Decision letter: An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Two-color fluorescence co-localization in 3D (three-dimension) has the potential to achieve accurate measurements at the nanometer length scale. Here, we optimized a 3D fluorescence co-localization method that uses mean values for chromatic aberration correction to yield the mean separation with ~10 nm accuracy between green and red fluorescently labeled protein epitopes within single human kinetochores. Accuracy depended critically on achieving small standard deviations in fluorescence centroid determination, chromatic aberration across the measurement field, and coverslip thickness. Computer simulations showed that large standard deviations in these parameters significantly increase 3D measurements from their true values. Our 3D results show that at metaphase, the protein linkage between CENP-A within the inner kinetochore and the microtubule-binding domain of the Ndc80 complex within the outer kinetochore is on average ~90 nm. The Ndc80 complex appears fully extended at metaphase and exhibits the same subunit structure in vivo as found in vitro by crystallography. https://doi.org/10.7554/eLife.32418.001 Introduction Confocal light microscopy (LM) is widely used for understanding protein localization, architecture, and functions in cells, but the maximum resolution without any image processing is still only ~200 nm in the x-y image plane and ~400 nm along the z-axis. The scale of many protein complexes is smaller than the diffraction limit of LM. In order to deduce the function of protein complexes, it is critical to develop a method to determine the three-dimensional (3D) locations of proteins within a complex in their native environment. Kinetochores are protein assemblies at the periphery of centromeric chromatin that have vital functions for achieving accurate chromosome segregation during mitosis and meiosis. A modified histone H3, CENP-A, within nucleosomes at centromeric chromatin marks the site of kinetochore assembly. CENP-A recruits ~25 different kinds of core-structural proteins including Constitutive Centromere Associated Protein Network (CCAN) proteins and outer kinetochore proteins called the KMN network (Knl1, Mis12 complex, and Ndc80 complex) (Cheeseman et al., 2006; McKinley and Cheeseman, 2016; Musacchio and Desai, 2017; Pesenti et al., 2016; Takeuchi and Fukagawa, 2012). Two important linkers in human kinetochores are CENP-C and CENP-T because they connect between centromeric chromatin and the KMN network proteins (Huis In 't Veld et al., 2016; Klare et al., 2015; Musacchio and Desai, 2017; Nishino et al., 2013; Nishino et al., 2012; Screpanti et al., 2011; Suzuki et al., 2015; Suzuki et al., 2014). The highly conserved Ndc80 complex (Hec1(Ndc80), Nuf2, Spc24, Spc25) is one of the best structurally characterized kinetochore proteins, and it has a major role in kinetochore microtubule (kMT) attachment (Ciferri et al., 2008; Musacchio and Desai, 2017). We previously reported a nm-scale map of how human kinetochore proteins are organized on average along the inner to outer kinetochore axis at metaphase, obtained using the ‘Delta’ two-color fluorescence co-localization method (Wan et al., 2009). The Delta method provided local chromatic aberration (CA) correction in the x-y image plane by measurements along the K-K axis between pairs of sister kinetochores. More recently, Fuller and Straight (2012) developed a 3D method (termed CICADA [Co-localization and In-situ Correction of Aberration for Distance Analysis]) to measure the mean separation of red and green fluorescent centroids at individual kinetochores while locally correcting for CA in the x, y, and z directions using a third color (Fuller and Straight, 2012). Their method yielded an identical value to what was obtained by the Delta method (Fuller and Straight, 2012; Suzuki et al., 2014; Wan et al., 2009). In contrast, a recent report by Smith et al., 2016 used a 3D measurement method to obtain mean separation values for protein domains within kinetochores that were significantly greater than our Delta values and the 3D value obtained by the CICADA method (Fuller and Straight, 2012; Smith et al., 2016; Suzuki et al., 2014; Wan et al., 2009). This group questioned the accuracy and significance of the Delta values because they were ‘1D measurements and neglected z-axis information’. In this paper, we have tested the above conclusion by optimizing a 2D and 3D fluorescence co-localization method to obtain the mean separation between the centroids of green and red foci with ~10 nm accuracy (Figure 1). The method depends on correcting CA based on mean values from a fluorescent standard, a method commonly used for fluorescence co-localization, but not previously optimized and rigorously analyzed for sources of error, as performed here. Computer simulations were used to understand how well the measurement method works and to find how accuracy is limited in our method. This study revealed major sources of error produced by standard deviations (SDs) in fluorescent centroid determination, CA correction, and coverslip thickness, as well as addressing several issues concerning fixation methods. We found that our 3D measurements applied to human kinetochores were similar to the Delta measurements and much shorter than those reported by Smith et al., 2016. Their larger measurements may be explained by much larger SDs in centroid determination and CA correction than typical of our experiments. We also used the known structure of the Ndc80 complex as a ‘Molecular Ruler’ to verify the accuracy of our measurement method, and to determine its subunit structure and conformation within the kinetochore. Figure 1 Download asset Open asset Principle of procedure used in this study. (A) Fixation: 3% PFA in PHEM buffer without any detergent was used as standard fixation in this study (Details in Text and Methods). (B) Staining: Fixed samples were immunofluorescently labeled with antibodies to enhance S/N. Samples were usually mounted with Prolong Gold. (C) Imaging was performed by spinning disk confocal microscopy (See Materials and methods for details). (D) For CA correction, Hec1-9G3 monoclonal antibody was labeled by green and red tagged secondary antibodies (Hec1-9G3-GR). (E) Measurements: X, Y, Z coordinates of the centroids of green and red kinetochore fluorescence were determined by 3D Gaussian fitting. Details of equations for separation measurements are in Text, Figures, and Methods. (F) Error analysis: computer simulations were used for error analysis of the method. The sources of potential errors in separation measurements were highlighted in pink underline with *. https://doi.org/10.7554/eLife.32418.002 Figure 1—source data 1 Pipeline for obtaining mean 3D separation measurements with nm-scale accuracy. Critical factors were: (1) prepare bright high signal to noise samples to reduce standard deviation (SD) of centroid determination; (2) use mounting media like ProLong Gold to suppress z-axis spherical aberration; (3) use 0.17 mm thick coverslips for all measurements including CA measurement; (4) align imaging system and image using only a small region within the center of camera to suppress SD of CA; and (5) use warmed PFA fixation without pre-extraction or detergent in fixation solution. https://doi.org/10.7554/eLife.32418.003 Download elife-32418-fig1-data1-v2.xlsx Results Principles of the method Sample preparation HeLa cells were grown on #1.5 coverslips, the design value for the microscope objective, to prevent Z-axis CA (chromatic aberration) (Figure 1A). Cells were fixed with paraformaldehyde (PFA) without pre-extraction or supplement of detergent in the PFA solution, and stained with primary antibodies using a protocol from previous studies (Figure 1A) (Suzuki et al., 2015; Suzuki et al., 2014; Suzuki et al., 2011). The concentration of green and red secondary antibodies was adjusted to give nearly equal fluorescence intensity (Figure 1B). Integrated kinetochore fluorescence above background (>100,000 photon counts per kinetochore) was important to minimize the SD of centroid determination (CDsd) close to 5 nm (Thompson et al., 2002). Labeled samples were typically mounted with ProLong Gold mounting media. For the cellular CA correction standard, a monoclonal antibody (9G3) was used to label Hec1 (hsNdc80) protein at a site just inside of its MT binding CH domain, and this antibody was double labeled with green and red fluorescence by secondary antibodies (called Hec1-9G3-GR). Imaging Imaging was performed using a confocal light microscope with a high NA objective lens (100x/1.4 NA) and a camera with small pixel size (64 nm, less than 1/3 resolution limit) (Figure 1C). Metaphase cells were positioned in the central field of view of the camera. Images were recorded at 200 nm intervals along the z-axis beginning just before the coverslip surface and extending to the top of the spindle to measure equal numbers of kinetochores below and above the middle of the spindle. Image analysis We selected sister kinetochores for measurement not only for Delta values (Figure 1D–E), but also to obtain the direction of the sister-sister kinetochore axis (K-K). For each kinetochore, we obtained the x, y, z coordinates for the centroids of green (Xg, Yg, Zg) and red (Xr, Yr, Zr) using 3D Gaussian fitting methods (Wan et al., 2009). The Sx, Sy, and Sz coordinates of the separation vector S between green and red foci were calculated from Sx = Xg-Xr-meanCAx (CAmx), Sy = Yg-Yr-meanCAy (CAmy), and Sz = Zg-Zr-meanCAz (CAmz) (Figure 1E), where the mean values of CAx, CAy, and CAz were obtained from kinetochores labeled with Hec1-9G3-GR within our measurement volume (Figure 1D). ‘Raw’ values for the mean separation vector lengths in 2D and 3D were calculated for each pair of green-red kinetochore labels as S(2D) = mean ((Sx2 + Sy2)0.5) and S(3D) = mean ((Sx2 + Sy2 + Sz2)0.5) from measurements of 200 to 400 kinetochores obtained from 5 to 20 different cells (Figure 1E). The mean of these ‘raw’ separation values (S(2D), and S(3D)) were over-estimates (Churchman et al., 2006; Churchman et al., 2005). In order to obtain more accurate mean separation values, a maximum likelihood method was applied to the raw separation data (called ML2D and ML3D) (Churchman et al., 2006; Churchman et al., 2005). Error analysis Potential errors from different embedding media, the wrong coverslip thickness, kinetochore tilt, and different fixation methods were examined experimentally (Listed in Figure 1—source data 1). A computer simulation of the measurement method was used to evaluate the significance of these errors, as well as errors produced by the SDs in centroid determination (CDsd) and CA (CAsd) over the field of measurement (Figure 1F). Chromatic aberration (CA) measurements from a cellular standard CA within our measurement was determined from CAx = Xg-Xr, CAy = Yg-Yr, and CAz = Zg-Zr measured for Hec1-9G3-GR labeled kinetochores of metaphase cells (Figures 1D and 2). The mean values of CAx, CAy, and CAz plus their standard deviations (SDs) are listed in Table 1 for several different preparations. Note the mean values for CAx, CAy, and CAz were each very similar between different preparations. We included one preparation where the embedding solution was PBS. PBS has a refractive index near water, 1.33, and far from the refractive index of the coverslip (~1.52). The mean CAx, CAy, and CAz values were similar to those using the ProLong Gold embedding medium (Table 1). The major difference was a much larger SD for CAz compared to ProLong Gold (48 nm vs ~18 nm, Table 1). This occurs because the lower refractive index induces spherical aberration that increases with z-axis depth from the coverslip surface (Figure 2—figure supplement 1). We also tested one sample with glutaraldehyde (GA) fixation and embedding with ProLong Gold. The mean CA values were nearly identical to the results obtained from PFA fixation (Table 1). Figure 2 with 3 supplements see all Download asset Open asset CA (Chromatic aberration) measurements using Hec1-9G3-GR. Schematic model of Hec1-9G3-GR. Hec1-9G3 antibody was labeled by secondary antibodies with Rhodamine Red-X and Alexa 488 (left). Representative Hec1-9G3-GR images in samples fixed by PFA or GA mounted with Prolong Gold or PBS. https://doi.org/10.7554/eLife.32418.004 Table 1 Mean and SD values of CAx, CAy, CAz for different sample preparations. https://doi.org/10.7554/eLife.32418.008 FixationMMGreenRedCAx (nm)CAy (nm)CAz (nm)NUse for correctionPFAPL9G39G313.1 ± 9.115.8 ± 7.53.5 ± 17.6166PFAPL9G39G314.7 ± 11.413.2 ± 12.11.5 ± 17.0256PFAPL9G39G315.7 ± 12.515.6 ± 8.2−4.8 ± 20.2206Use for correctionPFAPBS9G39G313.2 ± 10.718.7 ± 10.4−2.7 ± 47.5168Use for correctionGAPL9G39G319.2 ± 10.316.0 ± 9.0−6.8 ± 18.8170 A small measurement region in the center of the camera detector (~300×200 pixels) was used for the entire analysis in this study (Figure 2—figure supplement 2A–B). In the measurement region, there was a gradient for CAx in the x direction (~30 nm/ 280 pixels (19 µm)), but not in the y or z directions. There was a gradient for CAy in the y direction (~21 nm/180 pixels (11.5 µm)), but not in the x or z directions. There was no noticeable gradient for CAz in either the x, y, or z directions compared to the SD (Figure 2—figure supplement 3). These gradients in CA had no more effect on measurement accuracy than randomly distributed CA values with the same mean and SD (see computer simulations below). Validation of our 2D and 3D co-localization method for known separations within the kinetochore We initially measured the 2D and 3D separations between an antibody to CENP-T (CENP-T (M), Gascoigne et al., 2011) and Hec1-9G3 because previous studies reported values of Delta and 3D mean separations (by the CICADA method) using these same antibodies (Fuller and Straight, 2012; Suzuki et al., 2014; Wan et al., 2009). The reported 1D and 3D values for CENP-T (M) and Hec1-9G3 were ~60 and~62 nm respectively. Our mean separation ±SD between CENP-T (M) and Hec1-9G3 for Delta = 60 ± 7.5 nm, S(2D)=63 ± 11.2 nm, ML2D = 62 ± 11.3 nm, S(3D)=69 ± 12.3 nm, and ML3D = 66 ± 12.5 nm (Figure 3A–B), almost identical to the previous studies (Fuller and Straight, 2012; Suzuki et al., 2014; Wan et al., 2009). Figure 3 Download asset Open asset Mean Delta, 2D, and 3D separations between Hec1-9G3 and CENP-T (M). (A) Representative immunofluorescent (IF) images (top) and plots of Depth verses Delta, S(2D) and S(3D) for the separation between CENP-T(M) and Hec1-9G3 (bottom). (B) Histograms of maximum likelihood fit used to calculate ML2D and ML3D. (C) Orientation of the S(3D) separation vector relative to the sister K-K axis (x-axis in this diagram). Angle α is the rotation of the S(2D) vector component in the x-y plane away from the K-K (x-axis) and angle β is the rotation of the S(3D) vector away from the x-y plane and toward the z-axis (schematic on left). Histograms of α and β values (Right). n > 600 individual kinetochores. All values were mean ±SD. Note, CENP-T(M) is a polyclonal antibody against whole CENP-T protein, and its epitope is not precisely known (Gascoigne et al., 2011). https://doi.org/10.7554/eLife.32418.009 The orientation of the separation vector at each kinetochore was also calculated relative to the K-K axis. For CENP-T(M) to Hec1-9G3, the mean vector angle to the K-K axis in the X-Y plane, angle α = −0.3 ± 20.1 degrees, while the mean vector angle relative to the Z-axis (angle β) was −1.8 ± 23.3 degrees (Figure 3C). A significant fraction of the SD in these angles may be due to errors from CDsd and CAsd (see computer simulations below). To further test the accuracy of our 2D/3D co-localization method, we examined the mean separation of different domains within the Ndc80 complex. We chose the Ndc80 complex as a molecular ruler, since its protein structure is well known. The Ndc80 complex contains a dimer of Ndc80/Nuf2 and a dimer of Spc24/Spc25 (Cheeseman et al., 2006; Ciferri et al., 2008; Musacchio and Desai, 2017). Most of the length of the Ndc80 complex is alpha helical coiled-coil, and its contour length is 55 ~ 60 nm, as measured in vitro by electron microscopy and crystallography (Ciferri et al., 2008; Huis In 't Veld et al., 2016; Valverde et al., 2016; Wei et al., 2005). To reveal the cellular Ndc80 complex structure, we developed HeLa cells stably expressing EGFP-Ndc80, Ndc80-EGFP, EGFP-Nuf2, Nuf2-EGFP, EGFP-Spc24, Spc24-EGFP, EGFP-Spc25, or Spc25-EGFP (Figure 4A). Figure 4B and Figure 4—source data 1 show the separation values relative to Hec1-9G3 to different ends of the Ndc80, Nuf2, Spc24, and Spc25 proteins, tagged in each case with EGFP. For example, the mean separation measurements between Spc25-EGFP and Hec1-9G3 were Delta = 49 ± 5 nm, S(2D)=52 ± 10 nm, S(3D)=55 ± 10 nm, ML2D = 51 ± 10 nm, and ML3D = 53 ± 10 nm. The Ndc80 complex is likely extended along kMTs since the Delta and ML3D values for the length of the Ndc80 complex are nearly identical. Also, overall, our Delta, 2D, and 3D mean separation measurements yielded a close fit to a Ndc80 complex structure that was determined in vitro (Ciferri et al., 2008; Valverde et al., 2016). As expected, the C-terminus of Spc24 is located a few nm inside of the C-terminus of Spc25, and Spc24 is longer than Spc25 (Figure 4—figure supplement 1) (Valverde et al., 2016). The CH-domain of Nuf2 is located a few nm inside of the N-terminal CH domain of Ndc80, and Ndc80 is longer than Nuf2 (Figure 4—figure supplement 1) (Ciferri et al., 2008; Valverde et al., 2016). Unexpectedly, in vivo, the C-terminus of Ndc80 locates much closer to the globular domains of Spc24/Spc25 (Figure 4B). This difference might be caused by structural restrictions on the position of the EGFP tag, which was tethered to the protein end by a nine amino acid linker. Figure 4 with 1 supplement see all Download asset Open asset 2D/3D co-localization measurements of a structural standard, the extended Ndc80 complex. (A) Representative immunofluorescent images of Hec1-9G3 and EGFP fused to different protein domains along the Ndc80 complex (See Text and Materials and methods). (B) Schematic summary of the ML3D Ndc80 complex measurements at metaphase. All values were mean ±SD. n > 200 kinetochores for each measurement. Detail values are listed in Figure 4—figure supplement 1. https://doi.org/10.7554/eLife.32418.010 Figure 4—source data 1 The list for Ndc80 complex measurements. List of mean values of Delta, S(2D), S(3D), ML2D, ML3D between EGFP-Ndc80, Ndc80-EGFP, EGFP-Nuf2, Nuf2-EGFP, EGFP-Spc24, Spc24-EGFP, EGFP-Spc25, or Spc25-EGFP and Hec1-9G3. All values listed are mean ±S.D. https://doi.org/10.7554/eLife.32418.012 Download elife-32418-fig4-data1-v2.xlsx Sister kinetochore pairs were used for the majority of measurements in this study to directly compare separations obtained by Delta and 2D/3D fluorescent co-localization methods using the same data sets. To test if selection of sister pairs influenced our measurements, we measured 120 kinetochores selected randomly in a single metaphase cell. This produced a mean separation value nearly identical to the mean value for single kinetochore measurements of sister pairs obtained from six metaphase cells (Table 2). Table 2 Comparison of measurements for kinetochores that were part of sister pairs and kinetochores selected at random from a single metaphase cell. https://doi.org/10.7554/eLife.32418.013 MethodsGreenRedDelta (nm)S(2D) (nm)S(3D) (nm)ML2D (nm)ML3D (nm)nSister pairSpc25-EGFP9G349.4 ± 4.651.5 ± 9.655.2 ± 10.050.6 ± 9.753.3 ± 10.2280 (140 pairs) from 6 cellsRandomSpc25-EGFP9G3N.A.50.8 ± 6.755.3 ± 7.750.3 ± 6.754.2 ± 7.8120 from single cell The mean human kinetochore protein architecture at metaphase measured by the Delta method and our 3D method are similar Figure 5 shows how the centroids of protein labels are separated on average along the inner-outer kinetochore axis based on our 2D/Delta measurements (Top) and 3D measurements (bottom). All of our 2D and Delta values are nearly identical (usually less than 3 nm difference). In general, our 3D measurements were only ~5 nm larger than the Delta or 2D values (Figure 5 and the Figure 5—source data 1). Unlike the 3D kinetochore protein architecture proposed by (Smith et al., 2016), our data showed no ~70 nm gap between CCAN protein domains and the Ndc80 complex. The inner kinetochore proteins are much closer to the outer kinetochore, corresponding to the previous studies using Delta measurements (Suzuki et al., 2014). For example, our 3D mean separation between CENP-A and Hec1-9G3 is ~90 nm, which is nearly identical to values obtained by Delta (~84 nm) and ML2D (~85 nm). In Figure 5, the Ndc80 complex and protein linkage to the inner kinetochore are shown aligned with the axis of kMTs because the ML3D mean separation values are only slightly greater than their corresponding Delta values, and the Delta value is a projection of the ML3D value along the K-K axis. Figure 5 Download asset Open asset Schematic core-kinetochore drawing measured by 2D/Delta or 3D co-localization method. Schematic picture of the mean positions of protein labels within the core kinetochore protein architecture measured using ML2D/Delta (top) and ML3D (bottom) methods. N or C are the mean positions of N-terminal or C-terminal EGFP fusions or the mean position measured using polyclonal antibodies are not precisely known. Figure 5—source data 1 List for core-kinetochore protein measurements. List of mean values of Delta, S(2D), S(3D), ML2D, and for core kinetochore The between ML3D and ML2D ML3D and are also All values listed are mean ±S.D. Download Figure 5—source data 2 List of primary List of antibodies used in this study with and Download greater and less than 0.17 mm z-axis chromatic aberration of depth the value of for each preparation was within nm of (Figure 5—source data 1). significantly large or values which the 3D measurement much larger than for small The for one sample stained for CENP-T(M) and nm (Table which the S(3D) from to nm (Table this large was from the Sz the mean with value close to the value with near nm (Table This that the was of the mean separation between green and red This also that there was preparation in z-axis Table 3 (A) List of Delta, S(2D), S(3D), and for the mean separation between CENP-T(M) and in samples with or without large (B) List of Delta, S(2D), S(3D), and in samples with and The value was calculated from the S(3D) data of the mean in the middle of the spindle from the Sz values. All values were mean ±SD. ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± To we used a analysis reported previously and This analysis relative to the design values for the objective, in for light from a fluorescent as a function of the refractive index and of the media, the coverslip thickness, and as well as showed that the of z-axis CA is highly to in the of the We tested this by using different of We of our reported in this using We measured their with a and the mean value was 0.17 mm with a of to (Figure n > For coverslips, the mean was mm and for coverslips it was mm (Figure The mean Sz for CENP-T(M) to Hec1-9G3 showed nm for #1.5 coverslips, to nm for coverslips, and to nm for 2 (Table The in Figure supplement 1 the of to coverslip thickness. A is measured values in Table are as a function of the mean we measured for #1.5 and on these we that large values nm) measured in our (Figure 5—source data 1) were produced by coverslip much or than 0.17 Figure 6 with 2 supplements see all Download asset Open asset Accuracy of separation measurements depends critically on mean z-axis CA produced by coverslip from standard of coverslip within and In our we were to similar numbers of kinetochores above and below the spindle This is because kinetochores and their kMTs with their away from the spindle axis (Figure supplement (Smith et al., et al., 2009). the inner and outer domains of metaphase kinetochores typically have their to their kMTs (Wan et al., 2009). kinetochores are only measured on the of the the mean value of Sz not be because the kinetochore in the lower spindle is not by the from kinetochores in the (Figure supplement 2A–B). This kinetochore had no effect on measured 3D mean separation since values from only kinetochores in the of the spindle yielded similar results as from kinetochores in the lower of the spindle not Unlike 3D the mean angle β not angle was to numbers of kinetochores above and below the spindle The mean angle β is near in the middle of the spindle and mean values of degrees at the and top of the spindle (Figure supplement PFA fixation not but pre-extraction or detergent in PFA solution of cellular structure A recent study the that PFA fixation compared to GA fixation et al., 2016). GA has and more fixation compared to PFA of this GA fixation is usually used for electron the GA fixation exhibits and induces significantly less between cellular and antibodies compared to PFA For a as or is used to antibody the cell or is how these cellular For example, et PFA fixation they in their PFA fixation solution. Our standard method was primary fixation by PFA without detergent one in Table a method significantly different than the PFA fixation method used by et al., 2016). Table 4 of fixation methods used in Figure for the different fixation methods used in Figure 1 is the