The Experts below are selected from a list of 38211 Experts worldwide ranked by ideXlab platform

Travis I Moore - One of the best experts on this subject based on the ideXlab platform.

  • measuring integrin conformational change on the cell surface with super resolution Microscopy
    Cell Reports, 2018
    Co-Authors: Travis I Moore, Jesse Aaron, Tengleong Chew, Timothy A Springer
    Abstract:

    Summary We use super-resolution interferometric photoactivation and localization Microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron Microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence Polarization Microscopy.

  • direction of actin flow dictates integrin lfa 1 orientation during leukocyte migration
    Nature Communications, 2017
    Co-Authors: Pontus Nordenfelt, Shalin B Mehta, Travis I Moore, Joseph Mathew Kalappurakkal, Vinay Swaminathan, Nobuyasu Koga, Talley J Lambert, David Baker
    Abstract:

    Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence Polarization Microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, A-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

  • direction of actin flow dictates integrin lfa 1 orientation during leukocyte migration
    bioRxiv, 2017
    Co-Authors: Pontus Nordenfelt, Shalin B Mehta, Travis I Moore, Joseph Mathew Kalappurakkal, Vinay Swaminathan, Nobuyasu Koga, Talley J Lambert, David Baker, Jennifer C Waters, Rudolf Oldenbourg
    Abstract:

    Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin head, known from crystal structures, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid head of the full integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin, and measure orientation with fluorescence Polarization Microscopy. Dependent on coupling to the cytoskeleton, integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. We have developed a method that places atomic, ~A structures of cell surface receptors in the context of functional, cellular length-scale, ~μm measurements and shows that rotation and tilt of cell surface receptors relative to the membrane plane can be restrained by interactions with other cellular components.

Rudolf Oldenbourg - One of the best experts on this subject based on the ideXlab platform.

  • quantification of collagen organization in histopathology samples using liquid crystal based Polarization Microscopy
    Biomedical Optics Express, 2017
    Co-Authors: Adib Keikhosravi, Amitabh Verma, Rudolf Oldenbourg, Yuming Liu, Cole R Drifka, Kaitlin M Woo, Kevin W Eliceiri
    Abstract:

    A number of histopathology studies have utilized the label free Microscopy method of Second Harmonic Generation (SHG) to investigate collagen organization in disease onset and progression. Here we explored an alternative label free imaging approach, LC-PolScope that is based on liquid crystal based polarized light imaging. We demonstrated that this more accessible technology has the ability to visualize all fibers of interest and has a good to excellent correlation between SHG and LC-PolScope measurements in fibrillar collagen orientation and alignment. This study supports that LC-PolScope is a viable alternative to SHG for label free collagen organization measurements in thin histology sections.

  • direction of actin flow dictates integrin lfa 1 orientation during leukocyte migration
    bioRxiv, 2017
    Co-Authors: Pontus Nordenfelt, Shalin B Mehta, Travis I Moore, Joseph Mathew Kalappurakkal, Vinay Swaminathan, Nobuyasu Koga, Talley J Lambert, David Baker, Jennifer C Waters, Rudolf Oldenbourg
    Abstract:

    Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin head, known from crystal structures, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid head of the full integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin, and measure orientation with fluorescence Polarization Microscopy. Dependent on coupling to the cytoskeleton, integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. We have developed a method that places atomic, ~A structures of cell surface receptors in the context of functional, cellular length-scale, ~μm measurements and shows that rotation and tilt of cell surface receptors relative to the membrane plane can be restrained by interactions with other cellular components.

  • multifocus Polarization microscope mf polscope for 3d Polarization imaging of up to 25 focal planes simultaneously
    Optics Express, 2015
    Co-Authors: Sara Abrahamsson, Molly Mcquilken, Shalin B Mehta, Amitabh Verma, Johannes Larsch, Rob Ilic, Rainer Heintzmann, Cornelia I Bargmann, Amy S Gladfelter, Rudolf Oldenbourg
    Abstract:

    We have developed an imaging system for 3D time-lapse Polarization Microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different Polarization states. Due to the number of images necessary to collect both multiple Polarization states and multiple focal planes, 3D Polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus Polarization imaging of biological samples, including a protein assembly study in budding yeast cells.

Timothy A Springer - One of the best experts on this subject based on the ideXlab platform.

  • measuring integrin conformational change on the cell surface with super resolution Microscopy
    Cell Reports, 2018
    Co-Authors: Travis I Moore, Jesse Aaron, Tengleong Chew, Timothy A Springer
    Abstract:

    Summary We use super-resolution interferometric photoactivation and localization Microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron Microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence Polarization Microscopy.

Hernando Sosa - One of the best experts on this subject based on the ideXlab platform.

  • structural model for tubulin recognition and deformation by kinesin 13 microtubule depolymerases
    Cell Reports, 2013
    Co-Authors: Ana B Asenjo, Chandrima Chatterjee, Vania Depaoli, Dongyan Tan, William J Rice, Ruben Diazavalos, Mariena Silvestry, Hernando Sosa
    Abstract:

    Summary To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron Microscopy (EM) and fluorescence Polarization Microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

  • mechanism of kinesin 13 binding to microtubules as revealed by single molecule fluorescence Polarization Microscopy
    Biophysical Journal, 2012
    Co-Authors: Chandrima Chatterjee, Ana B Asenjo, Vania M De Paoli, Hernando Sosa
    Abstract:

    The kinesin superfamily of motor proteins are involved in diverse cellular processes, including intracellular organelle transport, cell division and cytoskeletal dynamics. The widely studied conventional kinesin (or kinesin-1) translocates along the microtubule (MT) by utilizing the energy generated from ATP hydrolysis. In contrast, members of the Kinesin-13 family do not walk along the MT lattice, but use their catalytic core to rapidly target microtubule ends by the process of one-dimensional diffusion (ODD) and promote depolymerization upon reaching there. However, the reason for such a significant difference in the behavior of the structurally conserved motor domain is not clearly understood. In order to reveal the mechanistic details of the kinesin-13 action, fluorescence Polarization Microscopy (FPM) has been employed to probe the configuration and mobility of BSR-labeled KLP10A (Drosophila m. Kinesin-13) molecules interacting with microtubules in the presence of different nucleotides. Experiments are being performed with KLP10A constructs of variable lengths to identify the potent mediators of diffusive motility. Preliminary results emerging from single-molecule FPM measurements have suggested that the motor core itself can undergo ODD without the assistance from the positively-charged neck domain. In addition, data acquired at both ensemble and single-molecule levels have revealed that the orientation of the KLP10A molecule relative to the MT filament is altered by mutating the crucial residues in the tubulin-binding sites on the motor domain. The structural and functional information extracted from analyses of our experimental findings will be discussed in details.

  • ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence Polarization Microscopy.
    Nature structural biology, 2001
    Co-Authors: Hernando Sosa, Erwin J.g. Peterman, W. E. Moerner, Lawrence S.b. Goldstein
    Abstract:

    Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence Polarization Microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.

Juntao Gao - One of the best experts on this subject based on the ideXlab platform.

  • advances of super resolution fluorescence Polarization Microscopy and its applications in life sciences
    Computational and structural biotechnology journal, 2020
    Co-Authors: Long Chen, Xingye Chen, Xusan Yang, Miaoyan Wang, Juntao Gao
    Abstract:

    Fluorescence Polarization Microscopy (FPM) analyzes both intensity and orientation of fluorescence dipole, and reflects the structural specificity of target molecules. It has become an important tool for studying protein organization, orientational order, and structural changes in cells. However, suffering from optical diffraction limit, conventional FPM has low orientation resolution and observation accuracy, as the Polarization information is averaged by multiple fluorescent molecules within a diffraction-limited volume. Recently, novel super-resolution FPMs have been developed to break the diffraction barrier. In this review, we will introduce the recent progress to achieve sub-diffraction determination of dipole orientation. Biological applications, based on Polarization analysis of fluorescence dipole, are also summarized, with focus on chromophore-target molecule interaction and molecular organization.

  • super resolution imaging of fluorescent dipoles via polarized structured illumination Microscopy
    Nature Communications, 2019
    Co-Authors: Karl Zhanghao, Wenhui Liu, Chunyan Shan, Xingye Chen, Yiqiong Liu, Yiming Wang, Sha Luo, Xiao Wang, Hao Xie, Juntao Gao
    Abstract:

    Fluorescence Polarization Microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination Microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

  • super resolution fluorescence Polarization Microscopy
    Journal of Innovative Optical Health Sciences, 2018
    Co-Authors: Karl Zhanghao, Juntao Gao, Dayong Jin, Xuedian Zhang
    Abstract:

    Fluorescence Polarization is related to the dipole orientation of chromophores, making fluorescence Polarization Microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. Several recent super resolution techniques have been applied to fluorescence Polarization Microscopy, achieving dipole measurement at nanoscale. In this review, we summarize both diffraction limited and super resolution fluorescence Polarization Microscopy techniques, as well as their applications in biological imaging.