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Biomechanics

Biomechanics - Poster Session D

Poster D2 - Loss of vimentin-nuclear connectivity impairs fibroblast morphology and nuclear positioning

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The cytoskeleton is comprised of actin, microtubules, and intermediate filaments. It is well established that actin and microtubules contribute to cellular attachment, spreading, morphology, and migration. Notably, all three components of the cytoskeleton are physically coupled to the nucleus via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. In this project, we investigated the role of intermediate filaments’ connection to the nucleus in shaping cell morphology and positioning. Specifically, we examined the impact of removing nesprin-3, a LINC complex protein that links intermediate filaments to the nucleus. We chose to study this in NIH3T3 fibroblasts, a common model cell line for cell spreading and migration. The major intermediate filament protein in fibroblasts is vimentin. We hypothesized loss of nesprin-3 in these cells would impair cell morphology and alter migration by preventing vimentin from interacting with the nucleus.

Materials and

Methods:  To remove nesprin-3 from fibroblasts, we used CRISPR/cas9 to create a nesprin-3 KO NIH3T3 cell line. Loss of nesprin-3 in the knockout was confirmed using western blotting and immunofluorescence. To examine cell attachment and spreading, cells were seeded on fibronectin coated glass coverslips and analyzed by immunofluorescent staining via confocal microscopy. To assess nuclear positioning, we seeded cells onto crossbow micropatterns to simulate 2D cell migration morphology. In all experiments, cells were stained for actin, vimentin, and nucleus. The images were analyzed to determine differences in cell area, perimeter, and distribution of vimentin. Live cell imaging was used to assess migration speed and persistence of migration.

Results, Conclusions, and Discussions: Nesprin-3 KO cells were able to adhere and spread on fibronectin-coated surfaces. Quantification cell area showed no significant differences in the spread area of nesprin-3 KO cells compared to wild-type cells. However, we observed that nesprin-3 KO cells do have altered cellular morphology, with diminished cell protrusions.

Cells seeded on crossbow micropatterns showed nuclear positioning defects in nesprin-3 KO cells when compared with the wild type. Upon further analysis, we observed that the displacement of the nuclei was greater only in the horizontal direction, perpendicular to cell movement, and not vertically. Additionally, it was observed that cross sectional nuclear area was decreased in nesprin-3 KO cells compared to wild type cells. In summary, our data demonstrate that vimentin to nuclear connectivity is important for nuclear positing and likely also regulates cellular protrusions.

Acknowledgements (Optional):