@article {47652, title = {Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.}, journal = {Blood}, volume = {119}, year = {2012}, month = {2012 Feb 23}, pages = {1848-55}, abstract = {

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

}, keywords = {Animals, Antigens, CD34, Antigens, CD82, Bone Marrow, Cell Cycle, Cell Line, Tumor, Cells, Cultured, Cytokines, Flow Cytometry, Fluorescent Antibody Technique, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Humans, Interleukin Receptor Common gamma Subunit, Leukemia, Membrane Microdomains, Mice, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Microscopy, Confocal, Receptors, CXCR4, Tetraspanins, Transplantation, Heterologous}, issn = {1528-0020}, doi = {10.1182/blood-2011-08-371583}, author = {Larochelle, Andre and Gillette, Jennifer M and Desmond, Ronan and Ichwan, Brian and Cantilena, Amy and Cerf, Alexandra and Barrett, A John and Wayne, Alan S and Lippincott-Schwartz, Jennifer and Dunbar, Cynthia E} } @article {47659, title = {Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.}, journal = {Blood}, volume = {119}, year = {2012}, month = {2012 Feb 23}, pages = {1848-55}, abstract = {

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

}, keywords = {Animals, Antigens, CD34, Antigens, CD82, Bone Marrow, Cell Cycle, Cell Line, Tumor, Cells, Cultured, Cytokines, Flow Cytometry, Fluorescent Antibody Technique, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Humans, Interleukin Receptor Common gamma Subunit, Leukemia, Membrane Microdomains, Mice, Mice, Inbred NOD, Mice, Knockout, Mice, SCID, Microscopy, Confocal, Receptors, CXCR4, Tetraspanins, Transplantation, Heterologous}, issn = {1528-0020}, doi = {10.1182/blood-2011-08-371583}, author = {Larochelle, Andre and Gillette, Jennifer M and Desmond, Ronan and Ichwan, Brian and Cantilena, Amy and Cerf, Alexandra and Barrett, A John and Wayne, Alan S and Lippincott-Schwartz, Jennifer and Dunbar, Cynthia E} } @article {47680, title = {Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics.}, journal = {Methods in enzymology}, volume = {475}, year = {2010}, month = {2010}, pages = {109-20}, abstract = {

Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues. Here we describe the method of single-molecule tracking photoactivated localization microscopy (sptPALM) and discuss how its use can contribute to a quantitative understanding of fundamental cellular processes.

}, keywords = {Fluorescent Dyes, Light, Membrane Proteins, Microscopy, Molecular Dynamics Simulation, Molecular Motor Proteins}, issn = {1557-7988}, doi = {10.1016/S0076-6879(10)75005-9}, author = {Manley, Suliana and Gillette, Jennifer M and Lippincott-Schwartz, Jennifer} } @article {927, title = {Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {106}, year = {2009}, month = {2009 Mar 3}, pages = {3125-30}, abstract = {

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

}, keywords = {Animals, Cell Line, Cercopithecus aethiops, Fluorescence, Humans, Imaging, Interference, Microscopy, Microtubules, Three-Dimensional}, issn = {1091-6490}, doi = {10.1073/pnas.0813131106}, author = {Shtengel, Gleb and Galbraith, James A and Galbraith, Catherine G and Lippincott-Schwartz, Jennifer and Gillette, Jennifer M and Manley, Suliana and Sougrat, Rachid and Waterman, Clare M and Kanchanawong, Pakorn and Davidson, Michael W and Fetter, Richard D and Hess, Harald F} } @article {2080, title = {High-density mapping of single-molecule trajectories with photoactivated localization microscopy. (With commentary)}, journal = {Nature Methods}, volume = {5}, year = {2008}, month = {2008 Feb}, pages = {155-7}, abstract = {

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

Commentary: As a stepping stone to true live cell PALM (see above), our collaborator Jennifer Lippincott-Schwartz suggested using the sparse photoactivation principle of PALM to track the nanoscale motion of thousands of individual molecules within a single living cell. Termed single particle tracking PALM (sptPALM), Jennifer{\textquoteright}s postdocs Suliana Manley and Jen Gillette used the method in our PALM rig to create spatially resolved maps of diffusion rates in the plasma membrane of live cells. sptPALM is a powerful tool to study the active cytoskeletal or passive diffusional transport of individual molecules with far more measurements per cell than is possible without sparse photoactivation.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Fluorescence, Image Enhancement, Membrane Proteins, Microscopy}, issn = {1548-7105}, doi = {10.1038/nmeth.1176}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18193054}, author = {Manley, Suliana and Gillette, Jennifer M and Patterson, George H and Shroff, Hari and Hess, Harald F and Betzig, Eric and Lippincott-Schwartz, Jennifer} }