Insights into nuclear dynamics using live‐cell imaging approaches

Bigley, Rachel B.; Payumo, Alexander Y.; Alexander, Jeffrey M.; Huang, Guo N.

doi:10.1002/wsbm.1372

Cited by 3 publications

(2 citation statements)

References 68 publications

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“…In contrast, live cell imaging allows us to visualize active processes in living cells, organs, whole embryos and whole animals from a temporospatial perspective and enables cellular, subcellular and tissue behavior to be quantified as a function of time. Live cell imaging is increasingly being used in various model systems to obtain quantitative insight into intracellular and extracellular processes, such as organelle transport, mitochondrial function, autophagy and energy metabolism, assembly and reorganization of the ECM as well as to study embryonic development/morphogenesis and stem cell function (Eils and Athale, 2003; Friedl, 2004; Kulesa, 2004; Dallas et al, 2006; Sivakumar et al, 2006; Zamir et al, 2008; Lo Celso et al, 2009; Tiede et al, 2009; Xie et al, 2009; Appelhans and Busch, 2017; Bell, 2017; Bigley et al, 2017; Ratnayake and Currie, 2017; Lu et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization

Shiflett

Tiede-Lewis

Xie

et al. 2019

Front. Cell Dev. Biol.

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Bone formation, remodeling and repair are dynamic processes, involving cell migration, ECM assembly, osteocyte embedding, and bone resorption. Using live-cell imaging, we previously showed that osteoblast assembly of the ECM proteins fibronectin and collagen is highly dynamic and is integrated with cell motility. Additionally, osteoblast-to-osteocyte transition involved arrest of cell motility, followed by dendrite extension and retraction that may regulate positioning of embedding osteocytes. To further understand how osteocytes differentiate and embed in collagen, mice were generated that co-expressed GFPtopaz-tagged collagen with a Dmp1-Cre-inducible tdTomato reporter targeted to preosteocytes/osteocytes. Dual live-cell imaging of collagen and osteocyte dynamics in mineralizing primary calvarial cell cultures showed that Dmp1-Cre/tdTomato turned on in early bone nodule forming regions, demarcated by foci of concentrated GFP-collagen bundles that appeared structurally distinct from the surrounding collagen. Dmp1-Cre/tdTomato-positive cells were post-mitotic and were continuously induced throughout the 2 week timecourse, whereas the majority of collagen was assembled by day 7. GFP-collagen fibrils showed global (tissue-level) motions, suggesting coordinated cell layer movement, and local fibril motions mediated by cell-generated forces. Condensation of collagen fibril networks occurred within bone nodules prior to mineralization. Intravital imaging confirmed a similar structural appearance of GFP-collagen in calvarial bone, with analogous global motions of mineralizing areas adjacent to sutures. In early (unmineralized) calvarial cell cultures, Dmp1-Cre/tdTomato-positive cells were motile (mean velocity 4.8 μm/h), moving freely in and around the forming bone nodule, with a small number of these cells embedded in collagen, constraining their motion. In mineralizing cultures, the average velocity of Dmp1-Cre/tdTomato-positive cells was significantly reduced (0.7 μm/h), with many immobilized in the mineralizing nodule. Three apparent mechanisms for embedding of Dmp1-Cre/tdTomato-positive cells were observed. In some cases, a previously motile Dmp1-Cre/tdTomato-positive cell became immobilized in collagen fibril networks that were newly assembled around the cell, thereby entrapping it. In other cases, a motile Dmp1-Cre/tdTomato-positive cell moved into an already formed “collagen lacuna,” arrested its motility and became embedded. Alternatively, some cells switched on tdTomato expression in situ within a lacuna. These data provide new insight into the dynamic process of bone collagen assembly and suggest multiple mechanisms for osteocyte entrapment in collagen matrix.

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Section: Introductionmentioning

confidence: 99%

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization

Shiflett

Tiede-Lewis

Xie

et al. 2019

Front. Cell Dev. Biol.

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“…For many decades, the division and migration of fungal nuclei has attracted the interest of researchers [69][70][71][72]. Structural imaging analysis under non-living cell conditions, as examined via scanning electron microscopy (SEM), highlights the complexity of nuclear structure [73]. However, the lack of living cell biology hinders researchers' understanding of nuclear dynamics during processes such as spore germination and hyphal extension and fusion [63].…”

Section: Discussionmentioning

confidence: 99%

Analysis of Nuclear Dynamics in Nematode-Trapping Fungi Based on Fluorescent Protein Labeling

Zhou,

He,

Zhang

et al. 2023

JoF

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Nematophagous fungi constitute a category of fungi that exhibit parasitic behavior by capturing, colonizing, and poisoning nematodes, which are critical factors in controlling nematode populations in nature, and provide important research materials for biological control. Arthrobotrys oligospora serves as a model strain among nematophagous fungi, which begins its life as conidia, and then its hyphae produce traps to capture nematodes, completing its lifestyle switch from saprophytic to parasitic. There have been many descriptions of the morphological characteristics of A. oligospora lifestyle changes, but there have been no reports on the nuclear dynamics in this species. In this work, we constructed A. oligospora strains labeled with histone H2B–EGFP and observed the nuclear dynamics from conidia germination and hyphal extension to trap formation. We conducted real-time imaging observations on live cells of germinating and extending hyphae and found that the nucleus was located near the tip. It is interesting that the migration rate of this type of cell nucleus is very fast, and we speculate that this may be related to the morphological changes involved in the transformation to a predatory lifestyle. We suggest that alterations in nuclear shape and fixation imply the immediate disruption of the interaction with cytoskeletal mechanisms during nuclear migration. In conclusion, these findings suggest that the signal initiating nuclear migration into fungal traps is generated at the onset of nucleus entry into a trap cell. Our work provides a reference for analysis of the dynamics of nucleus distribution and a means to visualize protein localization and interactions in A. oligospora.

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Nucleosome Crowding in Chromatin Slows the Diffusion but Can Promote Target Search of Proteins

Kanada

Terakawa

Kenzaki

et al. 2019

Biophysical Journal

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Dynamics of nuclear proteins in crowded chromatin has only been poorly understood. Here, we address the diffusion, target search, and structural dynamics of three proteins in a model chromatin using coarse-grained molecular simulations run on the K computer. We prepared two structures of chromatin made of 20 nucleosomes with different nucleosome densities and investigated dynamics of two transcription factors, HMGB1 and p53, and one signaling protein, ERK, embedded in the chromatin. We found fast and normal diffusion of the nuclear proteins in the low-density chromatins and slow and subdiffusional movements in the high-density chromatin. The diffusion of the largest transcription factor, p53, is slowed by high-density chromatin most markedly. The on rates and off rates for DNA binding are increased and decreased, respectively, in the high-density chromatin. To our surprise, the DNA sequence search was faster in chromatin with high nucleosome density, though the diffusion is slower. We also found that the three nuclear proteins preferred to bind on the linker DNA and the entry and exit regions of nucleosomal DNA. In addition to these regions, HMGB1 and p53 also bound to the dyad.

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Insights into nuclear dynamics using live‐cell imaging approaches

Cited by 3 publications

References 68 publications

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization

Analysis of Nuclear Dynamics in Nematode-Trapping Fungi Based on Fluorescent Protein Labeling

Nucleosome Crowding in Chromatin Slows the Diffusion but Can Promote Target Search of Proteins

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