Intracellular Protein Dynamics Research Articles

Intracellular protein dynamics as a mathematical problem

The present paper provides a mathematical analysis of the model of intracellular protein dynamics proposed in [14]. The model describes protein and mRNA transport inside a cell and takes into account diffusion in the nucleus and cytoplasm as well as active transport of protein molecules along microtubules in the cytoplasm. The model is a complex system of nonlinear PDEs with appropriate boundary conditions. The model reproduces, at least in numerical simulations, the oscillatory changes in protein concentration observed in the experimental data. To our knowledge this is the first paper that, in the multidimensional case, deals with a rigorous mathematical analysis of a model of intracellular dynamics with active transport on microtubules. In particular, in the present paper, we prove the existence and uniqueness result for the model in arbitrary space dimension. The model may be adapted to other signaling pathways.

Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.

Nanoscale label-free bioprobes to detect intracellular proteins in single living cells.

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

Mathematical modeling of the intracellular protein dynamics: The importance of active transport along microtubules

In this paper we propose a mathematical model of protein and mRNA transport inside a cell. The spatio-temporal model takes into account the active transport along microtubules in the cytoplasm as well as diffusion and is able to reproduce the oscillatory changes in protein concentration observed in many experimental data. In the model the protein and the mRNA interact with each other that allows us to classify the model as a simple gene regulatory network. The proposed model is generic and may be adapted to specific signaling pathways. On the basis of numerical simulations, we formulate a new hypothesis that the oscillatory dynamics is allowed by the mRNA active transport along microtubules from the nucleus to distant locations.

Chapter 18 - Analysis of focal adhesion turnover: A quantitative live-cell imaging example

Recent advances in optical and fluorescent protein technology have rapidly raised expectations in cell biology, allowing quantitative insights into dynamic intracellular processes like never before. However, quantitative live-cell imaging comes with many challenges including how best to translate dynamic microscopy data into numerical outputs that can be used to make meaningful comparisons rather than relying on representative data sets. Here, we use analysis of focal adhesion turnover dynamics as a straightforward specific example on how to image, measure, and analyze intracellular protein dynamics, but we believe this outlines a thought process and can provide guidance on how to understand dynamic microcopy data of other intracellular structures.

Analysis of protein dynamics with tandem fluorescent protein timers.

Fluorescent timers (FTs) are fluorescent proteins that change color with time. FTs can be used as tags to follow protein dynamics in living cells. Recently we described a novel class of FTs called tandem fluorescent protein timers (tFTs). Each tFT is a tandem fusion of two different conventional fluorescent proteins having distinct kinetics of fluorophore maturation. tFTs suitable for studying protein dynamics on different scales can be generated from a broad range of commonly used fluorescent proteins. Here we describe how to establish new tFTs and consider potential pitfalls. We detail a protocol for quantitative fluorescence microscopy imaging and analysis of intracellular protein dynamics with tFTs in the budding yeast Saccharomyces cerevisiae.

Enhancement of Renal Epithelial Cell Functions through Microfluidic-Based Coculture with Adipose-Derived Stem Cells

Current hemodialysis has functional limitations and is insufficient for renal transplantation. The bioartificial tubule device has been developed to contribute to metabolic functions by implanting renal epithelial cells into hollow tubes and showed a higher survival rate in acute kidney injury patients. In healthy kidney, epithelial cells are surrounded by various types of cells that interact with extracellular matrices, which are primarily composed of laminin and collagen. The current study developed a microfluidic coculture platform to enhance epithelial cell function in bioartificial microenvironments with multiple microfluidic channels that are microfabricated by polydimethylsiloxane. Collagen gel (CG) encapsulated with adipose-derived stem cells (CG-ASC) was injected into a central microfluidic channel for three-dimensional (3D) culture. The resuspended Madin-Darby canine kidney (MDCK) cells were injected into nascent channels and formed an epithelial monolayer. In comparison to coculture different cells using the commercial transwell system, the current coculture device allowed living cell monitoring of both the MDCK epithelial monolayer and CG-ASC in a 3D microenvironment. By coculture with CG-ASC, the cell height was increased with columnar shapes in MDCK. Promotion of cilia formation and functional expression of the ion transport protein in MDCK were also observed in the cocultured microfluidic device. When applying fluid flow, the intracellular protein dynamics can be monitored in the current platform by using the time-lapse confocal microscopy and transfection of GFP-tubulin plasmid in MDCK. Thus, this microfluidic coculture device provides the renal epithelial cells with both morphological and functional improvements that may avail to develop bioartificial renal chips.

Subcellular Positioning: Unstable Filaments On the Move

A key question in cell biology is how proteins and entire protein complexes localize to defined subcellular positions in non-compartmentalized cells or within cell compartments. A recent report involving computational modeling and live-cell imaging suggests that dynamically unstable protein filaments provide an adaptable and versatile positioning system.

An Integrative Model for Phytochrome B Mediated Photomorphogenesis: From Protein Dynamics to Physiology

BackgroundPlants have evolved various sophisticated mechanisms to respond and adapt to changes of abiotic factors in their natural environment. Light is one of the most important abiotic environmental factors and it regulates plant growth and development throughout their entire life cycle. To monitor the intensity and spectral composition of the ambient light environment, plants have evolved multiple photoreceptors, including the red/far-red light-sensing phytochromes.Methodology/Principal FindingsWe have developed an integrative mathematical model that describes how phytochrome B (phyB), an essential receptor in Arabidopsis thaliana, controls growth. Our model is based on a multiscale approach and connects the mesoscopic intracellular phyB protein dynamics to the macroscopic growth phenotype. To establish reliable and relevant parameters for the model phyB regulated growth we measured: accumulation and degradation, dark reversion kinetics and the dynamic behavior of different nuclear phyB pools using in vivo spectroscopy, western blotting and Fluorescence Recovery After Photobleaching (FRAP) technique, respectively.Conclusions/SignificanceThe newly developed model predicts that the phyB-containing nuclear bodies (NBs) (i) serve as storage sites for phyB and (ii) control prolonged dark reversion kinetics as well as partial reversibility of phyB Pfr in extended darkness. The predictive power of this mathematical model is further validated by the fact that we are able to formalize a basic photobiological observation, namely that in light-grown seedlings hypocotyl length depends on the total amount of phyB. In addition, we demonstrate that our theoretical predictions are in excellent agreement with quantitative data concerning phyB levels and the corresponding hypocotyl lengths. Hence, we conclude that the integrative model suggested in this study captures the main features of phyB-mediated photomorphogenesis in Arabidopsis.

Novel Agents on the Horizon for Cancer Therapy

Although cancer remains a devastating diagnosis, several decades of preclinical progress in cancer biology and biotechnology have recently led to successful development of several biological agents that substantially improve survival and quality of life for some patients. There is now a rich pipeline of novel anticancer agents in early phase clinical trials. The specific tumor and stromal aberrancies targeted can be conceptualized as membrane-bound receptor kinases (HGF/c-Met, human epidermal growth factor receptor and insulin growth factor receptor pathways), intracellular signaling kinases (Src, PI3k/Akt/mTOR, and mitogen-activated protein kinase pathways), epigenetic abnormalities (DNA methyltransferase and histyone deacetylase), protein dynamics (heat shock protein 90, ubiquitin-proteasome system), and tumor vasculature and microenvironment (angiogenesis, HIF, endothelium, integrins). Several technologies are available to target these abnormalities. Of these, monoclonal antibodies and small-molecule inhibitors have been the more successful, and often complementary, approaches so far in clinical settings. The success of this target-based cancer drug development approach is discussed with examples of recently approved agents, such as bevacizumab, erlotinib, trastuzumab, sorafenib, and bortezomib. This review also highlights the pipeline of rationally designed drugs in clinical development that have the potential to impact clinical care in the near future.

Digital differential interference contrast autofocus for high‐resolution oil‐immersion microscopy

Continued advances in cellular fluorescent biosensors enable studying intracellular protein dynamics in individual, living cells. Autofocus is valuable in such studies to compensate for temperature drift, uneven substrate over multiple fields of view, and cell growth during long-term high-resolution time-lapse studies of hours to days. Observing cellular dynamics with the highest possible resolution and sensitivity motivates the use of high numerical aperture (NA) oil-immersion objectives, and control of fluorescence exposure to minimize phototoxicity. To limit phototoxicity, to maximize light throughput of the objective for biosensor studies, and because phase contrast is distorted by the meniscus in microtiter plates, we studied autofocus in differential interference contrast (DIC) microscopy with a 60x 1.45 NA oil objective after removing the analyzer from the fluorescent light path. Based on a study of the experimental DIC modulation transfer function, we designed a new bandpass digital filter for measuring image sharpness. Repeated tests of DIC autofocus with this digital filter on 225 fields-of-view resulted in a precision of 8.6 nm (standard deviation). Autofocus trials on specimens with thicknesses from 9.47 to 33.20 mum, controlled by cell plating density, showed that autofocus precision was independent of specimen thickness. The results demonstrated that the selected spatial frequencies enabled very high-precision autofocus for high NA DIC automated microscopy, thereby potentially removing the problems of meniscus distortion in phase contrast imaging of microtiter plates and rendering the toxicity of additional fluorescence exposure unnecessary.

Real-time imaging nuclear translocation of Akt1 in HCC cells

Akt is one of the critical mediators in cellular signaling, and overactivation of Akt related pathway frequently occurs in hepatocellular carcinoma (HCC). In this study, we presented that Akt was upregulated in HCC cell lines, and its active phosphorylated form was mainly located in the nucleus. Employing the laser confocal techniques for imaging intracellular protein dynamics, we monitored the transnuclear movement of GFP-tagged wild-type Akt1 (Akt1-WT-GFP) and its inactive mutant (Akt1-T308A/S473A-GFP) in live SMMC-7721 HCC cells, and both of fusion proteins were found to distribute over the cytoplasm and nucleus. Moreover, it was found that platelet derived growth factor (PDGF) was able to accelerate the nuclear translocation of wild-type Akt1 in HCC cells but failed to speed up the motion of the mutant. It was demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt1 facilitated the nuclear translocation of Akt1, but the phosphorylation at threonine 308 and serine 473 was not prerequisite.

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We detemined average time constants of 51 s and 125 s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively.

G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.

Intracellular Protein and DNA Dynamics in Competent Bacillus subtilis Cells

We have found that two DNA repair/recombination proteins localize differentially to the cell poles in competent Bacillus subtilis cells. RecA protein colocalized with competence protein ComGA, and its polar localization largely depended on ComGA and ComK activity, while RecN oscillated between the poles in a minute time frame, independent of any competence factor. Oscillation of RecN arrested upon addition of external DNA, suggesting that an interaction with incoming single-stranded (ss) DNA favors the localization of RecN at the pole containing the competence machinery. In agreement with this model, purified RecN protein showed ATP-dependent binding to ssDNA. Addition of DNA resulted in the formation of RecA threads emanating from the competence machinery. Our data show that in competent bacteria there exists a specifically positioned and dynamic ssDNA binding apparatus that accepts ssDNA taken up through the polar competence machinery and processes ssDNA for recombination with chromosomal DNA via extended RecA filaments.

The dynamics of the proteome: Strategies for measuring protein turnover on a proteome-wide scale

Quantitative proteomics captures the steady-state amount of a protein in a cell but does not explain how a change in protein amount is manifest -- whether through a change in synthesis or a change in degradation. If we are to understand the changes in the proteome, we will need to define such processes. In this brief review, strategies for the determination of intracellular protein dynamics on a proteome-wide scale are discussed.

A photoactivatable GFP for selective photolabeling of proteins and cells.

We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.

Mitochondria‐targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells

Little is known concerning the heterogeneity of mitochondrial shape, size, number, cytoplasmic distribution, and motility in planta. Ultrastructural studies using the electron microscope have shown a variety of mitochondrial shapes and sizes within fixed cells, however, it is not possible to dismiss the possibility that any heterogeneity observed resulted from preparation or fixation artefacts. Unambiguous demonstration of the extent and nature of mitochondrial heterogeneity in vivo necessitates the use of a truly in vivo mitochondrial detection system. Green fluorescent protein is an excellent in vivo marker for gene expression and protein localization studies. It is particularly useful for real-time spatiotemporal analysis of intracellular protein targeting and dynamics and as such is an ideal marker for analysing mitochondria in planta. Stably transformed Arabidopsis lines have been generated with GFP targeted to the mitochondria using either of two plant mitochondrial signal sequences from the beta-ATPase subunit or the mitochondrial chaperonin CPN-60. Mitochondrially targeted GFP, which is easily detectable using an epifluorescent or confocal microscope, highlights heterogeneity of mitochondrial shape, size, position, and dynamic within living plant cells.