[IME Membership] [New Faces at Penn and the IME][Educational Initiatives][Recent Awards and Honors to IME Members][IME Seminar Series][IME Newsletter Table of Contents][IME Home Page]Rapid Displacement of Vimentin Intermediate Filaments in Living Endothelial Cells Exposed to Flow
Brian P. Helmke, Robert D. Goldman, and Peter F. Davies
When blood flows through an artery how does the flow mechanically stress the cell lining? Hemodynamic shear stress at the endothelial cell surface induces acute and chronic intracellular responses that regulate vessel wall biology. The cytoskeleton is implicated by acting both as a direct connector to local surface deformation and as a distribution network for mechanical forces throughout the cell. In a paper published in Circulation Research in April 2000 (86:745-752), Brian Helmke, Peter F. Davies, and their collaborator from Northwestern University demonstrated rapid deformation of the intermediate filament (IF) network in living endothelial cells under flow. Spatial and temporal dynamics of green fluorescent proteinvimentin IF in confluent endothelial cells were analyzed using time lapse optical sectioning and deconvolution microscopy within the first three minutes after the introduction of flow (shear stress 12 dyn/cm2). The imposition of shear stress significantly increased the variability of IF movement throughout the cell in the x-, y- and z-directions compared to the constitutive dynamics noted in the absence of flow. The magnitude and direction of flow-induced IF displacement was heterogeneous at the subcellular level. These qualitative and quantitative data demonstrate that shear stress acting at the luminal surface of the endothelium results in rapid deformation of a stable cytoskeleton network throughout the cell.
Muscle Cell Peeling from Micropatterned Collagen: Direct Probing of Focal and Molecular Properties of Matrix Adhesion
Hyun J. Ra, Catherine Picart, Huisheng Feng, H. Lee Sweeney, and Dennis E. Discher
Muscle cells of all types, including cardiomyocytes, smooth muscle cells, and skeletal muscle cells, must transmit their contractions through viable cell attachments. To understand this adhesion, Dennis Discher and coworkers cultured skeletal muscle cells for controlled peeling from narrow strips of collagen-coated glass. The geometric influence on differentiation minimized lateral cell contact and cell branching, enabling detailed study of myocyte-matrix adhesion. A micropipette was used to pull back one end of a single, quasi-cylindrical cell with detailed observation of the non-equilibrium detachment process. Peeling velocities fluctuated as focal roughness, microns in scale, was encountered along the detachment front. Nonetheless, peeling velocity (~µm/second) generally increased with detachment force (~nano-Newton), consistent with forced disruption of adhesion bonds. Immunofluorescence of b1-integrins correlated with the focal roughness and appeared clustered in axially-extended focal contacts. In addition, the peeling forces and rates were found to be moderately well-described by Demboıs dynamical peeling model for receptor-based adhesion. Estimates were thereby obtained for the spontaneous, molecular off-rate (kooff <~ 10 seconds-1) and the receptor complex stiffness (k ~ 10-5 to 10-6 Newton/meter) of adherent myocytes. Interestingly, the local stiffness is in the range of flexible proteins of the spectrin superfamily, including dystrophin, which is membrane-localized and implicated in Muscular Dystrophies. The study was published in the Journal of Cell Science (112:1425-36, 1999).
A New Bioengineering Confocal/Multiphoton Microscopy Core Facility
A new Bioengineering Confocal/Multiphoton Microscopy Core Facility opened in July to support innovative research into cell and tissue morphology using the latest in confocal and multiphoton imaging technologies. Funded by a Major Research Instrumentation grant from the National Science Foundation (PI: IME member Susan Margulies, Associate Professor of Bioengineering), this new inverted Nikon/BioRad microscope system with a tunable near-infrared Coherent laser is available for time-lapse imaging of live or fixed cells, tissues, and thick preparations. The facility is located at 388 Towne Building (220 S. 33rd St.), convenient to the Schools of Medicine, Arts and Sciences, and Engineering. Consultation and training is provided, and is tailored to individual investigator needs and experience level. Contact Director Susan Margulies, Ph.D. (215-898-0882, margulies@seas.upenn.edu) or Co-Director James F. Sanzo, Ph.D. (jfs@seas.upenn.edu) for additional information.
Cross-section of Normal Human Retina. Micrograph by J.F. Sanzo, Pen Bioengineering. Used by permission of A.H. Milano, S.K. John, and J.E. Smith, Scheie Eye Institute, Penn, and A. Swaroop, Kellogg Eye Center, Univ. of Michigan.
Nuclear Targeting Peptide Scaffolds for Lipofection of Nondividing Mammalian Cells
Ajit Subramanian, P. Ranganathan, Scott L. Diamond
While viruses are often very efficient in delivering genes to non-dividing cells, they can be difficult to manufacture and use. In contrast, plasmid DNA made in bacteria is extremely cost effective as a therapeutic agent. However, plasmids delivered to the cytoplasm of a cell have great difficulty traversing the nuclear pore complex to gain access to the nucleus. With this in mind, Dr. Diamondıs laboratory has developed a nuclear targeting scaffold to bind DNA and help deliver it across the nuclear pore.
"A nuclear targeting scaffold is an example of tissue engineering for the inside of a cell. We wish to graft recognition sequences to polymers in order to exploit very specific intracellular binding events inside the cell. That way we can target therapeutic agents to the desired intracellular compartment." says Scott Diamond, associate professor of chemical engineering. In a paper published in Nature Biotechnology, (17(9):873-7, 1999), Dr. Diamond and coworkers reported that, using the M9 peptide sequence from the heteronuclear ribonuclear protein (hnRNP A1), the normal carrier for RNA in the cell, they were able to target karyopherin beta-2 (transportin-1) to help shuttle plasmid into the nucleus of non-dividing endothelial cells. The method provided a transfection level of 83%, with a 63-fold enhancement of marker transgene expression.
Notch1 Expression in Early Lymphopoiesis Influences B versus T Lineage Determination
John C. Pui, David Allman, Lanwei Xu, Susan DeRocco Fredrick G. Karnell, Sonia Bakkour, Julia Y. Lee, Tom Kadesch, Richard R. Hardy, Jon C. Aster, and Warren S. Pear
Notch genes encode transmembrane receptors that regulate fate decisions in many cells. A recent study by Warren S. Pear and colleagues at Penn, the Fox Chase Cancer Center, and Harvard Medical School (Immunity, 11:21-31, 1999) showed that Notch1 provides a key regulatory signal to influence lineage commitment of hematopoietic cells and determine whether a common lymphoid progenitor cell will differentiate into a T- or a B-lymphocyte. In this study the authors reconstituted the hematopoietic system of lethally irradiated mice with bone marrow transduced with retroviruses encoding a constitutively active form of Notch1. While neither granulocyte nor monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced bone marrow contained immature CD4+/CD8+ T-cells in the bone marrow, indicating that the activated Notch1 protein induces T cell development in a thymus-independent manner. Simultaneously, activated Notch1 blocked B-lymphopoiesis at the earliest pro-B stage, possibly by the repression of the transcription factor E2A. These findings suggest that Notch may be the earliest signal that instructs a lymphoid stem cell to become a B or T cell. These findings may have important implications for stem cell biology, cancer (where Notch is implicated in T cell leukemia) and some immunodeficiencies where patients lack B or T cells.
Transport of Torsional Stress in DNA
Philip Nelson
DNA can be regarded as a linear repository of sequence information, or as a chemical compound subject to various modifications, and each of these viewpoints is important for understanding some aspects of gene function and regulation. However, many other important processes require an appreciation of DNA as an elastic object in a viscous environment. For example, the action-at-a-distance between eukaryotic promoters and their enhancers involves an effective concentration of bound enhancer units depending on both torsional and bend rigidity of DNA.
It is well known that transcription can induce torsional stress in DNA, affecting the activity of nearby genes or even inducing structural transitions in the DNA duplex. It has long been assumed that the generation of significant torsional stress requires the DNA to be anchored, forming a limited topological domain, since otherwise it would spin almost freely about its axis. Previous estimates of the rotational drag have, however, neglected the role of small natural bends in the helix backbone. In a paper published in the Proceedings of the National Academy of Sciences, USA (96: 14342-14347, 1999), Physics professor Philip Nelson, showed, how these bends can increase the drag several thousand-fold relative to prior estimates, allowing significant torsional stress even in linear, unanchored DNA. The model helps explain several puzzling experimental results on structural transitions induced by transcription of DNA.
Total and Non-Recoverable Strain Fields of the Glenohumeral Joint Capsule under Shoulder Subluxation
Louis J. Soslowsky, David M. Malicky, Cameron Mouro, Michael J. Bey, Juan C. Frisancho, Steven R. Lindholm, John E. Kuhn
This paper received the Charles S. Neer Award for Excellence in Basic Science Research from the American Shoulder and Elbow Surgeons. The paper was selected from over 300 papers submitted to the ASES Specialty Day meeting (Orlando, March 2000). This is the second consecutive year that a paper from Dr. Soslowskyıs lab received this award!
Instability of the shoulder is related to deformations of the ligaments and capsule, though the nature of this relationship is largely unknown. In the position of 90° arm abduction and external rotation, the inferior glenohumeral ligament (IGHL), located throughout the anteroinferior capsule (AIC), is an important stabilizer. In a study presented in March 2000 (American Shoulder and Elbow Surgeons, Paper #32), Dr. Louis J. Soslowsky and co-workers measured the total and non-recoverable strain fields in the AIC in an anteroinferiorly subluxed shoulder. To do so, seven shoulders were dissected down to the ligaments and rotator cuff tendons and rotator cuff muscles were loaded. A stereoradiogrammetric procedure and mathematical reconstruction was used to calculate the peak and mean maximum principal strains of the glenohumeral capsule. This study quantitated the two-dimensional strain field in a three-dimensional structure such as the glenohumeral capsule. Interestingly, strains computed were neither uniaxial, nor aligned with the anatomical ligaments. This is the first study to show the existence of pre-failure, non-recoverable strain due to joint subluxation. Clinically, large subluxations may produce non-recoverable, permanent strain, which is likely to aggravate joint instability.
Relationship between Selectin-Mediated Rolling of Hematopoietic Stem and Progenitor Cells and Progression in Hematopoietic Development
Adam W. Greenberg, William G. Kerr, Daniel Hammer
Hematopoietic stem and progenitor cells (HSPC) give rise to all mature blood cells and can be used to restore the blood and immune systems of cancer patients following high-dose chemotherapy, and to treat autoimmune, metabolic, and genetic diseases. IME faculty member Daniel A. Hammer and his graduate student, Adam W. Greenberg, in collaboration with William G. Kerr (formerly in the IHGT, Penn), have discovered a mechanism of HSPC adhesion that explains the ability of these cells to traffic throughout the body. In a recent paper in Blood (95(2):478-86, 2000), they showed that HSPC which display the surface marker CD34 and are best able to regenerate blood cells, adhere better than CD34- cells to selectin adhesion molecules under flow. Selectins support the trafficking of leukocytes around the body, suggesting that HSPC and leukocytes share a common mechanism for trafficking. HSPC further fractionated by different surface markers are very potent blood cell regenerators. Interestingly, the most potent CD34+CD38- subpopulation shows the most avid binding to selectin adhesion molecules under flow, suggesting that the effectiveness of HSPC subpopulations to regenerate blood cells is directly correlated with the ability of these cells to adhere to selectin adhesion molecules under flow and hence their ability to interact with the vascular wall in the bone marrow microcirculation. This work holds promise for understanding the molecular mechanisms of stem cell homing during bone marrow transplantation, and provides a means by which stem cells can be purified by differential adhesion to selectin-coated surfaces. Such a separation has major advantages over current separation technologies for HSPC that require many steps and the use of antibodies and other conjugated markers that must be removed prior to transplantation.
Fibrin Glues from Salmon Blood
Fibrinogen and other clotting factors are used to make fibrin glues for a range of surgical procedures and for formulation on bandages for treatment of acute injuries. A collaboration between IME faculty and Physiology Professor Paul Janmey and Dr. Evelyn Sawyer of Sea Run Holdings, at Conners Aquaculture in Eastport Maine, led to the production of fibrin glue prepared from the blood of farmed Atlantic salmon. The new sealant provides a great advantage over current products: blood from coldwater fish provides a source of well-controlled natural material that poses lower probability of viral or bacterial infection than fibrin glues made from human or bovine proteins thanks to the low body temperature of Atlantic fish. However, the large difference in body temperature presented interesting problems in protein chemistry and material properties, and protein purification protocols designed to function at 37oC had to be modified to preserve the function of proteins adapted to work at 10oC. These glues are now being tested in vivo.
