Cover image for Vol. 16 Issue 9

Edited By: Michael S. Marks, Trina A. Schroer, Tom H. Stevens, Sharon A. Tooze

Online ISSN: 1600-0854

Virtual Issue Toolbox

ToolboxVirtual Issue Toolbox

Traffic has become a premier journal for the publication of papers reporting new discoveries in cell biology, including methodological papers/reviews and other searchable resources. The Toolbox virtual issue collects all of the papers in one place for your easy reference.

These are the articles available for the Traffic Toolbox series:

Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates

T Schmiedinger, GF Vogel, O Eiter, K Pfaller, WA Kaufmann, A Flörl, K Gutleben, S Schönherr, B Witting, TW Lechleitner, HL Ebner, T Seppi and MW Hess

A novel assay for measurement of membrane-protein surface expression using a β-lactamase reporter
VM Lam, P Beerepoot, S Angers and A Salahpour

Analysis of ER resident proteins in S. cerevisiae: Implementation of H/KDEL retrieval sequences

CL Young, DL Raden, AS Robinson

Novel systems for dynamically assessing insulin action in live cells reveals heterogeneity in the insulin response
JG Burchfield, J Lu, DJ Fazakerley, SX Tan, Y Ng, K Mele, MJ Buckley, W Han, WE Hughes and DE James

A Sensor of Protein O-Glycosylation Based on Sequential Processing in the Golgi Apparatus
Collin Bachert, Adam D. Linstedt
Traffic, Early View, Abstract
Protein O-glycosylation is important in numerous processes including the regulation of proteolytic processing sites by O-glycan masking in select newly synthesized proteins. To investigate O-glycan-mediated masking using an assay amenable to large-scale screens, we generated a fluorescent biosensor with an O-glycosylation site situated to mask a furin cleavage site. The sensor is activated when O-glycosylation fails to occur because furin cleavage releases a blocking domain allowing dye binding to a fluorogen activating protein...

Investigating Endocytic Pathways to the Endoplasmic Reticulum and to the Cytosol Using SNAP-Trap
Roger Geiger, Stefania Luisoni, Kai Johnsson, Urs F. Greber, Ari Helenius
Traffic, Early View, Abstract
Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP-tag...

Simplified Equation to Extract Diffusion Coefficients from Confocal FRAP Data
Minchul Kang, Charles A. Day, Anne K. Kenworthy, Emmanuele DiBenedetto
Traffic, Volume 13, Issue 12: 1589–1600, Abstract
Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking...

Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET
Tien-Hung Lan, Qiuju Liu, Chunman Li, Guangyu Wu, Nevin A. Lambert
Traffic, Volume 13, Issue 11:1450–1456, Abstract
Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image-based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment-targeted BRET partners can report subcellular location and movement of membrane proteins in live cells...

NEX-TRAP, a novel method for in vivo analysis of nuclear export of proteins
Verena Raschbichler, Diana Lieber, Susanne M. Bailer
Traffic, Volume 13, Issue 10:1326–1334, Abstract
Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin alpha- and beta-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remains tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): ...

A Novel Approach for Intracellular 3D Immuno-Labeling for Electron Tomography
Nuria Jiménez and Jan Andries Post
Traffic, Volume 13, Issue 7: 926–933, Abstract
Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in ...

SNAP-tag Based Proteomics Approach for the Study of the Retrograde Route
Getao Shi, Michel Azoulay, Florent Dingli, Christophe Lamaze, Damarys Loew, Jean-Claude Florent and Ludger Johannes
Traffic, Volume 13, Issue 7: 914–925, Abstract
Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative....

Protein Ligation in Living Cells Using Sortase
Karin Strijbis, Eric Spooner and Hidde L. Ploegh
Traffic, Volume 13, Issue 6: 780–789, Abstract
Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca2+-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal ...

Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions
Lindsey M. Costantini, Matteo Fossati, Maura Francolini and Erik Lee Snapp
Traffic, Volume 13, Issue 5: 643–649, Abstract
Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we ...

Auto-Align – Multi-Modality Fluorescence Microscopy Image Co-registration
William T. E. Pitkeathly, Natalie S. Poulter, Ela Claridge and Joshua Z. Rappoport
Traffic, Volume 13, Issue 2: 204–217, Abstract
Multi-modality microscopes incorporate multiple microscopy techniques into one module, imaging through a common objective lens. Simultaneous or consecutive image acquisition of a single specimen, using multiple techniques, increases the amount of measurable information available. In order to benefit from each modality, it is necessary to accurately co-register data sets. Intrinsic differences in the image ...

Protein Trafficking Rates Assessed by Quantum Dot Quenching with Bromocresol Green
Cathleen D. Valentine, A.S. Verkman and Peter M. Haggie
Traffic, Volume 13, Issue 1: 25–29, Abstract
Quantum dots are bright, photostable fluorophores used extensively to investigate biological processes. In this study, we report that bromocresol green (BCG) at low micromolar concentrations rapidly, efficiently and reversibly quenches the fluorescence of commercial quantum dots having a wide range of functionalities. The broad utility of BCG quenching of quantum dots in cell biology is showed in ...

Semi-Automated Analysis of Organelle Movement and Membrane Content: Understanding Rab-Motor Complex Transport Function
Alistair N. Hume, Miranda S. Wilson, Dmitry S. Ushakov, Michael A. Ferenczi and Miguel C. Seabra
Traffic, Volume 12, Issue 12: 1686–1701, Abstract
Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the ...

A Weak Base-Generating System Suitable for Selective Manipulation of Lysosomal pH
Luciene R. Carraro-Lacroix, Valentin Jaumouillé, Gregory D. Fairn and Sergio Grinstein
Traffic, Volume 12, Issue 11: 1490–1500, Abstract
pH varies widely among the different intracellular compartments. The establishment and maintenance of a particular pH appears to be critical for proper organellar function. This has been deduced from experiments where intraorganellar pH was altered by means of weak acids or bases, ionophores or inhibitors of the vacuolar H+-ATPase (V-ATPase). These manipulations, however, are not ...

In situ Measurement of the Electrical Potential Across the Lysosomal Membrane Using FRET
Mirkka Koivusalo, Benjamin E. Steinberg, David Mason and Sergio Grinstein
Traffic, Volume 12, Issue 8: 972–982, Abstract
The progressive acidification of the endocytic pathway is generated by H+ pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion ...

VIS2FIX: A High-Speed Fixation Method for Immuno-Electron Microscopy
Matthia A. Karreman, Elly G. van Donselaar, Hans C. Gerritsen, C. Theo Verrips and Arie J. Verkleij
Traffic, Volume 12, Issue 7: 806–814, Abstract
Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation ...

Measuring the Hierarchy of Molecular Events During Clathrin-Mediated Endocytosis
Dinah Loerke, Marcel Mettlen, Sandra L. Schmid and Gaudenz Danuser
Traffic, Volume 12, Issue 7: 815–825, Abstract
A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used ...

Recombinant Heptameric Coatomer Complexes: Novel Tools to Study Isoform-Specific Functions
Monika C. Sahlmüller, Jeroen R. P. M. Strating, Rainer Beck, Priska Eckert, Vincent Popoff, Mathias Haag, Andrea Hellwig, Imre Berger, Britta Brügger and Felix T. Wieland
Traffic, Volume 12, Issue 6: 682–692, Abstract
COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies ...

Superfolder GFP Is Fluorescent in Oxidizing Environments When Targeted via the Sec Translocon
Deborah E. Aronson, Lindsey M. Costantini and Erik L. Snapp
Traffic, Volume 12, Issue 5: 543-548, Abstract
The ability to study proteins in live cells using genetically encoded fluorescent proteins (FPs) has revolutionized cell biology (1–3). Researchers have created numerous FP biosensors and optimized FPs for specific organisms and subcellular environments in a rainbow of colors (4,5). However, expressing FPs in oxidizing environments such as the eukaryotic endoplasmic reticulum ...

Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices
Sudip Mondal, Shikha Ahlawat, Kaustubh Rau, V. Venkataraman and Sandhya P. Koushika
Traffic, Volume 12, Issue 4: 372–385, Abstract
Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We ...

A Comparison of GFP-Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro
Anika Hoffmann, Philip N. Dannhauser, Stephanie Groos, Lars Hinrichsen, Ute Curth and Ernst J. Ungewickell
Traffic, Volume 11, Issue 9: 1129–1140, Abstract
Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)-tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has ...

Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras
Derek C. Prosser, Karen Whitworth, Beverly Wendland
Traffic, Volume 11, Issue 9: 1141–1150, Abstract
The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are ...

Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate based image segmentation
Daniel Wüstner, Ane Landt Larsen, Nils J. Færgeman, Jonathan R. Brewer, Daniel Sage
Traffic, Accepted Article, Abstract
The nematode Caenorhabditis elegans (C. elegans) is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE) is challenged by C. elegans' high autofluorescence in the same spectral region as emission of DHE. We present a method to detect DHE selectively based on its rapid bleaching kinetics compared to ...

Subcellular Distribution of Tail-Anchored Proteins in Arabidopsis
Verena Kriechbaumer, Rowena Shaw, Joy Mukherjee, Caroline G. Bowsher, Anne-Marie Harrison, Ben M. Abell
Traffic, Volume 10 Issue 12: 1753 - 1764, Abstract
Tail-anchored (TA) proteins function in key cellular processes in eukaryotic cells, such as vesicle trafficking, protein translocation and regulation of transcription. They anchor to internal cell membranes by a C-terminal transmembrane domain, which also serves as a targeting sequence. Targeting occurs post-translationally, via pathways that are specific to the precursor, which makes TA proteins a model system for investigating post-translational protein targeting ...

Live cell multi-color imaging of lipid droplets with a new dye, LD540
Johanna Spandl, Daniel J. White, Jan Peychl, Christoph Thiele
Traffic, Volume 10 Issue 11: 1579 - 1584, Abstract
A lipophilic dye based on the Bodipy fluorophore, LD540, was developed for microscopic imaging of lipid droplets. In contrast to previous lipid droplet dyes, it can spectrally be resolved from both green and red fluorophores allowing multicolor imaging in both fixed and living ...

Following the Fate In Vivo of COPI Vesicles Generated In Vitro
Christoph Rutz, Ayano Satoh, Paolo Ronchi, Britta Brügger, Graham Warren, Felix T. Wieland
Traffic, Volume 10 Issue 8: 994 - 1005, Abstract
COPI vesicles are a class of transport carriers that function in the early secretory pathway. Their fate and function are still controversial. This includes their contribution to bidirectional transport within the Golgi apparatus and their role during cell division. Here we describe a method that should address several open questions about the fate and function of COPI vesicles in vivo. To ...

Use of Kaede Fusions to Visualize Recycling of G Protein-Coupled Receptors
Antje Schmidt, Burkhard Wiesner, Klaus Weißhart, Katharina Schulz, Jens Furkert, Björn Lamprecht, Walter Rosenthal, Ralf Schülein
Traffic, Volume 10, Issue 1: 2-15, Abstract
The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere ...

A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos
Susan J. Nixon, Richard I. Webb, Matthias Floetenmeyer, Nicole Schieber, Harriet P. Lo, Robert G. Parton
Traffic, Volume 10, Issue 2: 131-136, Abstract
The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green ...

Nuclear Export Signal Consensus Sequences Defined Using a Localization-Based Yeast Selection System
Shunichi Kosugi, Masako Hasebe, Masaru Tomita, Hiroshi Yanagawa
Traffic, Volume 9, Issue 12: 2053 - 2062, Abstract
Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast ...

High Data Output and Automated 3D Correlative Light–Electron Microscopy Method
Giuseppe Vicidomini, Maria C. Gagliani, Michela Canfora, Katia Cortese, Fabio Frosi, Clara Santangelo, Pier Paolo Di Fiore, Patrizia Boccacci, Alberto Diaspro, Carlo Tacchetti
Traffic, Volume 9 Issue 11: 1828 - 1838, Abstract
Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to ...

BODIPY-Cholesterol: A New Tool to Visualize Sterol Trafficking in Living Cells and Organisms
Maarit Hölttä-Vuori, Riikka-Liisa Uronen, Jarmila Repakova, Emppu Salonen, Ilpo Vattulainen, Pertti Panula, Zaiguo Li, Robert Bittman, Elina Ikonen
Traffic, Volume 9 Issue 11: 1839 - 1849, Abstract
Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This ...

A Functional GFP Fusion for Imaging Clathrin-Mediated Endocytosis
Joshua Z. Rappoport, Sanford M. Simon
Traffic, Volume 9, Issue 8: 1250-1255, Abstract
The ability to localize proteins of interest in live cells through imaging inherently fluorescent protein tags has provided an unprecedented level of information on cellular organization. However, there are numerous cases where fluorescent tags alter the localization and/or function of the proteins to which they are appended. Clathrin-mediated endocytosis from the plasma membrane is a ...

A Cryosectioning Procedure for the Ultrastructural Analysis and the Immunogold Labelling of Yeast Saccharomyces cerevisiae
Janice Griffith, Muriel Mari, Ann De Mazière and Fulvio Reggiori
Traffic, Volume 9, Issue 7: 1060–1072, Abstract
Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron

Integral and Associated Lysosomal Membrane Proteins
Bernd Schröder, Christian Wrocklage, Cuiping Pan, Ralf Jäger, Bernd Kösters, Helmut Schäfer, Hans-Peter Elsässer, Matthias Mann Andrej Hasilik
Traffic, Volume 8, Issue 12: 1676-1686, Abstract
We searched for novel proteins in lysosomal membranes, tentatively participating in molecular transport across the membrane and/or in interactions with other compartments. In membranes purified from placental lysosomes, we identified 58 proteins, known to reside at least partially in the lysosomal membrane. These included 17 polypeptides comprising or associated ...

Immunogold Labeling of Cryosections from High-Pressure Frozen Cells
Elly van Donselaar, George Posthuma, Dagmar Zeuschner, Bruno M. Humbel Jan W. Slot
Traffic, Volume 8, Issue 5: 471-485, Abstract
Immunogold Labeling of Cryosections from High-Pressure Frozen Cells ... Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. ... We have explored some new methods for applying immunogold labeling on cryosections from high-pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). ...

WatershedCounting3D: A New Method for Segmenting and Counting Punctate Structures from Confocal Image Data
Thomas J. Gniadek Graham Warren
Traffic, Volume 8, Issue 4: 339-346, Abstract
WatershedCounting3D: A New Method for Segmenting and Counting Punctate Structures from Confocal Image Data ... Because this is not always the case, we developed WatershedCounting3D, a program that uses a modified watershed algorithm to more accurately identify intracellular structures from confocal image data, even in the presence of an inhomogeneous background. ... However, as was shown in the blue-masked image in Figure 1B, our WatershedCounting3D algorithm does not suffer the same difficulty. ...

Location-Specific Depletion of a Dual-Localized Protein
Lee Shlevin, Neta Regev-Rudzki, Sharon Karniely Ophry Pines
Traffic, Volume 8, Issue 2: 169-176, Abstract
Location-Specific Depletion of a Dual-Localized Protein ... The lack of sensitive and suitable tools to address these issues has led us to develop a novel tool for functional detection of cytosolic/nuclear isoproteins in the cell, which we term location-specific depletion or subcellular knockout. ... However, in the search for a location-specific function of a distributing protein, knockout of the protein [e.g. chromosomal knockout of the gene or small interfering RNA (siRNA)] is not suitable since it results in depletion of all the isoenzymes, if they are derived from a single translation product. ...

Spectral Shift of Fluorescent Dye FM4-64 Reveals Distinct Microenvironment of Nuclear Envelope in Living Cells
Tomasz Zal, M. Anna Zal, Carina Lotz, Craig J. Goergen Nicholas R. J. Gascoigne
Traffic, Volume 7, Issue 12: 1607-1613, Abstract
We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocate...

Fast and Precise Protein Tracking Using Repeated Reversible Photoactivation
Dmitriy M. Chudakov, Tatyana V. Chepurnykh, Vsevolod V. Belousov, Sergey Lukyanov Konstantin A. Lukyanov
Traffic, Volume 7, Issue 10: 1304-1310, Abstract
Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we rep...

Detection and Quantification of Protein–Microtubules Interactions Using Green Fluorescent Protein Photoconversion
Stéphane Brunet, Timo Zimmermann, Emmanuel G. Reynaud, Isabelle Vernos, Eric Karsenti Rainer Pepperkok
Traffic, Volume 7, Issue 9: 1283-1289, Abstract
We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be ...

Extracting Sequence Motifs and the Phylogenetic Features of SNARE-Dependent Membrane Traffic
Akiyasu C. Yoshizawa, Shuichi Kawashima, Shujiro Okuda, Masashi Fujita, Masumi Itoh, Yuki Moriya, Masahiro Hattori and Minoru Kanehisa
Traffic, Volume 7, Issue 8: 1104-1118, Abstract
The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in ...

Subcellular Localization of Mammalian Type II Membrane Proteins
Rajith N. Aturaliya, J. Lynn Fink, Melissa J. Davis, Melvena S. Teasdale, Kelly A. Hanson, Kevin C. Miranda, Alistair R. R. Forrest, Sean M. Grimmond, Harukazu Suzuki, Mutsumi Kanamori, Chikatoshi Kai, Jun Kawai, Piero Carninci, Yoshihide Hayashizaki and Rohan D. Teasdale
Traffic, Volume 7, Issue 5: 613-625, Abstract
Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping ...

In vivo Selective Cytoskeleton Dynamics Quantification in Interphase Cells Induced by Pulsed Ultraviolet Laser Nanosurgery
Julien Colombelli,, Emmanuel G. Reynaud, Jens Rietdorf, Rainer Pepperkok and Ernst H.K. Stelzer
Traffic, Volume 6, Issue 12: 1093-1102, Abstract
We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells ...

The Steady-State Distribution of Glycosyltransferases Between the Golgi Apparatus and the Endoplasmic Reticulum is Approximately 90:10
Sung Wu Rhee, Tregei Starr, Kimberly Forsten-Williams and Brian Storrie
Traffic, Volume 6, Issue 11: 978-990, Abstract
The Steady-State Distribution of Glycosyltransferases Between the Golgi Apparatus and the Endoplasmic Reticulum is Approximately 90:10 ... Best-practice widefield fluorescence microscopy yields approximately 90:10 Golgi-to-ER steady-state distribution for Golgi glycosyltransferasesglycosyltransferasesTaking this approach, we found that endogenous, epitope-tagged and GFP-tagged Golgi glycosyltransferases all had an approximately 90:10 steady-state distribution between the Golgi apparatus and ER. ...

Translocation Biosensors to Study Signal-Specific Nucleo-Cytoplasmic Transport, Protease Activity and Protein–Protein Interactions
Shirley K. Knauer, Sabrina Moodt, Thorsten Berg, Urban Liebel, Rainer Pepperkok and Roland H. Stauber
Traffic, Volume 6, Issue 7: 594-606, Abstract
Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent ...

The Kinetics of Phagosome Maturation as a Function of Phagosome/Lysosome Fusion and Acquisition of Hydrolytic Activity
Robin M. Yates, Albin Hermetter and David G. Russell
Traffic, Volume 6, Issue 5, Page 413-420, Abstract
Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation rema...

The Nanopore Connection to Cell Membrane Unitary Permeability
Reiner Peters
Traffic, Volume 6, Issue 3: 199-204, Abstract
Artificial nanopores have recently emerged as versatile tools for analyzing and sorting single molecules at high speed. However, the biological cell has already developed a large set of sophisticated protein nanopores that are able to selectively translocate all types of molecules through membranes. ...

VisBio: A Computational Tool for Visualization of Multidimensional Biological Image Data
Curtis Rueden, Kevin W. Eliceiri and John G. White
Traffic, Volume 5, Issue 6: 411-417, Abstract
New laser scanning microscopy techniques enable biologists to acquire larger, more complex image datasets. Emerging imaging modalities such as multispectral, harmonic, and fluorescence lifetime can generate data with six or more dimensions; however, existing software is not well suited ...

Traffic Jams II: An Update of Diseases of Intracellular Transport
Meir Aridor and Lisa A. Hannan
Traffic, Volume 3, Issue 11: 781-790, Abstract
As more details emerge on the mechanisms that mediate and control intracellular transport, the molecular basis for variety of human diseases has been revealed. In turn, disease pathology and physiology shed light on the intricate controls that regulate int...

Strategies for Prokaryotic Expression of Eukaryotic Membrane Proteins
Rico Laage and Dieter Langosch

Traffic, Volume 2, Issue 2: 99-104, Abstract
High-level heterologous expression of integral membrane proteins at full-length is a useful tool for their structural and functional characterization. Here, systems that have previously been used for efficient bacterial expression of eukaryotic membrane proteins are reviewed and novel vectors consisting of a modular  ...

Traffic Jam: A Compendium of Human Diseases that Affect Intracellular Transport Processes
Meir Aridor and Lisa A. Hannan
Traffic, Volume 1, Issue 11: 836-851, Abstract
As sequencing of the human genome nears completion, the genes that cause many human diseases are being identified and functionally described. This has revealed that many human diseases are due to defects of intracellular trafficking. This 'Toolbox' catalogs and briefly describes these diseases.

How to Convert a Traditional Electron Microscopy Laboratory to Digital Imaging: Follow the 'Middle Road'
John Heuser
Traffic, Volume 1, Issue 8: 614-621, Abstract
How to Convert a Traditional Electron Microscopy Laboratory to Digital Imaging: Follow the 'Middle Road' ... Today, electron microscopy (EM) is increasingly confronted by the revolution in image-processing technology provoked by modern computers. ... In this world, digital files are a 'must', of course, and issues of how to convert EMs into an optimal form for general Internet dissemination intersect with issues of how to prepare digital EMs for more traditional publication in printed journals. ...

The Production of 'Cell Cortices' for Light and Electron Microscopy
John Heuser
Traffic, Volume 1, Issue 7: 545-552, Abstract
The Production of 'Cell Cortices' for Light and Electron Microscopy ...

Molecular Structures of Proteins Involved in Vesicle Fusion
Joel A. Ybe, Diane E. Wakeham, Frances M. Brodsky and Peter K. Hwang
Traffic, Volume 1, Issue 6: 474-479, Abstract
We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.

Molecular Structures of Proteins Involved in Vesicle Coat Formation
Diane E. Wakeham, Joel A. Ybe, Frances M. Brodsky and Peter K. Hwang
Traffic, Volume 1, Issue 5: 393-398, Abstract
This review includes 16 structures of vesicle coat components and accessory proteins and a description of their roles in vesicle budding or coat disassembly.