DOI: 10.5061/dryad.m905qfv7g
title: Microscopic images and schematics illustrating processes of microenvironment sensing and cortical actomyosin partitioning in T cells

description: The all-terrain motility of lymphocytes in tissues and tissue-like gels is
 best described as amoeboid motility. For amoeboid motility, lymphocytes do
 not require specific biochemical or structural modifications to the
 surrounding extracellular matrix. Instead, they rely on changing shape and
 steric interactions with the microenvironment. However, the exact
 mechanism of amoeboid motility remains elusive. Here we report that
 septins participate in amoeboid motility of T cells, enabling the
 formation of F-actin and alpha-actinin-rich cortical rings at the sites of
 cell cortex-indenting collisions with the extracellular matrix. Cortical
 rings compartmentalize cells into chains of spherical segments that are
 spatially conformed to the available lumens, forming transient
 ‘hourglass’-shaped steric locks onto the surrounding collagen fibers. The
 steric lock facilitates pressure-driven peristaltic propulsion of
 cytosolic content by individually contracting cell segments. Our results
 suggest that septins provide microenvironment-guided partitioning of
 actomyosin contractility and steric pivots required for amoeboid motility
 of T cells in tissue-like microenvironments. 
description: # Microscopic images and schematics illustrating processes of
 microenvironment sensing and cortical actomyosin partitioning in T cells
 [https://doi.org/10.5061/dryad.m905qfv7g](https://doi.org/10.5061/dryad.m905qfv7g) Dataset contains all source files for imaging data used in the manuscript. ## Description of the data and file structure Source files are organized by name, as used in the manuscript's illustrations. Additionally, each source file contains a short legend, describing cells, dyes, antibodies, or drugs used. ## Sharing/Access information NA ## Code/Software 1. **Analysis of spatial co-localization and cross-correlation for the cellular components of cortical rings and cell surface-indenting collagen fibers in hCD4+ T cell** **Ring\_Analysis.zip** **Main Scripts:** orientation\_alignment.m – step 1: aligning the ring plane to the coordinate system quantification\_ring\_positions.m – step 2: plotting end saving quantification of the overlap between (actin, septin), (actin, collagen), and (septin, collagen) **Auxiliary Scripts:** import\_cell\_data.m – a function for importing and processing 3D data by z-scoring the signal in each frame of each channel and resampling 3D data to get cubic voxel (equal pixel size in xy, xz, yz projections) projections.m – a function to plot the xy, xz, and yz projections of the 3D data opt\_rotation\_check.m – a function for checking how well the ring plane is aligned with the coordinate system and for determining the position of the ring plane plot\_ring\_overlap.m – plotting actin ring, septin ring, collagen ring, and their overlap plot\_3Dsurf.m – 3D rendering to check the difference between the original and rotated orientation of the ring plane movie\_3D.m – generates the movie to illustrate the overlap of actin, septin, and collagen rings 2. **Analysis of cortical non-muscle myosin 2A activity during CD4+ T-cell migration in 3D collagen matrix** **Myosin\_Analysis.zip** Myosin\_NM2A\_activity.m – the main script to plot visualizations, measurements, and save results as an Excel file