Dataset Information

Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

ABSTRACT: Although osteocytes have historically been viewed as quiescent cells, it is now clear that they are highly active cells in bone and play key regulatory roles in diverse skeletal functions, including mechanotransduction, phosphate homeostasis and regulation of osteoblast and osteoclast activity. Three dimensional imaging of embedded osteocytes and their dendritic connections within intact bone specimens can be quite challenging and many of the currently available methods are actually imaging the lacunocanalicular network rather than the osteocytes themselves. With the explosion of interest in the field of osteocyte biology, there is an increased need for reliable ways to image these cells in live and fixed bone specimens. Here we report the development of reproducible methods for 2D and 3D imaging of osteocytes in situ using multiplexed imaging approaches in which the osteocyte cell membrane, nucleus, cytoskeleton and extracellular matrix can be imaged simultaneously in various combinations. We also present a new transgenic mouse line expressing a membrane targeted-GFP variant selectively in osteocytes as a novel tool for in situ imaging of osteocytes and their dendrites in fixed or living bone specimens. These methods have been multiplexed with a novel method for labeling of the lacunocanalicular network using fixable dextran, which enables aspects of the osteocyte cell structure and lacunocanalicular system to be simultaneously imaged. The application of these comprehensive approaches for imaging of osteocytes in situ should advance research into osteocyte biology and function in health and disease.

SUBMITTER: Kamel-ElSayed SA

PROVIDER: S-EPMC4591054 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

Kamel-ElSayed Suzan A SA Tiede-Lewis LeAnn M LM Lu Yongbo Y Veno Patricia A PA Dallas Sarah L SL

Bone 20150317

Although osteocytes have historically been viewed as quiescent cells, it is now clear that they are highly active cells in bone and play key regulatory roles in diverse skeletal functions, including mechanotransduction, phosphate homeostasis and regulation of osteoblast and osteoclast activity. Three dimensional imaging of embedded osteocytes and their dendritic connections within intact bone specimens can be quite challenging and many of the currently available methods are actually imaging the ...[more]

PMID: 25794783

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Project description:Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones, forming foci within 5-15 minutes. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

Dataset Information

Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

Publications

Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

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