Preservation of circadian rhythms by the protein folding chaperone, BiP

Pickard, Adam; Chang, Joan; Alachkar, Nissrin; Calverley, Ben; Garva, Richa; Arvan, Peter; Meng, Qing Jun; Kadler, Karl E.

doi:10.1101/348078

Cited by 9 publications

(9 citation statements)

References 43 publications

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“…Likewise, the secretory pathway inhibitors Brefeldin A and Monensin also caused inhibition of NLuc-PC-I secretion ( Figure 5 A). Treatment with Brefeldin A, unlike Monensin, resulted in accumulation of intracellular NLuc-PC-I ( Figure 5 B), which is in line with the fact that Brefeldin A is known to induce a strong ER stress response [ 24 ]. Encouraged by these results, we next sought to determine if collagen-DyProQ could be used to evaluate the function of the known antifibrotic therapeutics Nintedanib [ 25 ] and Pirfenidone [ 26 ].…”

Section: Resultssupporting

confidence: 71%

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging

Calverley

Kadler²,

Pickard³

2020

Cells

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The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.

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Section: Resultssupporting

confidence: 71%

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging

Calverley

Kadler²,

Pickard³

2020

Cells

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“…High‐load eccentric resistance training can reduce pain and improve ECM structural organization in patients with tendinopathy, although this training does not lead to full symptomatic resolution for many patients (63, 64). Recent studies have demonstrated that tendons synthesize collagen during rest phases at night and assemble collagen into mature fibrils during the active day phases (65, 66). Combining the use of recombinant growth factors with properly timed mechanical loading interventions and appropriate rest periods could lead to the improved treatment of tendon disorders.…”

Section: Discussionmentioning

confidence: 99%

Insulin‐like growth factor 1 signaling in tenocytes is required for adult tendon growth

et al. 2019

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Tenocytes serve to synthesize and maintain collagen fibrils and other extracellular matrix proteins in tendon. Despite the high prevalence of tendon injury, the underlying biologic mechanisms of postnatal tendon growth and repair are not well understood. IGF1 plays an important role in the growth and remodeling of numerous tissues but less is known about IGF1 in tendon. We hypothesized that IGF1 signaling is required for proper tendon growth in response to mechanical loading through regulation of collagen synthesis and cell proliferation. To test this hypothesis, we conditionally deleted the IGF1 receptor (IGF1R) in scleraxis (Scx)‐expressing tenocytes using a tamoxifen‐inducible Cre‐recombinase system and caused tendon growth in adult mice via mechanical overload of the plantaris tendon. Compared with control Scx‐expressing IGF1R‐positive (Scx:IGF1R+) mice, in which IGF1R is present in tenocytes, mice that lacked IGF1R in their tenocytes [Scx‐expressing IGF1R‐negative (Scx:IGF1RΔ) mice] demonstrated reduced cell proliferation and smaller tendons in response to mechanical loading. Additionally, we identified that both the PI3K/protein kinase B and ERK pathways are activated downstream of IGF1 and interact in a coordinated manner to regulate cell proliferation and protein synthesis. These studies indicate that IGF1 signaling is required for proper postnatal tendon growth and support the potential use of IGF1 in the treatment of tendon disorders.—Disser, N. P., Sugg, K. B., Talarek, J. R., Sarver, D. C., Rourke, B. J., Mendias, C. L. Insulin‐like growth factor 1 signaling in tenocytes is required for adult tendon growth. FASEB J. 33, 12680–12695 (2019). http://www.fasebj.org

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“…The proteins that, respectively, control the entrances and exits of the ER and Golgi are 24 h rhythmic, including SEC61, TANGO1, PDE4D, and VPS33B [6,28]. In addition, Pickard et al show that BiP is under the control of the circadian clocks, which take part in the collagen secretion process and assist collagen folding (Figure 1) [29]. In the Clock Δ19 mice with a defective circadian clock, the numbers of collagen and the structure of fibrils are abnormal.…”

Section: The Spatial Structures and Temporalmentioning

confidence: 99%

Spatial-Temporal Patterns and Inflammatory Factors of Bone Matrix Remodeling

Wang

Guo

Chen

et al. 2021

Stem Cells International

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The bone extracellular matrix (ECM) contains organic and mineral constituents. The establishment and degradation processes of ECM connect with spatial and temporal patterns, especially circadian rhythms in ECM. These patterns are responsible for the physical and biological characteristics of bone. The disturbances of the patterns disrupt bone matrix remodeling and cause diverse bone diseases, such as osteogenesis imperfecta (OI) and bone fracture. In addition, the main regulatory factors and inflammatory factors also follow circadian rhythms. Studies show that the circadian oscillations of these factors in bone ECM potentially influence the interactions between immune responses and bone formation. More importantly, mesenchymal stem cells (MSCs) within the specific microenvironments provide the regenerative potential for tissue remodeling. In this review, we summarize the advanced ECM spatial characteristics and the periodic patterns of bone ECM. Importantly, we focus on the intrinsic connections between the immunoinflammatory system and bone formation according to circadian rhythms of regulatory factors in bone ECM. And our research group emphasizes the multipotency of MSCs with their microenvironments. The advanced understandings of bone ECM formation patterns and MSCs contribute to providing optimal prevention and treatment strategies.

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Preservation of circadian rhythms by the protein folding chaperone, BiP

Cited by 9 publications

References 43 publications

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging

Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging

Insulin‐like growth factor 1 signaling in tenocytes is required for adult tendon growth

Spatial-Temporal Patterns and Inflammatory Factors of Bone Matrix Remodeling

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