) Ratios of the diameter in each cell are plotted; ns, no significant difference. Next, to quantify the distribution of the amplitude at the centre in spatial ACF, 2D Gaussian function, including the orientation factor, was employed (Eq. 4; Fig. 3B). As the orientation of the cells was not unified during image acquisition, the orientation factor served to increase the accuracy of fitting the Gaussian function to the distribution of the peaks in spatial ACF. Using calculated major and minor standard deviations from the fitting and calibrated function obtained in Fig. 2D, the average major and minor diameters (dl and ds, respectively) of the foci and IBs of GFP-OPTN in living cells were determined. Both the dl and ds of foci of GFP-OPTN-E50K were longer than that of the WT and E478G mutant (Fig. 3C). The dl and ds of foci of the E478G mutant showed a tendency to be short (Fig. 3C), suggesting that this mutant may be difficult to assemble compared with WT. The dl and ds of IBs of the E478G mutant were dramatically longer than the foci (Fig. 3C), suggesting that the IBs may contain large amounts of OPTN compared with the foci. The ratio between the dl and ds of the foci showed an increasing tendency (Fig. 3D); this was considered to occur because a portion of the foci was adjoined, even though the shape of the foci was almost uniform. Moreover, a proportion of the IBs of the E478G mutant showed a large ratio between dl and ds, and there were few cases where the ratio showed values close to 1 (Fig. 3D), suggesting that the shape of the IBs of the E478G mutant may not be a uniform sphere. Discussion In the present work, we established a spatial fluorescence fluctuation-based procedure to determine the average size of intracytoplasmic foci or IBs of OPTN in cells. The procedure may be adapted to other proteins that form granules, foci or IBs, in fixed or living cells. The method additionally enables the quantitative comparison of the sizes of foci and IBs in a facile and rapid manner. In particular, it is effective for rapid determination of average size of multiple foci; thus, SICS analysis may be applied to high-throughput detection of the structure and shape of cells. Using images with dots of known sizes, the accuracy of the radii of the dots determined from SICS analysis was determined to be less than 0.2 pixel (Fig. 2). This corresponds to 11 nm in terms of pixel size, as shown in Fig. 3. Therefore, the accuracy is sufficient to determine the size at the sub-diffraction limit when using a confocal fluorescence microscope. OPTN is associated with vesicular trafficking processes such as the maintenance of Golgi apparatus, exocytosis and autophagy (9, 13). Almost all cells expressing OPTN-WT possessed cytoplasmic foci (Fig. 1); thus, foci may be involved in vesicular trafficking including autophagy. TBK1, a kinase for OPTN regulation, is known to colocalize with OPTN foci (11); thus, the foci are thought to provide an environment suited to effective enzymatic activity in the cytoplasm by facilitating concentration of the proteins. Almost all cells expressing WT and the E50K mutant of OPTN carried cytoplasmic foci; however, the proportion of cells was decreased in the E478G mutant; in addition, the size of E478G foci tended to be slightly smaller than that of the WT. As the E478G mutant binds more weakly to linear (M1)-linked poly-Ub than does the WT (20), the decrease in the proportion of the cells carrying the E478G foci may be attributed to the low binding efficiency between OPTN-E478G and the M1-linked Ub chain. Moreover, the E478G foci were not entirely lost, and the E478G mutant of OPTN retained the ability to bind the Lys63 (K63)-linked Ub chain (20). K63 chains act as proteasome independent signals for endocytosis, DNA damage responses and immune responses (21). Therefore, the E478G foci may represent the position of the scaffold associated with signal transduction via the K63-linked Ub chain, and the scaffold size would be smaller than that associated with the M1-linked chain. A proportion of cells expressing E478G mutant harboured structures with extremely large diameter (2.7–3.4 μm). The IBs of OPTN in motor neurons from patients with ALS colocalize with Ub and TDP-43 aggregates, even though the E478G mutant does not bind the M1-linked Ub chain. Hence, OPTN in the IBs may be misfolded, and the IBs are considered to sequestrate several protein aggregates. The diameter of the E50K foci was enlarged (1.2–1.3 μm); however, these foci were dramatically smaller than the IBs of the E478G mutant. The E50K mutation altered the oligomeric state of OPTN and induced its tetramerization (11). The enlargement of the E50K foci may occur because the oligomeric OPTN efficiently sequestrates several OPTN-binding proteins such as LC3, TBC1D17 and TBK1 (11, 15, 22). The increase in the ratio between the dl and ds of the foci (Fig. 3D) enables the proximity of the OPTN-WT foci to be determined. As OPTN is involved in vesicular trafficking and mitophagy in the cytoplasm (19, 23), the intermediate fusion and fission of the membrane vesicle containing OPTN may be visualized. However, individual E478G IBs were observed to possess an ellipse-like shape (Fig. 3C and D), suggesting that misfolded OPTN may accumulate non-uniformly in the IBs. The ellipse-like shape would be involved in the physical properties of aggregates in the IBs such as solid or gel phase. Accordingly, the ALS-associated E478G mutant and glaucoma-associated E50K mutant of OPTN may possess distinct functions, especially with regard to vesicular trafficking and/or signal transduction. The present method using SICS enables facile determination of the average size of foci such as granules, autophagosomes, endosomes and other physiological structures in the cell. In future, the function of the foci of OPTN should be clarified in detail. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We are grateful to Dr J. Yamamoto and Mr R. Fukushima for their assistance with SICS analysis. 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All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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The Journal of BiochemistryOxford University Press

Published: Apr 19, 2018

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