2.2. Methods:
2.2.6. Analysis software and application
MetaMorph Colocalization
Merged multi-channel 40X light microscopy images were analyzed using MetaMorph Offline Version 7.7.0.0 (Molecular Devices, Inc.). A threshold is set for each channel followed by the generation of a mask for all channels, in three areas of size 25 pixels long (representing 2 µm in the sample) and 4 pixels wide, per image. These area masks were then overlaid in the arithmetic tool and divided to generate a third mask containing only the population of signals in the
Methods mask that do colocalize. The amount of bassoon colocalized is a represented as a percentage of the bassoon colocalized population divided by total bassoon population.
Imaris MeasurementPro 8.1(Bitplane AG.) software
Merged TIFF light microscopy images or STED images were analyzed using Imaris for range of analyses such as, the probability of colocalization (Pearson’s correlation coefficient), amount of colocalization, signal sizes and their population, and distribution of signals.
For Figure 4—Figure 5, Figure 15—Figure 19, Figure 22—Figure 24, a free-hand drawn mask was drawn to exclude all signals in the image that were in the nucleus, outside the cell soma, or in the axons. To factor out the effect of the varying number of signals counted per size of hand-drawn mask in each image, the area in µm3 of the mask used, was divided by the signals counted per image.
Probability of colocalization
Images with or without masks were all then run through the ImarisColoc module that is integrated with a Costes P-Value approximation92 plugin, to generate automated thresholds for all the channels of all the images in a set. Also this module is integrated with Image J plugin: just another colocalization plugin that can calculate the colocalization Pearson’s and Manders’ correlation coefficient Constants in the image.
To ascertain the amount of colocalization, the signal sizes and distribution of the spot signals in the image, the Imaris Spots module was first used to generate objects for each spot of signal in the image, for all channels. These objects are generated upon applying the automated intensity threshold value calculated by the Costes P-Value approximation, signal diameter size range of 30—160nm for STED images and >200nm for light microscopy images, an automated splitting of cluster signals (defined as >120nm for STED images and >400nm for light microscopy images) based on intensity profile plots of signals within the cluster, in each channel.
Signal Sizes and their populations
Imaris Spots calculates a range of statistical data for the spot objects generated for each channel. One such automatically generated type of data is the diameter size (determined by the FWHM of the PSF) of each spot signal. These data were collected in Excel for Figure 5 for all the images in the set, and the total number of spots in the size range of 30—60nm, 60—90nm and 90—130nm were calculated and plotted using GraphPad Prism.
Amount of colocalization
The amount of colocalization was calculated using the spot objects generated for each channel and a MATLAB extension in the spots module called Spots
Methods colocalize, which calculates the colocalized population of all channels using the a distance threshold of 0—100nm (for STED images) and 0—350nm (for light microscopy images) from the spot centers.
Distribution of Signals
To analyze the distribution of AZP to a Golgi marker, the distance transformation MATLAB extension94 from the Imaris XT module was used. This extension exchanges the voxel intensities data of all signals in the Golgi marker channel into spot coordinates data and creates a new channel with this data. This channel indicates the shortest distance to the object border of the Golgi marker spot. The AZP channel is overlaid over the Golgi marker distance transformation channel to reveal the shortest distances between all the AZP signals and the border coordinates of the all the Golgi marker signals. The shortest distance value for every AZP spot to a Golgi marker spot can as easily be exported to excel, as well as the total number of AZP signals within 0—100nm or 101—100nm distance ranges.
GraphPad Prism 5.02
All resulting data were analyzed and graphically represented using GraphPad Prism 5.02. Comparisons between groups were statistically tested, and the data in the graphs are presented as mean ± SEM. Differences were considered significant (*p < 0.05), strongly significant (**p ≤ 0.01) and extremely significant (***p ≤ 0.001). For Figure 3—Figure 8, Figure 10—Figure 13, and Figure 15—
Figure 19, a one-way annova test was performed with a post-hoc test of Tukey’s multiple comparisons test (that compares the means of all columns). For every significant difference noted between the two relevant groups, an additional two-tailed, unpaired Student’s t test with different variances was also performed to reveal the same significant difference.
Results
Chapter 3
Results
This chapter details my results, in which I study the localization and ultrastructure of active zone proteins (AZPs) at various subcellular structures, on their journey from the soma of young developing neurons, to the presynaptic cytomatrix at the active zone (CAZ). A deeper understanding of how AZPs are oriented, organized, and transported, at various sites in the developing neuron, is required for unraveling the mechanisms at play during mammalian active zone assembly and CAZ maturation. To tackle these topics, I will here first study the ultrastructural localization of endogenous AZPs at Golgi substructures, in the soma, and during transport in the axons of young hippocampal neurons. I will then compare the endogenous AZP localizations to the localization of well-established recombinant AZP constructs, and will finally introduce, characterize, and use new tools made off the full-length Bassoon molecule. These second generation constructs have been optimized and used to study the orientation, organization, and the detailed localization of this AZP at the Golgi, the soma, on trafficking dense-core vesicles, and at synaptic sites.
Results Endogenous localization of AZPs at the Golgi
3.1. Localization of endogenous AZPs at the Golgi apparatus
Initial studies in the field have shown AZPs Bassoon and Piccolo to be localized in the somas of young neurons at Golgi substructures and transported to presynaptic sites on 80nm dense-core vesicles known as Piccolo-Bassoon Transport Vesicles (PTVs)30,68,69. Other AZPs, namely Munc13-1, RIM1𝛼, and ELKS2, were also isolated in light brain fraction immunoprecipitates containing PTVs69,33. Golgi-derived AZP transport carriers, which are either preassembled PTVs93 or small clusters of clear- and dense-core vesicles carrying AZPs71, are believed to sufficiently transport the entire CAZ scaffold to the presynaptic membrane for the generation of a functional synapse.