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Abstract Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and tilting of the metaphase plate (MP). To understand the effect of confinement on mitosis by fibers (ECM confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesions on fibers. We discover that the extent of confinement influences RF organization in 3D, forming triangular and band-like patterns on the cell cortex under low and high confinement, respectively. Our mechanistic analysis reveals that the patterning of RFs on the cell cortex is the primary driver of the MP rotation. A stochastic Monte Carlo simulation of the centrosome, chromosome, membrane interactions, and 3D arrangement of RFs recovers MP tilting trends observed experimentally. Under high ECM confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM confinement leads to low and high MP tilts, which we mechanistically show to depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in ECM-confining microenvironments in vivo. 

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages. 

The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse-intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy ( LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of -DCM remains incompletely understood. Using induced-pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA-mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared with healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggests that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM. 

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell’s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces. 

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS. 

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours. 

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear–cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell–cell adhesion and barrier function, as well as altered cell–matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH–expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.