Vimentin supports cell polarization by enhancing centrosome function and microtubule acetylation

Saldanha, Renita 1 ; Krishnan, Nikhila2; Ho Thanh, Minh Tri1; Hehnly, Heidi1; Patteson, Alison1

Published Apr 23, 2024 on Dryad. https://doi.org/10.5061/dryad.mcvdnck7d

Abstract

Cell polarity is important for controlling cell shape, motility, and cell division processes. Vimentin intermediate filaments are important for cell migration and cell polarization in mesenchymal cells and assembly of vimentin and microtubule networks is dynamically coordinated, but the precise details of how vimentin mediates cell polarity remain unclear. Here, we characterize the effects of vimentin on the structure and function of the centrosome and the stability of microtubule filaments in wild-type and vimentin-null mouse embryonic fibroblasts (mEFs). We find that vimentin mediates the structure of the pericentrosomal material, promotes centrosome-mediated microtubule regrowth, and increases the level of stable acetylated microtubules in the cell. Loss of vimentin also impairs centrosome repositioning during cell polarization and migration processes that occur during wound closure. Our results suggest that vimentin modulates centrosome structure and function as well as microtubule network stability, which has important implications for how cells establish proper cell polarization and persistent migration.

https://doi.org/10.5061/dryad.mcvdnck7d

Cell lines used in all data sets are

Vim+/+ = wild type (wt) cells - cells with vimentin

Vim-/- = vimentin null (vn) cells - cells without vimentin

All plots were made on Graph Pad Prism

All data was analyzed using FIJI-ImageJ software

Microtubule intensity near the centrosome- Fig 1d

The microtubules in wt and vn cells were stained using alpha tubulin antibody. A box of fixed area was drawn around the centrosome, to measure the intensity of microtubules near the centrosome. The units are AU.

Area of centrosome marked by CEP215 - Fig 1e

Area of centrosome marked by gamma-tubulin - Fig 1f

The area of the centrosome was marked using an antibody for gamma-tubulin centrosomal protein (Peri-centriolar material protein). The Area of the centrosome was traced by using a freehand tool from FIJI ImageJ. The units are µm squared.

Area of centrosome marked by Pericentrin - Fig 1g

The area of the centrosome was marked using an antibody for Pericentrin centrosomal protein (Peri-centriolar material protein). The Area of the centrosome was traced by using a freehand tool from FIJI ImageJ. The units are µm squared.

Centrin Area - Fig 2a

The area of the centrosome was marked by using an antibody for centrin centrosomal protein (centrioles protein). The Area was the centrosome was traced by using a freehand tool from FIJI ImageJ. The units are µm squared.

Cenexin Area - Fig 2b

The area of the centrosome was marked using an antibody for cenexin centrosomal protein (centriolar protein). The Area of the centrosome was traced by using a freehand tool from FIJI ImageJ. The units are µm squared.

Distance between paired centrioles marked by centrin - fig 2d

The centrosomes were marked with centrin centrosomal protein antibody. The distance between the two centrioles was obtained using the line tool from FIJI ImageJ. The unit is microns.

Area of regrowth- Nocodazole treatment - Fig 4

The Vim+/+ (wt) and Vim-/- (vn) cells were treated with 1 micromolar nocodazole for 30 mins and then the cells were washed with PBS. The 0 min case corresponds to wt and vn cells treated with nocodazole. For 1 min case, the wt and vn cells were treated with nocodazole and then washed with PBS, growth media was then added to these cells and they were incubated at 37 C for 1 min. Similarly, for the 2 cases after nocodazole treatment, growth media was added to the cells, and the cells were incubated for 2 mins. The expansion area (the area near the centrosome where you can see the microtubule regrowth) was then measured for 0,1,2 min wt and vn cells using FIJI ImageJ. The units are µm squared.

Contour length of microtubules for nocodazole washout cells - Fig 5

The Vim+/+ (wt) and Vim-/- (vn) cells were treated with 1 micromolar nocodazole for 30 mins. The contour length of the microtubules was then measured using the curve fit algorithm from FIJI ImageJ. The units are microns.

Mean Intensity of Acetylated Microtubules - Fig 6

The microtubules in vim+/+ and vim-/- cells were stained with acetylated tubulin antibody. The area of the cell was traced and the mean intensity was obtained using FIJI ImageJ. The units are AU.

Probability distribution function for wt and vn cells for 1,2,4 hrs near wound edge- Fig 7 d,e,f

The angle between the center of the nucleus and the center of the centrosome was measured in wt and vn cells using FIJI ImageJ for cells near the wound edge. A probability distribution function for the angle made by the centrosome wrt to the nucleus as the center was plotted for wt and vn cells for 1,2,4 hr respectively after wounding (scratch assay). The bin size used was 30 degrees. The unit is degree.

Total intensity of centrosome marked by CEP215 - Supplementary fig 1a

The area of the centrosome was marked using an antibody for CEP215 centrosomal protein (Peri-centriolar material protein). The Area of the centrosome was traced by using a freehand tool from FIJI ImageJ and the total intensity was calculated using the mean intensity and the area of the centrosome. The units are AU *µm squared.

Total intensity of centrosome marked by gamma-tubulin - Supplementary fig 1b

Total intensity of centrosome marked by Pericentrin - Supplementary fig 1c

Total intensity of centrosome marked by cenexin - Supplementary fig 1d

The area of the centrosome was marked using an antibody for Pericentrin centrosomal protein (centriolar protein). The Area of the centrosome was traced by using a freehand tool from FIJI ImageJ and the total intensity was calculated using the mean intensity and the area of the centrosome. The units are AU *µm squared.

Total intensity of centrosome marked by centrin - Supplementary fig 1e

The area of the centrosome was marked using an antibody for Centrin centrosomal protein (centriolar protein). The Area of the centrosome was traced by using a freehand tool from FIJI IMAGEJ and the total intensity was calculated using the mean intensity and the area of the centrosome. The units are AU *µm squared.

Percentage centriole number quantification - Supplementary fig 2b

The cell centrioles are marked by centriolar protein- centrin. The percentage of vim+/+ and vim-/- cells with 1,2 and more than 3 centrioles are plotted.

Cell Spread Area- Supplementary Fig 3

The cell spread area was measured using FIJI ImageJ for cells trained with acetylated microtubules. The units are µm squared

Probability distribution function for wt and vn cells for 1,2,4 hrs in jammed region- Supplementary fig 4 a,b,c

The angle between the center of the nucleus and the center of the centrosome was measured in wt and vn cells using FIJI ImageJ for cells far away from the wound edge. A probability distribution function for the angle made by the centrosome wrt to the nucleus as the center was plotted for wt and vn cells for 1,2,4 hr respectively after wounding (scratch assay). The bin size used was 30 degrees. The unit is degree.