Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

Olga Adamczyk,Maksymilian Szczypior,Zbigniew Baster,Zenon Rajfur

doi:10.3390/ijms22020960

Olga Adamczyk, Maksymilian Szczypior + Show 2 more

Open Access

https://doi.org/10.3390/ijms22020960

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Abstract

It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes. Confocal microscopy allows to observe changes in the 3D organization of the cytoskeleton in response to a precise mechanical stimulus applied with, for example, a bead trapped with optical tweezers. Optical tweezers-based method (OT) is a microrheological technique which employs a focused laser beam and polystyrene or latex beads to study mechanical properties of biological systems. Latex beads, functionalized with a specific protein, can interact with proteins located on the surface of the cellular membrane. Such interaction can significantly affect the cell’s behavior. In this work, we demonstrate that beads alone, placed on the cell surface, significantly change the architecture of actin, microtubule, and intermediate filaments. We also show that the observed molecular response to such stimulus depends on the duration of the cell–bead interaction. Application of cytoskeletal drugs: cytochalasin D, jasplakinolide, and docetaxel, abrogates remodeling effects of the cytoskeleton. More important, when cells are plated on elastic substrates, which mimic the mechanical properties of physiological cellular environment, we observe formation of novel, “cup-like” structures formed by the microtubule cytoskeleton upon interaction with latex beads. These results provide new insights into the function of the microtubule cytoskeleton. Based on these results, we conclude that rigidity of the substrate significantly affects the cellular processes related to every component of the cytoskeleton, especially their architecture.

Highlights

Micrometer-size beads are commonly used in biomedical studies
We discovered that novel cup-like structures were formed by microtubule cytoskeleton, which is not known for its active involvement in endocytosis, only on elastic substrates
Cells were stained against actin, α-tubulin, or vimentin and images of respective cytoskeleton components were captured using confocal microscopy

Summary

Introduction

Micrometer-size beads are commonly used in biomedical studies. One area, where such microspheres are extensively used, is to study the process of phagocytosis where they serve as a model for cellular phagocytosis [1]. Latex beads are employed in cellular mechanobiology studies, where they are usually used with optical or magnetic tweezers, that allow to manipulate the beads. These techniques can be applied to stimulate cells mechanically [2] as well as to measure various parameters of cells, such as cytoskeleton elasticity [3] or membrane viscoelasticity [4]. The experiment bases on attaching a bead to the outer membrane of a cell and pulling a thin, membrane-derived thread out of it [9] This technique was used to estimate cell membrane properties such as its viscosity or the actin cytoskeleton–cell membrane interaction [5,9]. Optical tweezers-pulled tethers are used to study nanotubular protrusions like tubular nanotubes or filopodia [10,11]

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