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Articles by Gregory A. Gibson in JoVE

 JoVE Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

JoVE 50386

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Other articles by Gregory A. Gibson on PubMed

Robust Systemic Transduction with AAV9 Vectors in Mice: Efficient Global Cardiac Gene Transfer Superior to That of AAV8

It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

Effective Immunotherapy Against Murine Gliomas Using Type 1 Polarizing Dendritic Cells--significant Roles of CXCL10

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

IL-6 Adsorption Dynamics in Hemoadsorption Beads Studied Using Confocal Laser Scanning Microscopy

Sepsis is characterized by a systemic inflammatory response caused by infection, and can result in organ failure and death. Removal of inflammatory mediators such as cytokines from the circulating blood is a promising treatment for severe sepsis. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using biocompatible, polymer sorbent beads. In this study, we used confocal laser scanning microscopy (CLSM) to directly examine adsorption dynamics of a cytokine (IL-6) within hemoadsorption beads. Fluorescently labeled IL-6 was incubated with sorbent particles, and CLSM was used to quantify spatial adsorption profiles of IL-6 within the sorbent matrix. IL-6 adsorption was limited to the outer 15 microm of the sorbent particle over a relevant clinical time period, and intraparticle adsorption dynamics was modeled using classical adsorption/diffusion mechanisms. A single model parameter, alpha = q(max) K/D, was estimated by fitting CLSM intensity profiles to our mathematical model, where q(max) and K are Langmuir adsorption isotherm parameters, and D is the effective diffusion coefficient of IL-6 within the sorbent matrix. Given the large diameter of our sorbent beads (450 microm), less than 20% of available sorbent surface area participates in cytokine adsorption. Development of smaller beads may accelerate cytokine adsorption by maximizing available surface area per bead mass.

Characterization of an Lhx1a Transgenic Reporter in Zebrafish

The LIM-domain containing transcription factor, Lhx1, is involved in the regulation of early gastrulation cell movements, kidney organogenesis and other processes in vertebrate model organisms. To follow the expression of this gene in live embryos, we created transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of lhx1a regulatory regions. Tg(lhx1a:EGFP)(pt303) recapitulates the expression of endogenous lhx1a beginning at early gastrula stages through 72 hours of development with only few exceptions. In addition, over-expression of the Nodal ligand, ndr1, results in the concomitant expansion of the transgene and endogenous lhx1a expression. Treatment of Tg(lhx1a:EGFP)(pt303) embryos with the small molecule SB-431542, an inhibitor of Nodal signaling, results in the loss of both transgene and endogenous lhx1a expression. These experiments suggest that Tg(lhx1a:EGFP)(pt303) is regulated in a manner similar to endogenous lhx1a. Therefore, this reporter can be utilized not only for monitoring lhx1a expression, but also for numerous applications, including chemical genetics screening.

The Second Extracellular Loop Dictates Occludin-mediated HCV Entry

Recent findings have implicated tight junction (TJ) protein Occludin (OCLN) as an essential factor for hepatitis C virus (HCV) to enter human hepatocytes. To gain insights into OCLN-mediated HCV entry, we created a panel of OCLN deletion mutants and found that without impairing OCLN's cell surface localization, removal of the extracellular loop 2 (EL2) from OCLN abolished both its ability to mediate HIV-HCV pseudotypes' (HCVpp) entry as well as its ability to coprecipitate HCV glycoprotein E2. Recombinant OCLN EL2, however, failed to robustly bind soluble E2 (sE2) in pull-down assays. Subsequent studies revealed that OCLN formed complex with Dynamin II, an important GTPase for endocytosis, in an EL2-dependent fashion. HCVpp, as well as cell culture grown HCV (HCVcc), was sensitive to Dynamin knockdown or inhibition. We conclude that OCLN EL2 dictates the Dynamin-dependent HCV entry. Furthermore, OCLN could function to bridge virions to Dynamin-dependent endocytic machineries.

Rapid Host Defense Against Aspergillus Fumigatus Involves Alveolar Macrophages with a Predominance of Alternatively Activated Phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c(+) alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.

Direct Visualization of HIV-1 with Correlative Live-cell Microscopy and Cryo-electron Tomography

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.

Mechanism of Transfer of Functional MicroRNAs Between Mouse Dendritic Cells Via Exosomes

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

A Theoretical and Non-destructive Experimental Approach for Direct Inclusion of Measured Collagen Orientation and Recruitment into Mechanical Models of the Artery Wall

Gradual collagen recruitment has been hypothesized as the underlying mechanism for the mechanical stiffening with increasing stress in arteries. In this work, we investigated this hypothesis in eight rabbit carotid arteries by directly measuring the distribution of collagen recruitment stretch under increasing circumferential loading using a custom uniaxial (UA) extension device combined with a multi-photon microscope (MPM). This approach allowed simultaneous mechanical testing and imaging of collagen fibers without traditional destructive fixation methods. Fiber recruitment was quantified from 3D rendered MPM images, and fiber orientation was measured in projected stacks of images. Collagen recruitment was observed to initiate at a finite strain, corresponding to a sharp increase in the measured mechanical stiffness, confirming the previous hypothesis and motivating the development of a new constitutive model to capture this response. Previous constitutive equations for the arterial wall have modeled the collagen contribution with either abrupt recruitment at zero strain, abrupt recruitment at finite strain or as gradual recruitment beginning at infinitesimal strain. Based on our experimental data, a new combined constitutive model was presented in which fiber recruitment begins at a finite strain with activation stretch represented by a probability distribution function. By directly including this recruitment data, the collagen contribution was modeled using a simple Neo-Hookean equation. As a result, only two phenomenological material constants were required from the fit to the stress stretch data. Three other models for the arterial wall were then compared with these results. The approach taken here was successful in combining stress-strain analysis with simultaneous microstructural imaging of collagen recruitment and orientation, providing a new approach by which underlying fiber architecture may be quantified and included in constitutive equations.

Quantitative Proteomics Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes

ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection.

Mitochondrial Hyperfusion Induced by Loss of Fission Protein Drp1 Causes ATM-dependent G2/M Arrest and Aneuploidy Through DNA Replication Stress

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we have identified that inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by hyperfused mitochondrial structure and unscheduled expression of cyclin E in G2 phase. This persistent replication stress then induces an ATM-dependent activation of G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are the causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and ROS generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.

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