Time-lapse Experiment Research Articles

Vector correlation of amplitude variation with azimuth and curvature in a post-hydraulic-fracture Barnett Shale survey

Knowledge of induced fractures can help to evaluate the success of reservoir stimulation. Seismic P-waves through fracturing media can exhibit azimuthal variation in traveltime, amplitude, and thin-bed tuning, so amplitude variation with azimuth (AVAz) can be used to evaluate the hydraulic-fracturing-caused anisotropy. The Barnett Shale of the Fort Worth Basin was the first large-scale commercial shale gas play. We have analyzed two adjacent Barnett Shale seismic surveys: one acquired before hydraulic fracturing and the other acquired after hydraulic fracturing by more than 400 wells. Although not a rigorous time-lapse experiment, comparison of AVAz anisotropy of these two surveys provided valuable insight into the possible effects of hydraulic fracturing. We found that in the survey acquired prior to hydraulic fracturing, AVAz anomalies were stronger and highly correlated with major structural lineaments measured by curvature. In contrast, AVAz anomalies in the survey acquired after hydraulic fracturing were weaker and compartmentalized by rather than correlated to the most-positive curvature lineaments. We found in five microseismic experiments within the survey that these ridge lineaments form fracture barriers. These findings suggested that future time-lapse experiments may be valuable in mapping the modified horizontal stress field to guide future drilling and in recognizing zones of bypassed pay.

Oxidative Stress Resistance in Metastatic Prostate Cancer: Renewal by Self-Eating.

Resistant cancer phenotype is a key obstacle in the successful therapy of prostate cancer. The primary aim of our study was to explore resistance mechanisms in the advanced type of prostate cancer cells (PC-3) and to clarify the role of autophagy in these processes. We performed time-lapse experiment (48 hours) with ROS generating plumbagin by using multimodal holographic microscope. Furthermore, we also performed the flow-cytometric analysis and the qRT-PCR gene expression analysis at 12 selected time points. TEM and confocal microscopy were used to verify the results. We found out that autophagy (namely mitophagy) is an important resistance mechanism. The major ROS producing mitochondria were coated by an autophagic membrane derived from endoplasmic reticulum and degraded. According to our results, increasing ROS resistance may be also accompanied by increased average cell size and polyploidization, which seems to be key resistance mechanism when connected with an escape from senescence. Many different types of cell-cell interactions were recorded including entosis, vesicular transfer, eating of dead or dying cells, and engulfment and cannibalism of living cells. Entosis was disclosed as a possible mechanism of polyploidization and enabled the long-term survival of cancer cells. Significantly reduced cell motility was found after the plumbagin treatment. We also found an extensive induction of pluripotency genes expression (NANOG, SOX2, and POU5F1) at the time-point of 20 hours. We suppose, that overexpression of pluripotency genes in the portion of prostate tumour cell population exposed to ROS leads to higher developmental plasticity and capability to faster respond to changes in the extracellular environment that could ultimately lead to an alteration of cell fate.

Tudor Staphylococcal Nuclease (Tudor-SN), a Novel Regulator Facilitating G1/S Phase Transition, Acting as a Co-activator of E2F-1 in Cell Cycle Regulation

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein implicated in a variety of cellular processes. In the present study, we identified Tudor-SN as a novel regulator in cell cycle. Tudor-SN was abundant in proliferating cells whereas barely expressed in terminally differentiated cells. Functional analysis indicated that ectopic overexpression of Tudor-SN promoted the G1/S transition, whereas knockdown of Tudor-SN caused G1 arrest. Moreover, the live-cell time-lapse experiment demonstrated that the cell cycle of MEF(-/-) (knock-out of Tudor-SN in mouse embryonic fibroblasts) was prolonged compared with wild-type MEF(+/+). We noticed that Tudor-SN was constantly expressed in every cell cycle phase, but was highly phosphorylated in the G1/S border. Further study revealed that Tudor-SN was a potential substrate of Cdk2/4/6, supportively, we found the physical interaction of endogenous Tudor-SN with Cdk4/6 in G1 and the G1/S border, and with Cdk2 in the G1/S border and S phase. In addition, roscovitine (Cdk1/2/5 inhibitor) or CINK4 (Cdk4/6 inhibitor) could inhibit the phosphorylation of Tudor-SN, whereas ectopic overexpression of Cdk2/4/6 increased the Tudor-SN phosphorylation. The underlying molecular mechanisms indicated that Tudor-SN could physically interact with E2F-1 in vivo, and could enhance the physical association of E2F-1 with GCN5 (a cofactor of E2F-1, which possesses histone acetyltransferase activity), and promote the binding ability of E2F-1 to the promoter region of its target genes CYCLIN A and E2F-1, and as a result, facilitate the gene transcriptional activation. Taken together, Tudor-SN is identified as a novel co-activator of E2F-1, which could facilitate E2F-1-mediated gene transcriptional activation of target genes, which play essential roles in G1/S transition.

The Potential Impact of Biofield Treatment on Human Brain Tumor Cells: A Time-Lapse Video Microscopy

Study background: Glioblastoma (GBM) is the most common subtype of primary brain tumor in adults. The aim was to evaluate the impact of biofield treatment potential on human GBM and non-GBM brain cells using two time-lapse video microscopy technique.Methods: The human brain tumor, GBM cultured cells were divided into two groups viz. GBM control and GBM treatment. Similarly, human normal brain cultured cells (non-GBM) were taken and divided into two groups viz. non- GBM control and non-GBM treatment. The GBM and non-GBM treatment groups were given Mr. Trivedi’s biofield treatment for the assessment of its potential. Two time-lapse (10 hours prior; 10 hours after) video microscopy experiment was performed on tumor and non-tumor brain cells in six replicate (n=6). For each microscopic field, the total cell number was counted and each cell was tracked over the 20 hours period. The potential impact of biofield treatment was assessed by comparing cell death rate in both GBM and non-GBM cells before and after biofield treatment.Results: GBM control cells showed a basal level of cell death 10 hours prior and 10 hours after the biofield treatment, and the rate remained unchanged over the 20 hours period, while in treatment group of GBM, cell death rate was exponentially increased (41%) after biofield treatment as compared to control. The treated non-GBM cultured cells showed a significant reduction (64%) of cell death rate i.e. protective effects as compared to non-GBM control.Conclusion: Altogether, data suggests that biofield treatment has significantly increased the cell death rate of treated GBM cells and simultaneously boost the viability of normal brain cells. Therefore, biofield treatment could be a suitable alternate treatment strategy for cancer patients in near future.

The Investigational Aurora A Kinase Inhibitor Alisertib Exhibits Broad Activity in Preclinical Models of T-Cell Lymphoma and Is Highly Synergistic with Romidepsin

Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral T-cell lymphoma (PTCL), DLBCL and other hematologic malignancies. Herein we demonstrate the potent cytotoxicity and apoptotic effects of alisertib in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin.Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours, demonstrating the exquisite impact of time on effect. Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. A combinatorial experiment to identify potential synergistic interactions found no synergy with pralatrexate or proteasome inhibitors, but revealed potent synergy with romidepsin. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20, and IC30 demonstrated marked synergy only in TCL, but not B-cell lymphomas. The best synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1)We further evaluated this synergistic interaction through a time lapse experiment (0-72hr) by live cell imaging and found that the combined alisertib and romidepsin treatment in H9, a CTCL cell line, led to failure of cytokinesis leading to apoptosis. Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 and HH after 72 hours of exposure through increased Puma, Caspase 3 and PARP cleavage, and decreased BCL-xL and BCL2 expression; suggesting that cell death was apoptotic. AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis. In addition, cell cycle analysis was performed following 24 hour of treatment of romidepsin and alisertib as a single agent and in combination. Alisertib produced a G2/M arrest while the combination of both single agents induced polyploidy (up to 8N). This finding may also complement the increase in apoptosis in the combination treatment versus the single agents. Furthermore, preliminary results from an in-vivoxenograft demonstrate a synergistic interaction amongst the combined treatment of alisertib and romidepsin when compared to control cohort and both single agent cohorts.These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL. Mechanistic studies to better understand this synergistic interaction are ongoing. It is hypothesized that the combined alisertib and romidepsin treatment synergistically inhibit the activation of stat-3, leading to apoptosis and inhibition of cellular proliferation through BCL-xL and c-MYC.Abstract 4493. Table 1:Synergy Coefficients of Alisertib in Combination with Romidepsin at 72 Hours.[1]Drug CombinationDND41(T-ALL)J.CAM 1.6(T-ALL)HH(CTCL)H9(CTCL)C5MJ(HTLV+)Romidepsin [IC10-20] + Alisertib 50 nM0.960.811.051.11.53Romidepsin [IC10-20] + Alisertib 100nM0.510.560.680.660.58Romidepsin [IC10-20] + Alisertib 1000 nM0.400.200.400.460.70[1] 1=additive; <1=synergistic; >1= subadditive DisclosuresAmengual:Acetylon Pharmaceuticals: Research Funding. O’Connor:Millennium Pharmaceuticals: Consultancy; Celgene : Consultancy.

Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Methodology/Principal FindingsWe determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.

Visualization of G3BP stress granules dynamics in live primary cells.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Time-lapse wave-equation migration velocity analysis

Time-lapse (4D) analysis of seismic data acquired at different stages of hydrocarbon production or gas/fluid injection has been very successful at imaging detailed reservoir changes. Conventional time-domain analysis of 4D data sets usually assumes a linear perturbation about a reference baseline earth model. However, this assumption is violated when production/injection significantly alters the subsurface generating large 4D velocity changes, time shifts, and complicated 4D wavefield coda, necessitating a more robust 4D analysis involving prestack wave-equation depth migration and velocity analysis. We address these situations by extending conventional 3D waveequation migration velocity analysis (WEMVA) based on one-way wave-equations and single-scattering theory to 4D velocity estimation using a “parallel”inversion approach involving parallel solution of two separate inversion problems. Recognizing that the 4D WEMVA strategy requires precomputed baseline/monitor image-difference volumes, we develop an approximate 4D WEMVA technique that replaces these differences with a single weight function derived from the smooth background time-lapse image difference. We demonstrate the usefulness of the parallel and an approximate 4D WEMVA approach using a synthetic time-lapse CO2 geosequestration experiment that requires inverting for a thin-layer velocity change derived from CO2 injection in an analogue North Sea reservoir. The parallel 4D WEMVA solutions generate an excellent highresolution velocity estimates, whereas the approximate methods recover lower-resolution estimates with magnitudes that must be rescaled through a post-inversion gradient line-search.

Abstract C54: In vitro and in vivo anti-tumor activity of the antimacrophage stimulating 1-receptor antibody IMC-RON8 in breast and bladder cancer models.

Abstract Macrophage stimulating 1-receptor (RON) is a member of the c-Met receptor tyrosine kinase family. RON is normally expressed on macrophages and epithelial cells. However, it is overexpressed and activated in a large number of human tumors. The fully human anti-RON antibody, IMC-RON8, blocks the RON ligand, macrophage-stimulating protein (MSP), from binding to RON and has been demonstrated to have antitumor activity against human colon, lung, and pancreatic xenografts in mice. Overexpression of RON correlates with a worse clinical outcome for patients in at least two human cancer indications, namely breast and bladder. Given this correlation, we investigated the effect of IMC-RON8 in several in vitro and in vivo systems with the RON-positive breast and bladder cancer cell lines JIMT-1 and BFTC-905, respectively. Both of these were found from a screen of breast and bladder cancer cell lines designed to identify those that are RON positive and responsive to MSP through activation of the MAP Kinase signaling pathway. IMC-RON8 inhibited the phosphorylation of MAP Kinase in response to 5nM MSP stimulation for JIMT-1 and BFTC-905. IMC-RON8 also inhibited the MSP-induced cellular migration of JIMT-1 in a wound healing assay, completely preventing progression towards closing of the wound. As measured by flow cytometry, IMC-RON8 at 33.3nM induced the internalization of 38% and 31% cell-surface RON expressed on JIMT-1 and BFTC-905 cells, respectively, following 24 hours of treatment at 37°C. To investigate IMC-RON8-induced RON downmodulation, clonal Hela cells stably expressing a RON-GFP protein were generated. These cells were treated with MSP, a RON agonist antibody (RON2), or IMC-RON8 and the level of RON-GFP was then measured by confocal microscopy in a live-cell time lapse experiment. IMC-RON8 treatment actively induced receptor interalization and caused RON-GFP degradation (reduction of the GFP signal) relative to IgG controls, although to a lesser extent than MSP and RON2. In an in vivo JIMT-1 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 59 when administered at 60 mg/kg twice a week. Combination of IMC-RON8 with the chemotherapeutic agent, docetaxel at 12 mg/kg once per week, significantly improved the anti-tumor effects compared to either monotherapy with a %T/C value of 20. In an in vivo BFTC-905 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 58 when administered at 60 mg/kg twice a week. BFTC-905 tumors removed after the last IMC-RON8 dose (6 total doses over a 3 week study period), showed a decrease in the total level of RON receptor present in tumors relative to controls (p-value of &amp;lt;0.0001). These preclinical results therefore demonstrate that IMC-RON8 possesses antitumor activity in vitro and in vivo and may be effective as a single agent or in combination with a conventional cytotoxic therapy for the treatment of cancer. Taken together, these data suggest the mechanism of action for IMC-RON8 antitumor activity can be both ligand-blocking activity and receptor downmodulation; both leading to a decrease in ligand-induced tumor cell proliferation. IMC-RON8 is presently in Phase I studies to treat patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C54.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

Modelling the geoelectric and seismic reservoir response caused by carbon dioxide injection based on multiphase flow simulation: Results from the CO 2SINK project

Results from crosshole geoelectric and surface seismic monitoring for geological storage of carbon dioxide (CO 2) were investigated by forward modelling within the framework of the CO 2SINK project. Selected geological and petrophysical parameters reflect the conditions of the CO 2SINK site. CO 2 saturation distributions were derived from multiphase flow modelling, whereas the alteration of geophysical rock properties by CO 2 was obtained from laboratory experiments. Crosshole geoelectric modelling was performed for three electrode combinations and three time-dependent CO 2 migration scenarios with different reservoir permeabilities. The magnitude and alteration of modelled resistances were analyzed in the pre-inversion domain. Time-lapse alterations were observable on the synthetic data, with diverse characteristics dependent on applied electrode configuration. Analysis of the alterations showed the opportunity to differentiate migration scenarios within the constraints of the ambient noise level. The synthetic time-lapse seismic reflection experiment was performed for the anticline used for CO 2 storage. The geological model incorporates the structural framework, as determined from the seismic interpretation, and velocities derived from seismic processing and velocity logs. Common depth point (CDP) processing of synthetic shot gathers of a baseline and repeat experiment provided the data for a difference stack section exhibiting a CO 2 induced time-lapse signature. Interpretation of the signature in conjunction with the underlying CO 2 distribution has shown that lateral extent of the plume may be accurately detected. The vertical plume extent is concealed in the waveform coda and is unlikely to be retrievable from standard seismic processing.

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Analysis of biological time-lapse microscopic experiment from the point of view of the information theory [Micron Vol. 41 Issue 5

Analysis of biological time-lapse microscopic experiment from the point of view of the information theory [Micron Vol. 41 Issue 5

History-Dependent Catastrophes Regulate Axonal Microtubule Behavior

In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

Ex vivo live imaging of melanoblast migration in embryonic mouse skin

Ex vivo live imaging of melanoblast migration in embryonic mouse skin

Analysis of biological time-lapse microscopic experiment from the point of view of the information theory

Abstract We report objective analysis of information in the microscopic image of the cell monolayer. The process of transfer of information about the cell by the microscope is analyzed in terms of the classical Shannon information transfer scheme. The information source is the biological object, the information transfer channel is the whole microscope including the camera chip. The destination is the model of biological system. The information contribution is analyzed as information carried by a point to overall information in the image. Subsequently we obtain information reflection of the biological object. This is transformed in the biological model which, in information terminology, is the destination. This, we propose, should be constructed as state transitions in individual cells modulated by information bonds between the cells. We show examples of detected cell states in multidimensional state space. This space is reflected as colour channel intensity phenomenological state space. We have also observed information bonds and show examples of them.

A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution

We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO 2-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.

Advantages in multiplex and live-cell imaging using the CELLview™ cell culture dish

Advantages in multiplex and live-cell imaging using the CELLview™ cell culture dish

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.