Biology subjects

Kapania, R.

Publications and source records attributed to Kapania, R..

4 recordsLinked to original sources

bioRxiv2024

Confinement in fibrous environments positions and orients mitotic spindles

Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement 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 adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. 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 mechanistically 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 fibrous ECM-confining microenvironments in vivo.

biophysics↗

bioRxiv2021

Mitotic Outcomes in Fibrous Environments

During mitosis, cells round up and generate outward forces to create space and orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we recapitulate in vivo adhesion organization and confinement to interrogate mitotic outcomes 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 large 3D displacement while being held by retraction fibers. Increasing the number of parallel fibers increases cellular extremity FACs and retraction fiber-driven stability, leading to reduced 3D cell-body movement, metaphase plate rotations, 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 retraction fibers from two perpendicular suspended fibers. We develop a cortex-astral microtubule analytical friction and force model to capture retraction-fiber-driven stability of the metaphase plate rotations. We report that reduced orientational stability results in increased monopolar mitotic defects. In the case of cells attached to two parallel fibers, rounded mitotic cells can get confined between the suspended fibers, allowing estimation of the mitotic forces through measurement of the outward deflection of the fibers. Interestingly, confinement causes rotated mitotic spindles similar to those reported in dense tissues. Overall, we establish dynamics of mitosis in fibrous environments governed by fiber arrangement and architecture-driven differences in interphase cell shapes, adhesion geometries, and varying levels of mechanical confinement.

cell biology↗

bioRxiv2021

Sculpting rupture-free nuclear shapes in fibrous environments

Cytoskeleton-mediated force transmission regulates nucleus morphology. How nuclei shaping occurs in fibrous in vivo environments remains poorly understood. Here a suspended nanofiber assay of precisely-tunable (nm-m) diameters is used to quantify nucleus plasticity in fibrous environments mimicking the natural extracellular matrix. In contrast to the apical cap over the nucleus in cells on 2-dimensional surfaces, the cellular cytoskeleton of cells on fibers displays a uniform actin network caging the nucleus. The role of contractility-driven caging in sculpting nuclear shapes is investigated as cells spread on aligned single fibers, doublets, and multiple fibers of varying diameters. Cell contractility increases with fiber diameter due to increased focal adhesion clustering and density of actin stress fibers, which correlates with increased mechanosensitive transcription factor YAP translocation to the nucleus. Unexpectedly, large- and small-diameter fiber combinations lead to teardrop-shaped nuclei due to stress-fiber anisotropy across the cell. As cells spread on fibers, diameter-dependent invaginations that run the nucleuss length are formed at contact sites. The deepest and sharpest invaginations are insufficient to trigger nucleus rupture, often observed in 2D or confined systems. Overall, we describe the unknown adaptability of nuclei to fibrous environments and resultant sculpting of the nucleus shapes, with pathophysiological implications.

cell biology↗

bioRxiv2019

Nano-twines to twine-bridges: Role of force exerting perpendicular lateral protrusions in fibroblastic cell contraction

Aligned extracellular matrix fibers enable fibroblasts to undergo myofibroblastic activation and lead to elongated cell morphology. The fibroblasts in turn contract to cause alignment of the extracellular matrix. This feedback process is critical in pathological occurrences such as desmoplasia and is not well understood. Using engineered fiber networks that serve as force sensors, we identify lateral protrusions with specific functions and morphology that are induced by elongated fibroblastic cells and which apply extracellular fiber-deflecting contractile forces. Lateral projections, named twines, produce twine bridges upon interacting with neighboring parallel fibers. These mature into \"perpendicular lateral protrusions\" (PLPs) that enable cells to spread laterally and effectively contract. Using quantitative microscopy, we show that the twines originate from the stratification of cyclic actin waves traversing the entire length of the cell. The primary twines swing freely in 3D and engage neighboring extracellular fibers. Once engaged, a lamellum extends from the primary twine and forms a second twine, which also engages with the neighboring fiber. As the lamellum fills in the space between the two twines, a sheet-like PLP is formed to contract effectively. By controlling the geometry of extracellular networks we confirm that anisotropic fibrous environments enable PLP formation, and these force-generating PLPs are oriented perpendicular to the parent cell body. PLP formation kinetics indicated mechanisms analogous to other/known actin-based structures. Our identification of force-exerting PLPs in anisotropic fibrous environments suggests an explanation for cancer-associated desmoplastic expansion at single-cell resolution, providing possible new clinical intervention opportunities.

cell biology↗