DOI: 10.5061/dryad.fttdz096g
title: Cooperation of kindlin-3 and talin-1 in integrin activation during neutrophil arrest and inflammation

description: Neutrophil arrest requires the activation of beta2 integrins. Two FERM
 domain proteins, kindlin-3 and talin-1, are necessary for neutrophil
 arrest. In this project, we explored the molecular mechanisms of talin-1
 and kindlin-3 cooperation during neutrophil adhesion. Flow chamber
 experiments showed that talin-1 and kindlin-3 are simultaneously recruited
 to the plasma membrane of HL-60 cells during the transition from rolling
 to arrest. Using primary neutrophils expressing fluorescent fusion
 proteins of talin-1 or kindlin-3, we confirmed that both proteins are
 recruited to the plasma membrane at the time of arrest, supporting their
 coordinated role in neutrophil integrin activation. In a mouse cremaster
 muscle ischemia-reperfusion model, beta2 integrin activation was detected
 in neutrophils attached to the endothelium after ischemia-reperfusion.
 These findings support that kindlin-3 and talin-1 work together to induce
 high-affinity integrin activation during inflammation.
description: HL-60 cells were differentiated for 5-7 days in 1.3 % DMSO and
 flowed on P-selectin and ICAM-1 substrate in a microfluidic device. Mouse
 neutrophils were isolated by flushing mouse tibia or femur. Cells were
 perfused at 6 dyn/cm<sup>2</sup> wall shear stress through a
 microfluidic flow chamber and examined by total internal reflection
 fluorescence microscopy. Confocal microscopy was utilized to examine the
 antibody labelling in the cremaster muscle microcirculation of a male
 mouse before and after cremaster ischemia and reperfusion.
description: # Cooperation of kindlin-3 and talin-1 in integrin activation during
 neutrophil arrest and inflammation Dataset DOI:
 [10.5061/dryad.fttdz096g](https://doi.org/10.5061/dryad.fttdz096g) ##
 Description of the data and file structure This dataset is associated with
 the American Heart Association (AHA) sponsored Career Development Award
 (942098) “Cooperation of kindlin-3 and talin-1 in integrin activation
 during neutrophil arrest and inflammation”. The Data_deposit.zip contains
 four experimental efforts, separated into subfolders: 1. The monitoring of
 the two cytosolic proteins talin-1 and kindlin-3 during HL-60 cell
 adhesion. 2. The monitoring of talin-1 during mouse neutrophil adhesion.
 3. The monitoring of kindlin-3 during mouse neutrophil adhesion. 4.
 Confocal imaging of beta2 integrin activation in response to ischemia
 reperfusion injury (IRI).  The dataset includes zipped files, which
 contain multiple files that need to be uncompressed. The folders are:
 **Folder: HL-60.zip** File Description:  1. 3 - 1_XY1597092941_Z0_T000_C0:
 (fluorescence images of TagRFP-kindlin-3 in movie frames) 2. 3 -
 1_XY1597092941_Z0_T000_C2: (fluorescence images of mEGFP-Talin-1 in movie
 frames) 3. 3 - 1_XY1597092941_Z0_T000_C3: (fluorescence images of Cell
 Membrane Deep Red in movie frames) **Folder:
 mouse_neutrophils_mEGFP-T1.zip** File Description:  1. mEGFP-T1.tiff:
 shows the movie of mEGFP-talin-1 during neutrophil arrest from rolling on
 P-selectin and ICAM-1-coated coverslip. 2. mEGFP-T1.xlsx: shows the
 analysis of talin-1 kinetics during neutrophil arrest. The integrated
 fluorescence intensity (A.U.), which is the product of mean fluorescence
 intensity and area, at different time points were analyzed. 0s is the time
 of arrest. **Folder: mouse_neutrophils_mScarlet-K3.zip** 1.
 mScarlet-K3.tiff: shows the movie of mScarlet-kindlin-3 during neutrophil
 arrest from rolling on P-selectin and ICAM-1-coated coverslip. 2.
 mScarlet-K3.xlsx: shows the analysis of kindlin-3 kinetics during
 neutrophil arrest. The integrated fluorescence intensity (A.U.), which is
 the product of mean fluorescence intensity and area, at different time
 points were analyzed. **Folder: IRI.zip** File Description:  1.
 20220621_IRI_ITGB2KI-0001 m24.tiff (fluorescence image of mAb24 antibody
 labelling before IRI) 2. 20220621_IRI_ITGB2KI-0002 Ly.tiff (fluorescence
 image of Ly6G antibody labelling  before IRI) 3. 20220621_IRI_ITGB2KI-0003
 BF.tiff (brightfield image of the cremaster tissue before IRI) 4.
 20220621_IRI_ITGB2KI-0004 KIM.tiff (fluorescence image of KIM127 antibody
 labelling before IRI) 5. C1-20220621_IRI_ITGB2KI.lif - Series007-2
 m24.tiff (fluorescence image of mAb24 antibody labelling after IRI) 6.
 C2-20220621_IRI_ITGB2KI.lif - Series007-2 Ly.tiff (fluorescence image of
 Ly6G antibody labelling after IRI) 7. C3-20220621_IRI_ITGB2KI.lif -
 Series007-2 BF.tiff (brightfield image of the cremaster tissue after IRI)
 8. C4-20220621_IRI_ITGB2KI.lif - Series007-2 KIM.tiff (fluorescence image
 of KIM127 antibody labelling after IRI) 9. mAb24_KIM127 in IRI.xlsx
 (Analysis of mAb24 and KIM127 labelling, mean fluorescence intensity,
 before and after IRI) 10. Figure IRI.jpg (Description and Analysis of
 files 1-9) ## Code/software All .tiff files can be viewed by FIJI (ImageJ
 1.54p). All .xlsx files can be viewed by Microsoft Excel. All versions
 appear to be compatible.