Computational protocol: An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence

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Protocol publication

[…] In , a line scan (i1) of 3-pixel width containing both GFP signal and background was drawn along the nMT array using ImageJ software (National Institutes of Health, Bethesda, MD). A line of 6-pixel width (at the same location) was drawn in order to calculate the intensity of the surrounding background (i2). The real intensity (ir) was calculated as follows: ib = (i2 × 6) − (i1 × 3) and ir = i1 − (ib/3). We arbitrarily set the highest fluorescence value for each line scan to 100%. On the graph, depending on the directionality of the line scan, 0 or 100% fluorescence was set to 0 µm on the x axis.To measure MT length variation ( and Fig. S2), position of the SPB and the plus-end extremities of the MT structure (aMT or nMT array) were followed over time. Euclidian distances (D) between two positions were calculated as follows: D = √ ((x2 − x1)2 + (y2 − y1)2). In time-lapse series, the first measured length was set to zero and the variation was calculated according to this reference. SPB/centromere and SPB/kinetochore Euclidian distance measurements were done using the MTrackJ plugin in ImageJ, their respective positions being determined in the best focal plane.Fluorescence recovery was corrected for background noise and continuous photobleaching using ImageJ software, and then normalized to the fluorescence measured before bleach (). The fluorescence intensity of a region of interest (5 × 4 pixels) was measured on maximal projection of z-stacks. The fluorescence intensity Ir was calculated over time as follows: I = (Iregion of interest − Ibackground). Photobleaching was measured on unbleached nMT arrays, and the intensity was corrected using Ir = Iy × (Iunbleached first point / Iunbleached point y), where y is a specific time point over time.The relative position of the nucleolus versus the SPB was determined using a central symmetry axis along the nucleus draw from the SPB.To determine the location of Arp1/Dyn1 toward nuclear membrane, a line scan (i1) of 5-pixel width containing both GFP/RFP signal and background was drawn along the GFP/RFP signal using ImageJ software. A line of 10-pixel width (at the same location) was drawn in order to calculate the intensity of the surrounding background (i2). The real intensity (ir) was calculated as follows: ib = (i2 × 10) − (i1 × 5) and ir = i1 − (ib/5). Individual Arp1/Dyn1/Nup2 Gaussian fits were determined and aligned using GFP fits set to 0 on the x axis. GFP/RFP average fits are displayed in Fig. S3 H. […]

Pipeline specifications

Software tools ImageJ, MTrackJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae