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Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness.

ABSTRACT: In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a comprehensive approach. We studied two cell types-Schlemm's canal endothelial cells and mouse embryonic fibroblasts (MEFs)-using four different probe technologies: 1) atomic force microscopy (AFM) with sharp tip, 2) AFM with round tip, 3) optical magnetic twisting cytometry (OMTC), and 4) traction microscopy (TM). Perturbation of Schlemm's canal cells with dexamethasone treatment, ?-actinin overexpression, or RhoA overexpression caused increases in traction reported by TM and stiffness reported by sharp-tip AFM as compared to corresponding controls. By contrast, under these same experimental conditions, stiffness reported by round-tip AFM and by OMTC indicated little change. Knockout (KO) of vimentin in MEFs caused a diminution of traction reported by TM, as well as stiffness reported by sharp-tip and round-tip AFM. However, stiffness reported by OMTC in vimentin-KO MEFs was greater than in wild type. Finite-element analysis demonstrated that this paradoxical OMTC result in vimentin-KO MEFs could be attributed to reduced cell thickness. Our results also suggest that vimentin contributes not only to intracellular network stiffness but also cortex stiffness. Taken together, this evidence suggests that AFM sharp tip and TM emphasize properties of the actin-rich shell of the cell, whereas round-tip AFM and OMTC emphasize those of the noncortical intracellular network.

SUBMITTER: Vahabikashi A

PROVIDER: S-EPMC6369565 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness.

Vahabikashi Amir A Park Chan Young CY Perkumas Kristin K Zhang Zhiguo Z Deurloo Emily K EK Wu Huayin H Weitz David A DA Stamer W Daniel WD Goldman Robert D RD Fredberg Jeffrey J JJ Johnson Mark M

Biophysical journal 20190107 3

In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a co ...[more]

PMID: 30685055

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Project description:Background and aimsPenium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.MethodsMicrotubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.Key resultsThe cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.ConclusionsThe cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.

Dataset Information

Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness.

Publications

Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness.

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