Biology subjects

Nain, A. S.

Publications and source records attributed to Nain, A. S..

5 recordsLinked to original sources

bioRxiv2025

Observing biological spatio-angular structures and dynamics with statistical image reconstruction and polarized fluorescence microscopy

Understanding molecular orientation and density distributions is essential for exploring biological structure and function. Polarized fluorescence microscopy (PFM) provides insights into molecular architecture but struggles to resolve three-dimensional (3D) molecular orientation distributions, particularly in densely labeled or structurally complex specimens. To address this, we introduce the efficient generalized Richardson-Lucy (eGRL) algorithm, a robust framework for reconstructing 3D molecular density and orientation (spatio-angular) distributions from PFM data. By modeling the imaging process in spatio-angular hyperspace, we propose a maximum-likelihood solution enhanced by dimensionality reduction and angular domain transformation to overcome computational challenges. eGRL improves accuracy and efficiency across different PFM implementations, enabling use on standard platforms. We utilize our methods to resolve biological spatio-angular structures and dynamics otherwise impossible to resolve, including the tangential alignment of actin filaments in U2OS cells, nanowire-guided cytoskeletal organization in NIH3T3 cells, rotational actin patterns in live HeLa protrusions, and membrane tension-induced anisotropy in live macrophages.

bioengineering↗

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↗

bioRxiv2022

Fiber Diameter and Architecture Direct Three-Dimensional Assembly of Pericytes into Spheroids

Due to their physiological relevance, multicellular 3D spheroids are actively replacing standard 2D monolayer cultures. How spheroids are formed through the assembly of individual cells in natural fibrous environments that include a mix of diameters and architectures in vivo remains unknown. Here, we demonstrate that the spontaneous assembly of human vasa vasorum-derived pericytes in 3D spheroids depends on the fiber diameter and network architecture. A parallel arrangement of suspended fibers of all tested diameters (200, 500, and 800 nm) leads to the formation of spheroids, while on crosshatch networks, spheroid assembly on larger diameters is absent. The design of fibrous networks of a mix of diameters and architectures leads to the patterning of spheroids in desired locations. Fiber remodeling in parallel arrangements serves as force sensors providing mechanical insights into the assembly dynamics of spheroids and subsequent cell sprouting from spheroids. Translocation and merger of spheroids occur predominantly on parallel fiber networks, while on crosshatch networks, a cellular exchange is observed between spheroids connected with remodeled fibers. Rho kinase inhibition by Y27632 and subsequent wash-off leads to spheroid disintegration and reassembly, thus, highlighting the role of cell contractility in the assembly and integrity of 3D spheroids. Overall, using extracellular mimicking fiber networks of varying diameters and architectures, we report new insights into the 3D dynamics of spheroids which may inform pericytes role in vasculogenesis, and (patho)physiological angiogenesis

bioengineering↗

bioRxiv2020

Rules of Contact Inhibition of Locomotion for Cells on Suspended Nanofibers

Contact inhibition of locomotion (CIL), in which cells repolarize and move away from contact, is now established as a fundamental driving force in development, repair, and disease biology. Much of what we know of CIL stems from studies on 2D substrates that fail to provide an essential biophysical cue - the curvature of extracellular matrix fibers. We discover rules controlling outcomes of cell-cell collisions on suspended nanofibers, and show them to be profoundly different from the stereotyped CIL behavior known on 2D substrates. Two approaching cells attached to a single fiber do not repolarize upon contact but rather usually migrate past one another. Fiber geometry modulates this behavior: when cells are attached to two fibers, reducing their freedom to reorient, only one of a pair of colliding cells repolarizes on contact, leading to the cell pair migrating as a single unit. CIL outcomes also change when one cell has recently divided and moves with high speed- cells more frequently walk past each other. In collisions with division in the two-fiber geometry, we also capture rare events where a daughter cell pushes the non-dividing cell along the fibers. Our computational model of CIL in fiber geometries reproduces the core qualitative results of the experiments robustly to model parameters. Our model shows that the increased speed of post-division cells may be sufficient to explain their increased walk-past rate. Our results suggest that characterizing cell-cell interactions on flat substrates, channels, or micropatterns is not sufficient to predict interactions in a matrix - the geometry of the fiber can generate entirely new behaviors. SignificanceWhen cells heal a wound or invade a new area, they coordinate their motion. Coordination is often studied by looking at what happens after pairs of cells collide. Post-collision, cells often exhibit contact inhibition of locomotion- they turn around and crawl away from the point where they touched. Our knowledge of repolarization on contact comes from studies on flat surfaces, unlike cells in the body, which crawl along fibers. We discover that cells on single fibers walk past one another- but that cells in contact with multiple fibers stick to one another and move as pairs. This outcome changes to walk-past after cell division. Our experiments and models reveal how the environment regulates cell-cell coordination after contact.

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↗