The Experts below are selected from a list of 39132 Experts worldwide ranked by ideXlab platform

Timothy J Mitchison - One of the best experts on this subject based on the ideXlab platform.

  • A high-throughput Cell Migration Assay using scratch wound healing, a comparison of image-based readout methods
    BMC Biotechnology, 2004
    Co-Authors: Justin C Yarrow, Zachary E Perlman, Nicholas J Westwood, Timothy J Mitchison
    Abstract:

    Background Cell Migration is a complex phenomenon that requires the coordination of numerous Cellular processes. Investigation of Cell Migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current Migration Assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results We have adapted the commonly used scratch wound healing Assay of tissue-culture Cell monolayers to a 384 well plate format. By mechanically scratching the Cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect Cell Migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions The adaptation of a wound healing Assay to a 384 well format facilitates the study of aspects of Cell Migration, tissue reorganization, Cell division, and other processes that underlie wound healing. This Assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

  • a high throughput Cell Migration Assay using scratch wound healing a comparison of image based readout methods
    BMC Biotechnology, 2004
    Co-Authors: Justin C Yarrow, Zachary E Perlman, Nicholas J Westwood, Timothy J Mitchison
    Abstract:

    Cell Migration is a complex phenomenon that requires the coordination of numerous Cellular processes. Investigation of Cell Migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current Migration Assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. We have adapted the commonly used scratch wound healing Assay of tissue-culture Cell monolayers to a 384 well plate format. By mechanically scratching the Cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect Cell Migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. The adaptation of a wound healing Assay to a 384 well format facilitates the study of aspects of Cell Migration, tissue reorganization, Cell division, and other processes that underlie wound healing. This Assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

Alan Hall - One of the best experts on this subject based on the ideXlab platform.

  • cdc42 regulates gsk 3beta and adenomatous polyposis coli to control Cell polarity
    Nature, 2003
    Co-Authors: Sandrine Etiennemanneville, Alan Hall
    Abstract:

    Cell polarity is a fundamental property of all Cells. In higher eukaryotes, the small GTPase Cdc42, acting through a Par6-atypical protein kinase C (aPKC) complex, is required to establish Cellular asymmetry during epithelial morphogenesis, asymmetric Cell division and directed Cell Migration. However, little is known about what lies downstream of this complex. Here we show, through the use of primary rat astrocytes in a Cell Migration Assay, that Par6-PKCzeta interacts directly with and regulates glycogen synthase kinase-3beta (GSK-3beta) to promote polarization of the centrosome and to control the direction of Cell protrusion. Cdc42-dependent phosphorylation of GSK-3beta occurs specifically at the leading edge of migrating Cells, and induces the interaction of adenomatous polyposis coli (Apc) protein with the plus ends of microtubules. The association of Apc with microtubules is essential for Cell polarization. We conclude that Cdc42 regulates Cell polarity through the spatial regulation of GSK-3beta and Apc. This role for Apc may contribute to its tumour-suppressor activity.

  • cdc42 regulates gsk 3beta and adenomatous polyposis coli to control Cell polarity
    Nature, 2003
    Co-Authors: Sandrine Etiennemanneville, Alan Hall
    Abstract:

    Cell polarity is a fundamental property of all Cells. In higher eukaryotes, the small GTPase Cdc42, acting through a Par6–atypical protein kinase C (aPKC) complex, is required to establish Cellular asymmetry during epithelial morphogenesis, asymmetric Cell division and directed Cell Migration1,2,3,4,5. However, little is known about what lies downstream of this complex. Here we show, through the use of primary rat astrocytes in a Cell Migration Assay, that Par6–PKCζ interacts directly with and regulates glycogen synthase kinase-3β (GSK-3β) to promote polarization of the centrosome and to control the direction of Cell protrusion. Cdc42-dependent phosphorylation of GSK-3β occurs specifically at the leading edge of migrating Cells, and induces the interaction of adenomatous polyposis coli (Apc) protein with the plus ends of microtubules. The association of Apc with microtubules is essential for Cell polarization. We conclude that Cdc42 regulates Cell polarity through the spatial regulation of GSK-3β and Apc. This role for Apc may contribute to its tumour-suppressor activity.

Josephine Nalbantoglu - One of the best experts on this subject based on the ideXlab platform.

Yung-shin Sun - One of the best experts on this subject based on the ideXlab platform.

  • Quantifying the roles of random motility and directed motility using advection-diffusion theory for a 3T3 fibroblast Cell Migration Assay stimulated with an electric field
    BMC systems biology, 2017
    Co-Authors: Matthew J. Simpson, Yung-shin Sun
    Abstract:

    Directed Cell Migration can be driven by a range of external stimuli, such as spatial gradients of: chemical signals (chemotaxis); adhesion sites (haptotaxis); or temperature (thermotaxis). Continuum models of Cell Migration typically include a diffusion term to capture the undirected component of Cell motility and an advection term to capture the directed component of Cell motility. However, there is no consensus in the literature about the form that the advection term takes. Some theoretical studies suggest that the advection term ought to include receptor saturation effects. However, others adopt a much simpler constant coefficient. One of the limitations of including receptor saturation effects is that it introduces several additional unknown parameters into the model. Therefore, a relevant research question is to investigate whether directed Cell Migration is best described by a simple constant tactic coefficient or a more complicated model incorporating saturation effects. We study directed Cell Migration using an experimental device in which the directed component of the Cell motility is driven by a spatial gradient of electric potential, which is known as electrotaxis. The electric field (EF) is proportional to the spatial gradient of the electric potential. The spatial variation of electric potential across the experimental device varies in such a way that there are several subregions on the device in which the EF takes on different values that are approximately constant within those subregions. We use Cell trajectory data to quantify the motion of 3T3 fibroblast Cells at different locations on the device to examine how different values of the EF influences Cell motility. The undirected (random) motility of the Cells is quantified in terms of the Cell diffusivity, D, and the directed motility is quantified in terms of a Cell drift velocity, v. Estimates D and v are obtained under a range of four different EF conditions, which correspond to normal physiological conditions. Our results suggest that there is no anisotropy in D, and that D appears to be approximately independent of the EF and the electric potential. The drift velocity increases approximately linearly with the EF, suggesting that the simplest linear advection term, with no additional saturation parameters, provides a good explanation of these physiologically relevant data. We find that the simplest linear advection term in a continuum model of directed Cell motility is sufficient to describe a range of different electrotaxis experiments for 3T3 fibroblast Cells subject to normal physiological values of the electric field. This is useful information because alternative models that include saturation effects involve additional parameters that need to be estimated before a partial differential equation model can be applied to interpret or predict a Cell Migration experiment.

  • Quantifying the roles of random motility and directed motility using advection-diffusion theory for a 3T3 fibroblast Cell Migration Assay stimulated with an electric field
    2017
    Co-Authors: Matthew J. Simpson, Yung-shin Sun
    Abstract:

    Background Directed Cell Migration can be driven by a range of external stimuli, such as spatial gradients of: chemical signals (chemotaxis); adhesion sites (haptotaxis); or temperature (thermotaxis). Continuum models of Cell Migration typically include a diffusion term to capture the undirected component of Cell motility and an advection term to capture the directed component of Cell motility. However, there is no consensus in the literature about the form that the advection term takes. Some theoretical studies suggest that the advection term ought to include receptor saturation effects. However, others adopt a much simpler constant coefficient. One of the limitations of including receptor saturation effects is that it introduces several additional unknown parameters into the model. Therefore, a relevant research question is to investigate whether directed Cell Migration is best described by a simple constant tactic coefficient or a more complicated model incorporating saturation effects. Results We study directed Cell Migration using an experimental device in which the directed component of the Cell motility is driven by a spatial gradient of electric potential, which is known as electrotaxis. The electric field (EF) is proportional to the spatial gradient of the electric potential. The spatial variation of electric potential across the experimental device varies in such a way that there are several subregions on the device in which the EF takes on different values that are approximately constant within those subregions. We use Cell trajectory data to quantify the motion of 3T3 fibroblast Cells at different locations on the device to examine how different values of the EF influences Cell motility. The undirected (random) motility of the Cells is quantified in terms of the Cell diffusivity, D, and the directed motility is quantified in terms of a Cell drift velocity, v. Estimates D and v are obtained under a range of four different EF conditions, which correspond to normal physiological conditions. Our results suggest that there is no anisotropy in D, and that D appears to be approximately independent of the EF and the electric potential. The drift velocity increases approximately linearly with the EF, suggesting that the simplest linear advection term, with no additional saturation parameters, provides a good explanation of these physiologically relevant data. Conclusions We find that the simplest linear advection term in a continuum model of directed Cell motility is sufficient to describe a range of different electrotaxis experiments for 3T3 fibroblast Cells subject to normal physiological values of the electric field. This is useful information because alternative models that include saturation effects involve additional parameters that need to be estimated before a partial differential equation model can be applied to interpret or predict a Cell Migration experiment.

Nicholas J Westwood - One of the best experts on this subject based on the ideXlab platform.

  • A high-throughput Cell Migration Assay using scratch wound healing, a comparison of image-based readout methods
    BMC Biotechnology, 2004
    Co-Authors: Justin C Yarrow, Zachary E Perlman, Nicholas J Westwood, Timothy J Mitchison
    Abstract:

    Background Cell Migration is a complex phenomenon that requires the coordination of numerous Cellular processes. Investigation of Cell Migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current Migration Assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results We have adapted the commonly used scratch wound healing Assay of tissue-culture Cell monolayers to a 384 well plate format. By mechanically scratching the Cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect Cell Migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions The adaptation of a wound healing Assay to a 384 well format facilitates the study of aspects of Cell Migration, tissue reorganization, Cell division, and other processes that underlie wound healing. This Assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

  • a high throughput Cell Migration Assay using scratch wound healing a comparison of image based readout methods
    BMC Biotechnology, 2004
    Co-Authors: Justin C Yarrow, Zachary E Perlman, Nicholas J Westwood, Timothy J Mitchison
    Abstract:

    Cell Migration is a complex phenomenon that requires the coordination of numerous Cellular processes. Investigation of Cell Migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current Migration Assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. We have adapted the commonly used scratch wound healing Assay of tissue-culture Cell monolayers to a 384 well plate format. By mechanically scratching the Cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect Cell Migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. The adaptation of a wound healing Assay to a 384 well format facilitates the study of aspects of Cell Migration, tissue reorganization, Cell division, and other processes that underlie wound healing. This Assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.