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Nesprin-2 accumulates at the front of the nucleus during confined cell migration.

ABSTRACT: The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, which links the cytoskeleton to the nuclear interior. We demonstrate that nesprin-2 accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are mobile at time scales similar to the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient for this accumulation. Actin filaments are organized in a barrel structure around the nucleus in the direction of movement. Using two-photon ablation and cytoskeleton-inhibiting drugs, we demonstrate an actomyosin-dependent pulling force on the nucleus from the front of the cell. The elastic recoil upon ablation is dampened when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant toward the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

SUBMITTER: Davidson PM

PROVIDER: S-EPMC7332974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundProteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated.Methodology/principal findingsWe investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization.ConclusionsThese studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

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Nesprin-2 accumulates at the front of the nucleus during confined cell migration.

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