Figure 2. Knockout of KIF21B causes microtubule (MT) overgrowth in Jurkat cells.

(A–B) STED images of indicated Jurkat T cell lines. Cells were added to poly-D-lysine-coated coverslips with immobilized anti-CD3 and fixed 7 min after incubation. Cells were stained for α-tubulin and F-actin. Orange boxes indicate zoomed areas shown on the right. (B) Images were background subtracted and split into radial and non-radial components based on the MT orientation in relation to the centrosome. Radial intensity profiles were made from a 45° sector starting from the centrosome and directed toward the farthest removed portion of the cell periphery (white). The resulting heat maps were used to quantify the proportion of non-radially oriented MTs as described in Materials and methods. (C) Quantification of the proportion of non-radially oriented MTs analyzed per cell. n = 44, 45, and 52 cells from three independent experiments. ***p<0.001, ns = not significant (t test). (D–E) Volume renderings of indicated T cell lines stained for α-tubulin. T cells were fixed 2 min after activation on poly-D-lysine-coated coverslips with immobilized anti-CD3. Samples were expanded ~4.5 times following an Expansion Microscopy (ExM) protocol and MTs were imaged using on a confocal microscope. Imaris software was used to create volume renders and analysis. MTs emanating from the centrosome were analyzed using a spherical shell with a 10 μm inner diameter and a thickness of 1 μm (gray); filaments crossing the sphere are highlighted (red). Zooms of the centrosome are shown from two different angles. (E) Fluorescence signal was converted to a surface mask using Imaris software to analyze total tubulin intensity. Additional examples are shown in Figure 2—figure supplement 1B. (F) Quantification of the total number of MTs per cell emanating from the centrosome quantified at 5 μm distance from the centrosome of indicated Jurkat T cell lines. n = 8 cells for all conditions. From left to right: *p=0.0131, *p=0.0367, ns = not significant (t test). (G) Quantification of estimated MT length per cell in indicated Jurkat T cell lines. Fluorescence intensity of 1 μm filament length was measured for all MTs crossing a spherical shell of 1 um width with a 5 μm inner radius located around the centrosome, as shown in D. The average MT length per cell was calculated by correcting the total fluorescence intensity per cell by the number of MTs at the centrosome. n = 8 cells for all conditions. ***p=0.0002, **p=0.0013, ns = not significant (t test).

Figure 2—figure supplement 1. Characterization of microtubule (MT) network in polarizing T cells by Expansion Microscopy (ExM).

(A) Quantification of total estimated MT length per cell indicated in indicated Jurkat T cell lines. Values were obtained by multiplication of total MT numbers by the estimated average MT length per cell. n = 8 cells for all conditions. *p=0.0169, **p=0.0043, ns = not significant (t test). (B) Series of five exemplary volume renderings per indicated T cell lines stained for α-tubulin (related to Figure 2D, E). T cells were fixed 2 min after activation on poly-D-lysine-coated coverslips with immobilized anti-CD3. Samples were expanded ~4.5 times following an ExM protocol and MTs were imaged using on a confocal microscope. Imaris software was used to create volume renders. (C) Volume renderings of indicated T cell lines stained for α-tubulin to highlight examples suggesting that a MT stalk is formed during the process of centrosome polarization. Centrosome (cyan arrowheads) and MT stalks emanating from the centrosome toward the synapse are indicated (orange arrowheads).

Figure 2—video 1. 3D volume render of an Expansion Microscopy (ExM) imaged Jurkat control cell 2 min after activation.

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Volume rendering of a control Jurkat cell fixed 2 min after activation on anti-CD3 coated coverslips stained for α-tubulin. Samples were isotropically expanded using an ExM protocol and imaged on a confocal microscope. Images were processed and analyzed using Imaris software. Microtubules emanating from the centrosome were analyzed using a spherical shell with a 5 μm inner radius, filaments crossing the sphere are highlighted (red). Every square in the grid corresponds to 5 × 5 μm post expansion. Example corresponds to Figure 2D–E.

Figure 2—video 2. 3D volume render of an Expansion Microscopy (ExM) imaged Jurkat KIF21B-KO cell (KO #1) 2 min after activation.

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Volume rendering of a KIF21B-KO Jurkat cell (KO #1) fixed 2 min after activation on anti-CD3-coated coverslips stained for α-tubulin. Samples were isotropically expanded using an ExM protocol and imaged on a confocal microscope. Images were processed and analyzed using Imaris software. Microtubules emanating from the centrosome were analyzed using a spherical shell with a 5 μm inner radius, filaments crossing the sphere are highlighted (red). Every square in the grid corresponds to 5 × 5 μm post-expansion. Example corresponds to Figure 2D–E.

Figure 2—video 3. 3D volume render of an Expansion Microscopy (ExM) imaged Jurkat KIF21B-KO cell (KO #2) 2 min after activation.

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Volume rendering of a control KIF21B-KO Jurkat cell (KO #2) fixed 2 min after activation on anti-CD3-coated coverslips stained for α-tubulin. Samples were isotropically expanded using an ExM protocol and imaged on a confocal microscope. Images were processed and analyzed using Imaris software. Microtubules emanating from the centrosome were analyzed using a spherical shell with a 5 μm inner radius, filaments crossing the sphere are highlighted (red). Every square in the grid corresponds to 5 × 5 μm post expansion. Example corresponds to Figure 2D–E.