| 1. |
- Durrieu, Lucia, et al.
(författare)
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Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources
- 2023
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Ingår i: ISCIENCE. - : CELL PRESS. - 2589-0042. ; 26:1
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)-based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive trans-port, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.
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| 2. |
- Durrieu, Lucía, et al.
(författare)
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Quantification of nuclear transport in single cells
- 2014
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Regulation of nuclear transport is a key cellular function involved in many central processes, such as gene expression regulation and signal transduction. Rates of protein movement between cellular compartments can be measured by FRAP. However, no standard and reliable methods to calculate transport rates exist. Here we introduce a method to extract import and export rates, suitable for noisy single cell data. This method consists of microscope procedures, routines for data processing, an ODE model to fit to the data, and algorithms for parameter optimization and error estimation. Using this method, we successfully measured import and export rates in individual yeast. For YFP, average transport rates were 0.15 sec-1. We estimated confidence intervals for these parameters through likelihood profile analysis. We found large cell-to-cell variation (CV = 0.79) in these rates, suggesting a hitherto unknown source of cellular heterogeneity. Given the passive nature of YFP diffusion, we attribute this variation to large differences among cells in the number or quality of nuclear pores. Owing to its broad applicability and sensitivity, this method will allow deeper mechanistic insight into nuclear transport processes and into the largely unstudied cell-to-cell variation in kinetic rates.
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| 3. |
- Xu, Maojia, et al.
(författare)
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Chondrocytes Derived From Mesenchymal Stromal Cells and Induced Pluripotent Cells of Patients With Familial Osteochondritis Dissecans Exhibit an Endoplasmic Reticulum Stress Response and Defective Matrix Assembly
- 2016
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Ingår i: Stem Cells Translational Medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 5:9, s. 1171-1181
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Tidskriftsartikel (refereegranskat)abstract
- Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs). We also looked at cartilage derived from induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our results revealed several characteristics of the differentiated chondrocytes that help to explain the disease phenotype and susceptibility to cartilage injury. First, patient chondrogenic pellets had poor structural integrity but were rich in glycosaminoglycan. Second, it was evident that large amounts of aggrecan accumulated within the endoplasmic reticulum of chondrocytes differentiated from both BM-MSCs and iPSCs. In turn, there was a marked absence of aggrecan in the extracellular matrix. Third, it was evident that matrix synthesis and assembly were globally dysregulated. These results highlight some of the abnormal aspects of chondrogenesis in these patient cells and help to explain the underlying cellular pathology. The results suggest that FOCD is a chondrocyte aggrecanosis with associated matrix dysregulation. The work provides a new in vitro model of osteoarthritis and cartilage degeneration based on the use of iPSCs and highlights how insights into disease phenotype and pathogenesis can be uncovered by studying differentiation of patient stem cells.
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