December 2011: This Month in JoVE

Summary

Here are some highlights from the December 2011 Issue of Journal of Visualized Experiments (JoVE).

Protocol

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Joel R. Meyerson^{1, 2}, Tommi A. White¹, Donald Bliss³, Amy Moran³, Alberto Bartesaghi¹, Mario J. Borgnia¹, M. Jason V. de la Cruz¹, David Schauder¹, Lisa M. Hartnell¹, Rachna Nandwani^{1, 4}, Moez Dawood⁵, Brianna Kim⁶, Jun Hong Kim⁷, John Sununu⁸, Lisa Yang⁹, Siddhant Bhatia¹⁰, Carolyn Subramaniam¹, Darrell E. Hurt¹¹, Laurent Gaudreault¹², Sriram Subramaniam^1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , ³National Library of Medicine, National Institutes of Health, ⁴Massachusetts Institute of Technology, ⁵William Fremd High School, ⁶University of Virginia , ⁷Duke University , ⁸Yale University, ⁹University of Notre Dame , ¹⁰Washington University in St. Louis , ¹¹Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, ¹²Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Electrophysiological Measurements and Analysis of Nociception in Human Infants

L. Fabrizi^1,*, A. Worley^2,*, D. Patten¹, S. Holdridge¹, L. Cornelissen¹, J. Meek³, S. Boyd², R. Slater^{1, 4
1}Neuroscience, Physiology and Pharmacology, University College London, ²Department of Clinical Neurophysiology, Great Ormond Street Hospital, ³Elizabeth Garrett Anderson Obstetric Hospital, University College Hospital, ⁴Nuffield Department of Anaesthetics, University of Oxford
^* These authors contributed equally

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Planar and Three-Dimensional Printing of Conductive Inks

Bok Yeop Ahn¹, Steven B. Walker¹, Scott C. Slimmer¹, Analisa Russo¹, Ashley Gupta¹, Steve Kranz¹, Eric B. Duoss^{1, 2}, Thomas F. Malkowski^{1, 3}, Jennifer A. Lewis^1
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, ²Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, ³Presently at the Interdisciplinary Center for Wide Band-gap Semiconductors, University Of California Santa Barbara

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

Yu Huang^{1, 2,*}, Basheal Agrawal^3,*, Paul A. Clark³, Justin C. Williams^{1, 2, 3}, John S. Kuo^{3, 4
1}Department of Biomedical Engineering, University of Wisconsin-Madison, ²Materials Science Program, University of Wisconsin-Madison, ³Department of Neurological Surgery, University of Wisconsin-Madison, ⁴Carbone Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, University of Wisconsin-Madison
^* These authors contributed equally

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Julien Brechbühl¹, Gaëlle Luyet¹, Fabian Moine¹, Ivan Rodriguez², Marie-Christine Broillet^1
1Department of Pharmacology and Toxicology, University of Lausanne, ²Department of Genetics and Evolution, University of Geneva

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Selective Viral Transduction of Adult-Born Olfactory Neurons for Chronic In Vivo Optogenetic Stimulation

Gabriel Lepousez, Mariana Alonso, Sebastian Wagner, Benjamin W. Gallarda, Pierre-Marie Lledo
Laboratory for Perception and Memory, Institut Pasteur and Centre National de la Recherche Scientifique (CNRS)

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

Limei Ma¹, Sachiko Haga-Yamanaka¹, Qingfeng Elden Yu¹, Qiang Qiu¹, SangSeong Kim¹, C. Ron Yu^{1, 2
1}Stowers Institute for Medical Research, ²Department of Anatomy and Cell Biology, The University of Kansas School of Medicine

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Measurement of Cytosolic Ca²⁺ in Isolated Contractile Lymphatics

Flavia M. Souza-Smith, Kristine M. Kurtz, Jerome W. Breslin
Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center

We introduce an approach to evaluate the cytosolic Ca²⁺ concentration in isolated lymphatics to study Ca²⁺-dependent and Ca²⁺-sensitizing mechanisms of lymphatic smooth muscle contraction.