Trajectories and single-particle tracking data of intracellular vesicles loaded with either SNAP-Crb3A or SNAP-Crb3B

Using a combined approach of pulse chase labeling and single-particle tracking of Crb3A or 3B loaded vesicles we collected trajectories of different vesicle population in living podocyte cells and evaluated statistically their different mobility patterns. Differences in their intracellular mobility and in their directed transport correspond well to the role of Crb3A and 3B in renal plasma membrane sorting (Djuric et al., 2016) [1].


How data was acquired
Single Molecule Microscopy, Single-particle-tracking Data format Analyzed Experimental factors

Experimental features
Human podocytes (AB8) stably expressing either Crb3A or Crb3b fused with the SNAP tag were fluorescence labelled using a respective SNAP tag dye Data source location

University of Bonn, Germany
Data accessibility Data is within this article

Value of the data
Renal plasma membrane proteins Crb3A and Crb3B can be assigned to different vesicle subpopulations.
The different membrane proteins can be used as distinct vesicle marker to identify and track different vesicle populations.
Differences in mobility pattern and trafficking dynamics might be related to intracellular redistribution of Crb3A and Crb3B and their respective plasma membrane portion.

Data
Single vesicles loaded with different membrane proteins fused to a SNAP-tag were tracked in living cells after fluorescence labeling (Fig. 1). From single trajectories the momentum scaling spectrum was computed and its slope calculated [2,3] (Fig. 2). The slopes from many different trajectories were collected ( Fig. 3A and C) and compared to the slope of trajectories after respective drug treatment ( Fig. 3B and D). The distinct mobility patterns of Crb3A versus Crb3B vesicles can be best visualized by a histogram of all S MSS (slope of the momentum scaling spectrum) as it is shown in Djuric et al. [1].

Cell culture and SNAP-tag labeling
The SNAP-tag fusion protein expressing AB8 cells were seeded on cover slides and incubated in standard RPMI 1640 medium containing 10% FCS and supplements and 1% antibiotics (Pen/Strep). Directly before the measurements the cells were incubated with the fluorescent SNAP-tag substrate (BG594) according to the manufacturer's protocol. After incubation free dye was removed by washing several times.

Live cell Imaging and vesicle tracking
Cells were imaged using an inverted custom-built single-molecule microscope based on an Axiovert 200TV (Zeiss, Jena, Germany) equipped with a 63 Â NA 1.2 water immersion objective lens [5]. For excitation the 532 nm laser line of a Cobolt Laser (Cobolt Dual Calypso, 100 mW) was used and movies were recorded with an electron multiplying CCD camera (iXon BI DV-860, Andor Technologies, Belfast, Ireland) at 10 Hz frame rate. Tracking of single vesicles was done with ImageJ and the Particle Tracker 2D/3D plugin [6] using the unfiltered data.

Analysis of vesicle mobility
We followed an approach from Ewers et al. [2] who studied the different mobility patterns of virus like particles during infection. The x/y coordinates of the trajectories were exported to Origin8 (OriginLab Corp., Northhampton, USA) and further analyzed used a custom-built OriginC script, which allowed automated calculation of the so called momentum scaling spectrum (MSS) and its slope, S MSS .
For each trajectory i the moments of displacement (m ѵ,i ) were calculated for ѵ¼1, …, 6 as a function of time according to: Here, x i (n) designates the position vector of trajectory i at time nΔt, where Δt is the inverse frame rate and n a specific frame number: n ¼0, 1, …, M i À 1 (M i is the trajectory length). For ѵ¼2 this matches with the "classical" mean square displacement analysis. Using all momenta allowed construction of the MSS for each single trajectory [2,3]. Therefore m ѵ,i (nΔt) was plotted versus nΔt in a double logarithmic plot ( Fig. 1C and D). From these plots the so called scaling moments γ ѵ,i, can be derived assuming each moment m depends on the time shift according to m ѵ (nΔt) $ nΔt γѵ [2,3].
Plotting γ ѵ versus ѵ will finally give the respective MSS ( Fig. 2E and F). The MSS will show a straight line through the origin and its slope is an excellent measure for the type of movement. In case of free, unhindered diffusion the slope is 0.5, a slope 4 0.5 indicates directed motion or active transport, while a slope o0.5 implies retardation or immobilization.