31 research outputs found
Protein Dynamics in Individual Human Cells: Experiment and Theory
A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells
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Protein Dynamics in Individual Human Cells: Experiment and Theory
A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle–dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell–cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell–cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells
Human Muscle Progenitor Cells Overexpressing Neurotrophic Factors Improve Neuronal Regeneration in a Sciatic Nerve Injury Mouse Model
The peripheral nervous system has an intrinsic ability to regenerate after injury. However, this process is slow, incomplete, and often accompanied by disturbing motor and sensory consequences. Sciatic nerve injury (SNI), which is the most common model for studying peripheral nerve injury, is characterized by damage to both motor and sensory fibers. The main goal of this study is to examine the feasibility of administration of human muscle progenitor cells (hMPCs) overexpressing neurotrophic factor (NTF) genes, known to protect peripheral neurons and enhance axon regeneration and functional recovery, to ameliorate motoric and sensory deficits in SNI mouse model. To this end, hMPCs were isolated from a human muscle biopsy, and manipulated to ectopically express brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1). These hMPC-NTF were transplanted into the gastrocnemius muscle of mice after SNI, and motor and sensory functions of the mice were assessed using the CatWalk XT system and the hot plate test. ELISA analysis showed that genetically manipulated hMPC-NTF express significant amounts of BDNF, GDNF, VEGF, or IGF-1. Transplantation of 3 × 106 hMPC-NTF was shown to improve motor function and gait pattern in mice following SNI surgery, as indicated by the CatWalk XT system 7 days post-surgery. Moreover, using the hot-plate test, performed 6 days after surgery, the treated mice showed less sensory deficits, indicating a palliative effect of the treatment. ELISA analysis following transplantation demonstrated increased NTF expression levels in the gastrocnemius muscle of the treated mice, reinforcing the hypothesis that the observed positive effect was due to the transplantation of the genetically manipulated hMPC-NTF. These results show that genetically modified hMPC can alleviate both motoric and sensory deficits of SNI. The use of hMPC-NTF demonstrates the feasibility of a treatment paradigm, which may lead to rapid, high-quality healing of damaged peripheral nerves due to administration of hMPC. Our approach suggests a possible clinical application for the treatment of peripheral nerve injury
Dynamic Proteomics: a database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells
Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/
Dynamic proteomics reveals bimodal protein dynamics of cancer cells in response to HSP90 inhibitor
Evaluation of Groundwater Salinization Risk Following Application of Anti-Dust Emission Solutions on Unpaved Roads in Arid and Semiarid Regions
Unpaved roads could be a significant source of dust emission. A common and effective practice to suppress this emission is the application of brine solution on these roads. However, this application could increase the risk of water source salinization in arid and semiarid regions, such as Israel. The general objective of the present study was to investigate the potential effects of treated wastewater (TWW), fresh water (FW), and brine applications as anti-dust emission solutions on water source salinization in these regions. A rainfall simulator experiment and a mass balance model were used for this goal. The TWW loaded the highest amounts of Cl, Na, and Ca+Mg on the unpaved roads, while the brine loaded higher amounts of Cl and Ca+Mg than the FW, and ~0 Na. In the rainfall experiment, runoff was not formed, and ~100% of the loaded amounts were leached downwards by rain, indicating a negligible salinization risk to surface water. We estimated that the average increases in the Cl concentrations in the modeled aquifer, following TWW, brine, and FW applications, were low: 1.2–1.6, 0.58–0.8, and 0.32–0.4 mg L−1, respectively. Thus, the solution selection for preventing dust emission should be based on the total cost of the solution application
Fold-change Response of Photosynthesis to Step Increases of Light Level
Summary: Plants experience light intensity over several orders of magnitude. High light is stressful, and plants have several protective feedback mechanisms against this stress. Here we asked how plants respond to sudden rises at low ambient light, far below stressful levels. For this, we studied the fluorescence of excited chlorophyll a of photosystem II in Arabidopsis thaliana plants in response to step increases in light level at different background illuminations. We found a response at low-medium light with characteristics of a sensory system: fold-change detection (FCD), Weber law, and exact adaptation, in which the response depends only on relative, and not absolute, light changes. We tested various FCD circuits and provide evidence for an incoherent feedforward mechanism upstream of known stress response feedback loops. These findings suggest that plant photosynthesis may have a sensory modality for low light background that responds early to small light increases, to prepare for damaging high light levels. : Biological Sciences; Systems Biology; Plant Biology Subject Areas: Biological Sciences, Systems Biology, Plant Biolog
Generation of double-labeled reporter cell lines for studying co-dynamics of endogenous proteins in individual human cells.
Understanding the dynamic relationship between components of a system or pathway at the individual cell level is a current challenge. To address this, we developed an approach that allows simultaneous tracking of several endogenous proteins of choice within individual living human cells. The approach is based on fluorescent tagging of proteins at their native locus by directed gene targeting. A fluorescent tag-encoding DNA is introduced as a new exon into the intronic region of the gene of interest, resulting in expression of a full-length fluorescently tagged protein. We used this approach to establish human cell lines simultaneously expressing two components of a major antioxidant defense system, thioredoxin 1 (Trx) and thioredoxin reductase 1 (TrxR1), labeled with CFP and YFP, respectively. We find that the distributions of both proteins between nuclear and cytoplasmic compartments were highly variable between cells. However, the two proteins did not vary independently of each other: protein levels of Trx and TrxR1 in both the whole cell and the nucleus were substantially correlated. We further find that in response to a stress-inducing drug (CPT), both Trx and TrxR1 accumulated in the nuclei in a manner that was highly temporally correlated. This accumulation considerably reduced cell-to-cell variability in nuclear content of both proteins, suggesting a uniform response of the thioredoxin system to stress. These results indicate that Trx and TrxR1 act in concert in response to stress in regard to both time course and variability. Thus, our approach provides an efficient tool for studying dynamic relationship between components of systems of interest at a single-cell level
Generation of a fluorescently labeled endogenous protein library in living human cells
We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100–200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3′ rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format