Accompanying data for: Local negative feedback of Rac activity at the leading edge underlies a pilot pseudopod-like program for amoeboid cell guidance

This is the data underlying all of the figures in: Local negative feedback of Rac activity at the leading edge underlies a pilot pseudopod-like program for amoeboid cell guidance by Jason P. Town and Orion D. Weiner

Paper Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to “lock” onto a particular direction, limiting the ability of cells to reorient. We use spatially-defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance “decisiveness” needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals. 

All data can be processed using standard scientific python packages using the Jupyter Notebooks located here. To run the notebooks and reproduce the figures in the paper, download all of the zipped data files, unzip them, and arrange them into the following directory structure:

projectfolder

data

fig03data

fig04data

fig05data

figS01data

figS08data

figS10data

simpleStimdata

figure01.ipynb

figure02.ipynb

...

Microscopy data was collected using an automated, Pycromanager-based pipeline. The most recent version of the pipeline can be found here. 

This zipped folders contains microscopy data (images & metadata) in several directories:

"simpleStimData" contains data used in several figures in the paper. It shows the dynamic behaviors of neutrophils as they are subjected to local optogenetic activation of optoPI3K.

File folders are not entirely standardized, but typically contain

The data of the experiment (YYMMDD format)

The cell line used and the constructs expressed (35 is the optogenetic anchor, 52 is the optogenetic cargo, 97 is the PH-Akt-Halo PIP3 reporter, and mcPak is the Pak-PBD-mCherry Rac reporter)

A generic phrase 'Assays', 'VariousFrontStimulations', 'simpleStimScreen' that has no experimental meaning

An indicator of the pre-programmed assay "type" - either 'none, 'front', 'side', 'back', 'global', 'lateral45', 'lateral90',  '0Rad', '0.2 Rad', '0.39Rad', '0.79Rad' or '1.57Rad'

The intensity of blue LED light used in the experiment ('50000'). This did not turn out to be very important for the paper in this particular dataset, but cells do not display an all-or-none response to optogenetic PI3K acitvation.

And finally a replicate number for that assay that day (i.e. '000', '001', ...)

Within each file folder, there is a collection of images (Note that each image has a lot of extra information in the associated metadata)

Channel 0 is the PHAkt PIP3 reporter fluorescence data

Channel 1 is the Pak-PBD Rac reporter fluorescence data

Channel 2 is a reflected blue light dataset. A small degree of blue light reflected off the bottoms of coverslips and could be collected on the camera

Channel 3 is a binary segmentation channel generated on-the-fly during acquisition by our automated microscopy software

Channel 4 is another binary segmentation channel, but this one is a prediction of the real image of the optogenetic stimulus based on a calibrated affine transformation of the projected image onto the microscopy field of view. 

if present, Channels 5 and 6 are control images taken at the end of the experiment to verify the presence of optogenetic constructs 35 (405 channel) and 52 (488 channel)

There is a small amount of data here that was not directly used in the paper. For example, the 'frontToGlobal' experiments did not produce interesting results, but actually led us to develop the method shown in Figure 5 in our paper where we did something closer to a 'sideToGlobal' assay (See Fig 5 in the paper). 

"fig03Data" contains data used to generate Fig 3 and Fig S7B in the paper. This data is structured identically to the data in simpleStimData described above

"fig04Data" contains data used to generate Fig 4 and Fig S9D in the paper. Some of this is from older software, so the metadata (i.e. LED intensities) are stored in a separate file called intensities.txt. This text file contains time stamps and LED intensity data that can be matched with the temporal metadata from the microscopy images.  

"fig05Data" contains data used to generate Fig 5 and Fig S11. These are structured identically to the simpleStimData structure described above. (Though these files are stored in more organized subdirectories compared to simpleStimData). 

"figS01Data" contains the microscopy data used to generate Fig S1

"figS08Data" contains data used to generate Fig S8 in the paper. This is from older microscopy automation software, so the metadata (i.e. LED intensities) are stored in a separate file called metadata.txt. This text file contains time stamps and LED intensity changes that can be matched with the temporal metadata from the microscopy images.  

"figS10Data" contains data used to generate Fig S10 in the paper. This is a combination of data structures similar to that described above in "fig04Data" for the step inputs and "simpleStimData" for the turning assays.