Microscopic images and schematics illustrating processes of microenvironment sensing and cortical actomyosin partitioning in T cells
Data files
Dec 14, 2023 version files 118.79 GB
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Figure_10_-_source_file_(MsCD4__T_cell_-_NM2A-GFP).tif
2.30 GB
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Figure_1a_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_Clathrin_Collagen1)_.tif
1.17 GB
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Figure_1c_-_source_file_(HuCD4__T_cell_-_Hoechst_NM2A-Phalloidin_Collagen1).tif
562.12 MB
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Figure_2a_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_Actinin_Collagen1).tif
1.35 GB
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Figure_2b_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_Actinin_Collagen1).tif
872.49 MB
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Figure_2c_-_source_file_(HuCD4__T_cell_-_Hoechst_NM2A_Phalloidin_Collagen1).tif
360.74 MB
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Figure_3a_cell_1__3c_-_source_file_(HuCD4__T_cell_-_Hoechst_Septin_7_Phalloidin_Collagen1).tif
629.20 MB
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Figure_3a_cell_2_-_source_file_(HuCD4__T_cell_-_Hoechst_Septin_7_Phalloidin_Collagen1).tif
469.80 MB
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Figure_3a_cell_3_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_Septin_7_Collagen1).tif
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Figure_3a_cell_4_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_Septin_7_Collagen1).tif
282.31 MB
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Figure_3d_-_source_file_(DO-11-10_shSEPT7-GFP_cells_-_Brightfield_GFP_Hoechst).tif
5.94 GB
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Figure_4_(_Blebbistatin)_-_source_file_(HuCD4__T_cells__BF).tif
1.17 GB
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Figure_4_(_Calyculin_A)_-_source_file_(HuCD4__T_cells__BF).tif
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Figure_4_(_DMSO)_-_source_file_(HuCD4__T_cells__BF).tif
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Figure_4_(_Nocodazole)_-_source_file_(HuCD4__T_cells__BF).tif
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Figure_4_(_UR214-9)_-_source_file_(HuCD4__T_cells__BF).tif
2.04 GB
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Figure_5a_-_source_file_(HuCD4__T_cells_-_Hoechst_Septin7_Phalloidin_Collagen1).tif
210.43 MB
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Figure_5b_-_source_file_-_Actinin_CTRL_T_cell_2_w1iSIM640-Em.tif
872.49 MB
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Figure_5b_-_source_file_-_Actinin_CTRL_T_cell_4_w1iSIM640-Em.tif
805.37 MB
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Figure_5b_-_source_file_-_Collagen_405_GAM550_Tubulin_Phalloidin488V-Green_Centriolin647_CTRL_w1iSIM640-Em.tif
1.34 GB
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Figure_5b_-_source_file_-_CTRL_405Phal_488RbTub_550MsMLCK_647_GelSiRActin_1_w1iSIM640-Em.tif
1.13 GB
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Figure_5b_-_source_file_-_CTRL_405Phal_488RbTub_550MsMLCK_647_GelSiRActin_3_w1iSIM640-Em.tif
1.55 GB
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Figure_5b_-_source_file_-_Data_legend.xlsx
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Figure_5b_-_source_file_-_FAK_CTRL_T_cell_1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_FAK_CTRL_T_cell_2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G10_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G12_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G13_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G15_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G17_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G18_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G19_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G20_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G21_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G4_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G5_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G6_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G7_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_-H-P-2A-G8_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK1_w1iSIM640-Em.tif
990 MB
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK4_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_CTRL_HC-Pha-rTub-MLCK5_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Actub_Phal-Col_i_w1iSIM640-Em.tif
696.31 MB
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Figure_5b_-_source_file_-_T_cell_gel_H-Actub_Phal-Col_i1_w1iSIM640-Em.tif
528.53 MB
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Figure_5b_-_source_file_-_T_cell_gel_H-aTub_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Dynein_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Dynein_Phal-Col_i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Kin_Phal-Col_i_w1iSIM640-Em.tif
444.63 MB
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Figure_5b_-_source_file_-_T_cell_gel_H-NM2A_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-NM2A_Phal-Col_i1_w1iSIM640-Em.tif
360.74 MB
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Figure_5b_-_source_file_-_T_cell_gel_H-NM2A_Phal-Col_i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-NM2A_Phal-Col_i3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep10_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep12_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep12_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep2_Phal-Col_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep2_Phal-Col1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep2_Phal-Col2_w1iSIM640-Em.tif
629.20 MB
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep3_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep3_Phal-Col_i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep3_Phal-Col_i3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep4_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep4_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep5_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep5_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep6_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep6_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep6_Phal-Col_i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep6_Phal-Col_i3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep7_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep7_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep8_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep8_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep9_Phal-Col_i_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep9_Phal-Col_i1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep9_Phal-Col_i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cell_gel_H-Sep9_Phal-Col_i3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells__DAB2_Met__i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells__DAB2_PFA__i2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells__phAP2_Met__i8_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-AcTub-Phal-Gel_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-AcTub-Phal-Gel1_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-AcTub-Phal-Gel2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel2_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel3_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel4_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel5_w3iSIM488-Em.tif
503.36 MB
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel7_w1iSIM640-Em.tif
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Figure_5b_-_source_file_-_T_cells_CTRL_H-NM2A-Phal-Gel8_w3iSIM488-Em.tif
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Figure_6_-_source_file_-_hoe405_phall488_RbSept7-555_Col647_UR214_Tsx100obj1_w1iSIM640-Em-1.tif
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj10_w1iSIM640-Em.tif
587.25 MB
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj11_w1iSIM640-Em.tif
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj12_w1iSIM640-Em.tif
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj18_w1iSIM640-Em.tif
654.37 MB
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj3_w1iSIM640-Em.tif
528.53 MB
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj5_w1iSIM640-Em.tif
822.15 MB
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj6_w1iSIM640-Em.tif
570.47 MB
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Figure_6_-_source_file_-_hoe405_phall555_RbSept7-488_Col647_control_Tsx100obj9_w1iSIM640-Em-11.tif
2.10 GB
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Figure_7a_-_source_file_(MDA-MB-231_-_Hoechst-Septin7GFP_Tub_AcTub).tif
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Figure_7c_-_source_file_(NK-92_-_Phalloidin_Collagen1_Septin7_Hoechst).tif
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Figure_7d_-_source_file_(NK-92_-_Hoechst_Phalloidin_Septin7_Tub).tif
2.14 GB
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Figure_8_-_source_file_(NK-92_cell_-_Hoechst_Phalloidin_Tubulin_Collagen1).tif
612.46 MB
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Figure_9c_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_phSer19_Collagen1).tif
1.01 GB
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Figure_9f_-_source_file_(HuCD4__T_cell__BF__5spf).tif
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Figure_SI1__DMSO_sample_1_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__DMSO_sample_2_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__DMSO_sample_3_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__DMSO_sample_4_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__UR214-9_sample_1_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__UR214-9_sample_2_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__UR214-9_sample_3_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
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Figure_SI1__UR214-9_sample_4_-_source_file_(HuCD4_T_cell_-_Collagen1_Phalloidin_Hoechst).tif
906.04 MB
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Figure_SI2_-_source_file_(HuCD4_T_cell_-_SiR-Actin_Hoechst).tif
2.94 GB
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Figure_SI3e_-_source_file.pdf
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Figure_SI3f_-_source_file_-_2023_09_15_NK92_UR214-9_Septin7.pdf
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Figure_SI3f_-_source_file_-_2023_09_19_NK92_UR214-9_Septin7.pdf
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Figure_SI4a_(_DMSO)_-_source_file_(MDA-MB-231_-_Hoechst_GFP-Septin9_Phalloidin_Tubulin).tif
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Figure_SI4a_(_UR214-9)_-_source_file_(MDA-MB-231_-_Hoechst_GFP-Septin9_Phalloidin_Tubulin).tif
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Figure_SI4b_(_DMSO)_-_source_file_(MDA-MB-231__GFP-Septin9).tif
717.32 MB
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Figure_SI4b_(_UR214-9)_-_source_file_(MDA-MB-231__GFP-Septin9).tif
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Figure_SI4c_(_BLB)_-_source_file_(MDA-MB-231_-_Hoechst_Tubulin_Septin9-GFP_F-Actin).tif
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Figure_SI5_-_source_file_(MDA-MB-231_cells_-_Hoechst_Septin7GFP_acTub_Tub).tif
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Figure_SI6a_cell_1_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_phSer19_Collagen1).tif
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Figure_SI6a_cell_2_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_phSer19_Collagen1).tif
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Figure_SI6b_cell_1_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_phSer19_Collagen1).tif
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Figure_SI6b_cell_2_-_source_file_(HuCD4__T_cell_-_Hoechst_Phalloidin_phSer19_Collagen1).tif
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README.md
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Abstract
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.
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
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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 -
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