Imaging, analysis & sample preparation 49!

Fluorescence time-lapse imaging of H2B-GFP/mRFP-#-tubulin cells was carried out on a Personal Deltavision microscope (Applied Precision, LLC) using a 40 x NA 1.3 objective, an GFP (excitation 475/28, emission 525/50) and mCherry (excitation 575/25, emission 632/60) filter set with a Quad- mCherry dichroic mirror (reflection bands 381-401, 464-492, 561-590, 625-644, transmission bands 409-456, 500-553, 598-617, 652-700) (Chroma), Xenon light source and a CoolSNAP HQ2 camera (Roper Scientific). Image stacks (7 x 2 "m z-sections) were collected every 3 min for a total time of 10 h. These conditions were also used to image mitotic progression in CENP-Q- eGFP/mCherry-CENP-A cells (MC069) and mCherry-CENP-A cells (MC051), but without using the GFP channel. The GFP channel was not used as the signal was too weak to facilitate overnight image without considerable photo-toxic effects. However, the presence of CENP-Q-eGFP was confirmed before the start of the experiment. Maximum intensity projections of the acquired time series were then generated using SoftWorks (Applied Precision, LLC). For initial experiments

CHAPTER 2 – Materials & Methods

50 confirming the expression and localisation of CENP-Q-eGFP and eGFP-CENP-P relative to mCherry-CENP-A or mRFP-#-tubulin the same conditions were used as for H2B-GFP/mRFP-#- tubulin, with the exception of a 100 x NA 1.4 plan apochromatic objective and a reduced total imaging time of ~15 min. Kinetochore tracking movies and fate movies of eGFP-CENP-A/eGFP- Centrin1 cells were carried out on a Personal Deltavision microscope (Applied Precision, LLC) using a 100 x NA 1.4 plan apochromatic objective, a GFP (excitation 475/28, emission 525/50) filter set with a Quad-mCherry dichroic mirror (reflection bands 381-401, 464-492, 561-590, 625- 644, transmission bands 409-456, 500-553, 598-617, 652-700) (Chroma), Xenon light source and a CoolSNAP HQ2 camera (Roper Scientific). Image stacks (30 x 0.5 "m z-sections) were collected every 7.5 sec for a total time of 5 min total., The total experiment length was increased to 10 min for tracking unaligned chromosomes in unperturbed cells. These images were deconvolved using SoftWorks (Applied Precision, LLC); 8 iterations of ratio mode deconvolution with medium noise filtering.

2.2.2 Kinetochore tracking

Automatic kinetochore detection and tracking and data analysis was performed as previously described (Jaqaman et al., 2010), with the exception of the metaphase plate fitting step, which was modified by Edward Harry (McAinsh lab). This step was modified due to the high number of unaligned chromosomes present in CENP-Q depleted cells. The original plate fitting strategy would very often fail when there were multiple unaligned kinetochores, even though a metaphase plate structure was clearly visible. This was because the average nearest-neighbour distribution was similar for inliers as it was within each cluster of unaligned spots, therefore presenting no outliers to exclude. A more robust method of metaphase plate finding and fitting was therefore implemented. In each frame: (1) A graph was created between all spots with a cut-off distance of two microns. This creates a cluster of sub-graphs in which the metaphase plate structure (sometimes plus a few outliers) was one sub-graph. (2) Sub-graphs were identified by flood-filling the graph. Each flood-fill was continued until no more new nodes were being filled. If any unfilled nodes remained a new flood-fill was started on an empty node. All the nodes filled in each flood were taken to belong to a

51 new, separate sub-graph. (3) The original plate fitting algorithm (Jaqaman et al., 2010) was used on the sub-graph with the highest number of nodes (the metaphase plate structure is normally the sub-graph with the highest number of nodes). Spots not in the sub-graph were taken as outliers automatically. (4) If a plate was not successfully fitted the process was repeated on the next largest sub-graph until a correct plate fit was generated. (5) All spots in each frame were reclassified as inlier (aligned) or outlier (unaligned) based on their distance away from the fitted plate (> 2.5 SD) for outliers as previously described (Jaqaman et al., 2010). This plate fit modified version of the tracking code was used for all conditions.

2.2.3 Immunofluorescence sample preparation

Cover slips with adherent HeLa cells were placed into 3.5 cm 6 well plates, those already grown in 6 well plates were aspirated to remove DMEM. Taking care to ensure cells didn’t dry out 2 ml of pre-extraction buffer was added to each cover slip containing well for 1 min. Pre-extraction buffer contains the following: 20 mM PIPES pH 6.8 containing 10 mM EGTA, 1 mM MgCl2, 0.2% Triton X- 100. After 1 min the well was aspirated and PTEMF buffer was added to fixed cells. PTMEF buffer contains the following: 20 mM PIPES pH 6.8 containing 10 mM EGTA, 1 mM MgCl2, 0.2% Triton X- 100 and 4% formaldehyde. After 10 min the PTEMF buffer was aspirated out of each well and replaced with 2 ml of PBS and incubated for 10 min to wash, this step was repeated twice more. Following the wash, 2 ml of PBS with 3% bovine serum albumin (BSA) was added to each well to prevent non-specific antibody binding and incubated for 30 min. Cover slips were then removed from wells and excess liquid was removed by drying the edge on tissue paper. Cover slips were then placed on a parafilm covered glass tile and primary antibodies were added in 250 "l of PBS with 3% BSA. Primary antibodies (see Table 6) were incubated with cells for 1 hr covering with a plastic container to reduce evaporation. The cover slips were then aspirated to remove the primary antibodies and washed for 10 min with 300 µl PBS, this wash process was then repeated two more

times. Secondary fluorescent antibodies (see Table 6) along with DAPI stain (diluted 1 in 2000) were added to cover slips in 250 "l PBS with 3% BSA for 30 min covering with a plastic container

CHAPTER 2 – Materials & Methods

52 to reduce evaporation and foil to reduce photo bleaching. After 30 min secondary antibodies were aspirated off of cover slips and replaced with 300 µl of PBS and incubated for 10 min to wash, this

was step was repeated twice more. The cover slips were then placed face down on an 11 "l dot of Vectashield mountant (Vector Laboratories) and sealed with clear nail varnish (Superdrug). Slides were then visualised via fluorescence microscopy.

2.2.4 Imaging immunofluorescence experiments

Fixed cell imaging was conducted on a Deltavision Core microscope (Applied Precision, LCC) with an Olympus main body and objectives. Epi-fluorescent illumination was provided by a xenon light source with fibre-optic light guide with a DAPI-FITC-Rhod/TR-CY5 filter set. Images were captured on a CoolSNAP HQ2 camera (Roper) using 0.2 µm z-steps (between 10 and 18 "m total) using a 100X oil NA 1.4 objective. Exposure times were varied depending on fluorescence staining intensity but were fixed within each experiment to allow direct comparison. These images stacks were deconvolved using SoftWorx (Applied Precision, LLC); 8 iterations of ratio mode deconvolution with medium noise filtering. Intensity quantifications at the kinetochore taken from deconvolved image stacks were also carried out in SoftWorx. The average intensity was taken at the maximal intensity plane for the kinetochore within a 5x5 pixel box along with adjacent background. The intensity of the channel being measured was corrected by background subtraction and was calculated as a ratio against CENP-A as an internal control.

2.2.5 Kinetochore-monopole distance measurements

For analysis of distances from kinetochores to spindle poles the Volocity 3D image analysis software package (PerkinElmer) was used. The centre of mass of the monopolar spindle was determined in 3D using the anti- -tubulin signal., Kinetochores (stained with anti-CENP-A) were identified by the built in Velocity spot detection algorithm, this detection was confined to the chromatin region (DAPI signal). The distance from every kinetochore to the spindle monopole centre of mass was measured in 3D; this was repeated for a minimum of 8 cells per experiment.

53 Cumulative distribution frequencies (CDF) were calculated using MATLAB, the average of these CDFs was plotted for each condition, standard deviation was also calculated and is shown as a shaded area.

2.2.6 CENP-Q kinetochore positioning

To visualise the positioning of CENP-Q within the kinetochore relative to CENP-A and Hec1, we first had to correct for chromatic aberrations within the optical path. To achieve this, cells were stained with a mouse anti-CENP-A monoclonal antibody followed by a mix of 3 secondary anti- mouse antibodies with fluorophores emitting at 488, 594 and 647 nm. The triple tagged CENP-A cells were imaged in all 3 channels with high axial sampling (50-nm) and the x,y,z shifts in each channel were measured relative to the 488 nm channel in Huygens Professional (X11). Cells transiently transfected with eGFP-CENP-Q were fixed and stained with antibodies against CENP-A and Hec1. These cells were imaged as above, except that z sampling was every 100 nm. The resulting image stacks were then corrected for chromatic aberration using the earlier measured deviation. After correction, intensity line profiles were taken through kinetochore pairs using the open source ImageJ analysis package to identify peaks for each protein.

2.3 Molecular biology