Mitotic outcomes and errors in fibrous environments

Abstract

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex–astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries.

Confinement in fibrous environments positions and orients mitotic spindles

“…1e ). The observation that increased inter-kinetochore separation distance corresponds to faster mitotic durations matches our previous observations 9 . Cytokinesis events captured from live cell imaging revealed substantial reorientation of the division axis in cells under ECM-confinement ( Supplementary Fig.…”

“…In our previous work, we showed that cells attached to two fibers (12 µm apart) underwent division by balling up while being held by RFs originating from four adhesion sites on fibers (Fig. 1a(i), Supplementary Movie S1) 9 . Often the cells got trapped between the two fibers, causing them to bow outwards (Fig.…”

“…RFs are part of the cortical mechanosensory complex, which links the cell cortex to the astral microtubules to transmit the effects of external cues to organize the mitotic spindle 6,25 . Our prior work has shown that increased RF coverage (RFC) on the cell cortex limits rounding cell body movement, causing faster division and a higher incidence of multipolar defects 9 . Studies using flat tissue culture plates and fibronectin-coated micropatterns have shown that RFs formed on the coverslips promote spindle alignment parallel to the substrate 1,2 .…”

Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)

“…Previously, we showed that cellular shapes can be tuned to the underlying fiber network, with actin stress fibers aligning along ECM-fibers in cells attached to single and multiple parallel fibers, and intersecting actin networks in cells attached to a crosshatch network of fibers [49, 50]. In a subsequent study, the underlying actin networks were found to regulate mitotic outcomes through actin retraction fibers connecting the rounded mitotic cell body to interphase adhesion sites [51]. In both studies, while the patterning of actin networks matched the arrangement of underlying ECM networks, it was unclear how far the effect of adhesions on fibers was extended to shape the actin networks.…”

“…Fibers were spun on hollow 5 × 5 mm metal scaffolds. The first layer of fibers deposited were large diameter fibers ∼ 2 µ m ( M w = 15 × 10 6 g/mol) followed by an orthogonal layer of 200 nm ( M w = 2.5 × 10 6 g/mol) fibers with spacing varying from 7 to 20 µ m to achieve a variety of cell shapes (elongated on single fibers and parallel-shaped cells on two or more fibers) [51, 69, 70]. Additionally, crosshatch networks of 200 nm fiber diameters were also prepared with spacing varying from 7 to 20 µ m [50, 71] to achieve polygonal and kite-shaped cells on multiple fibers.…”