Cover image for Vol. 17 Issue 10

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

A Toolbox for Rapid Quantitative Assessment of Chronological Lifespan and Survival in Saccharomyces cerevisiae
Sarah R. Chadwick, Athanasios D. Pananos, Sonja E. Di Gregorio, Anna E. Park, Parnian Etedali-Zadeh, Martin L. Duennwald, and Patrick Lajoie
Traffic, Volume 17, Issue 6: 689-703, Abstract
The yeast Saccharomyces cerevisiae provides an unmatched genetic platform to study the mechanisms underlying the aging process. We present a rapid and efficient quantitative assay to measure chronological aging in yeast through fluorescent labeling of dead cells in multiwell plates. We designed an open-source software to rapidly quantify yeast survival under various conditions, including chronological aging. We employed our assays to study the role of different heat shock proteins in the extension of yeast chronological lifespan by caloric restriction.

Measuring Exocytosis Rate Using Corrected Fluorescence Recovery After Photoconversion
Nan Luo, An Yan, and Zhenbiao Yang
Traffic, Volume 17, Issue 5: 554-564, Abstract
An optical method is developed to measure the exocytosis rate of plasma membrane or extracellular matrix proteins. In this method, the protein-of-interest is tagged with a green-to-red photoconvertible fluorescent protein; after photoconverting a region-of-interest on the cell surface, exocytosis-dependent and -independent trafficking events are tracked simultaneously for accurate determination of exocytosis rate.

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells
Michelle S. Itano, Marina Bleck, Daniel S. Johnson, and Sanford M. Simon
Traffic, Volume 17, Issue 2: 179-186, Abstract
The trafficking of human immunodeficiency virus (HIV)-1 genomic RNAs from sites of replication in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane is critical for HIV-1 viral replication. We present a broadly accessible microscopy method that captures multiple focal planes simultaneously, enabling high-resolution 3D imaging of a single macromolecule. We show that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus.

LUCID: A Quantitative Assay of ESCRT-Mediated Cargo Sorting into Multivesicular Bodies
Daniel P. Nickerson, and Alexey J. Merz
Traffic, Volume 16, Issue 12: 1318-1329, Abstract
Selective targeting of transmembrane cargo proteins to luminal vesicles at endosomes is a process essential for correct regulation of cell signaling pathways and maintenance of cellular health. We present a convenient, microscale, quantitative assay system that monitors exposure of chimeric luciferase-transmembrane cargoes to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal depends upon luminal delivery of the reporter rather than proteolytic degradation.

Assessing Mitochondrial Movement Within Neurons: Manual Versus Automated Tracking Methods
Helena Bros, Anja Hauser, Friedemann Paul, Raluca Niesner, and Carmen Infante-Duarte
Traffic, Volume 16, Issue 8: 906-917, Abstract
We examined the suitability of automated tools for tracking mitochondria within neurons, compared with manual tracking (lines indicating mitochondrial movements are marked on each micrograph). The results from automated programs differed significantly from those obtained manually, and both correlation and agreement between methods were poor. However, automated tools could detect relative transport alterations induced by experimental interventions. Thus, automated mitochondrial tracking may be suitable for assessing relative differences in transport, but not for providing absolute quantification of mitochondrial dynamics.

Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans
Ophélie Nicolle, Agnès Burel, Gareth Griffiths, Grégoire Michaux, and Irina Kolotuev
Traffic, Volume 16, Issue 8: 893-905, Abstract
The cryo-sectioning procedure developed by Tokuyasu enables efficient protein localization at the ultrastructural level of diverse samples but has proven to be technically difficult for asymmetric samples. Here, we describe a method that enables reproducible cryo-sectioning of Caenorhabditis elegans larvae and embryos. The procedure considerably simplifies manipulation and lateral orientation. In addition to the standard chemical fixation-based procedure, we have improved the hybrid cryo-immobilization–rehydration technique. Ultrastructure preservation was efficient using both approaches, but immuno-electron localization of different C. elegans specific proteins gave better results with the fast-hybrid procedure. Our method can be successfully used to prepare small asymmetric samples for immunogold labeling.

A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases
Torben Mentrup, Robert Häsler, Regina Fluhrer, Paul Saftig, and Bernd Schröder
Traffic, Volume 16, Issue 8: 871-892, Abstract
The intramembrane proteases signal peptide peptidase-like 2a/b (SPPL) liberate intracellular domains (ICDs) from type II transmembrane proteins. We describe a cell-based assay employing β-galactosidase fragment complementation for quantifying the activity of SPPL2 or other intramembrane proteases based on detecting nuclear translocation of released substrate ICDs. In this system, we demonstrate nuclear translocation of CD74-, TNF- and ITM2B-ICDs and characterize SFRP2 as potential transcriptional target of the CD74-ICD. The assay will allow to screen for novel inhibitors and to analyze the regulation of intramembrane proteases.

Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP
Seong-In Hyun, Liliana Maruri-Avidal, and Bernard Moss
Traffic, Volume 16, Issue 7: 787-795, Abstract
The split green fluorescent protein (GFP) system offers advantages for quickly determining topology of intracellular proteins: the S11 tag is similar in length to epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae
Steven M. Markus, Safia Omer, Kaitlyn Baranowski, and Wei-Lih Lee
Traffic, Volume 16, Issue 7: 773-786, Abstract
In budding yeast, integration of fluorescent protein (FP) at the TUB1 locus causes synthetic interactions with bik1Δ or bim1Δ deletion. Here, we show that the synthetic defects are due to uncoupling of the 3′-untranslated sequence from the TUB1 gene upon integration of the FP. We have designed a new set of FP-Tub1-tagging plasmids, with a variety of colors and selection markers, that overcome this issue and are improved compared with older FP-Tub1 plasmids for visualizing spindle and astral microtubules.

High-Resolution Membrane Capacitance Measurements for Studying Endocytosis and Exocytosis in Yeast
Lucia Carrillo, Bayram Cucu, Vera Bandmann, Ulrike Homann, Brigitte Hertel, Stefan Hillmer, Gerhard Thiel, and Adam Bertl
Traffic, Volume 16, Issue 7: 760-772, Abstract
With patch-clamp recording, we detect in yeast protoplasts individual exo- and endocytotic events as discrete steps in membrane capacitance. The high-resolution data show that exo- and endocytotic vesicles undergo, similar to other eukaryotes, permanent and transient fusion/fission with a bias for transient events. The electrical data are a good representation of the membrane dynamics of a growing yeast cell.

SpatTrack: An Imaging Toolbox for Analysis of Vesicle Motility and Distribution in Living Cells
Frederik W. Lund, Maria Louise V. Jensen, Tanja Christensen, Gitte K. Nielsen, Christian W. Heegaard, and Daniel Wüstner
Traffic, Volume 15, Issue 12: 1406-1429, Abstract
The endocytic pathway has been studied by fluorescence microscopy for decades, with increasing sophistication of imaging instrumentation. Reliable analysis of the complex imaging data for characterization of vesicle distribution and dynamics is challenging. We present SpatTrack, an open access program allowing for spatiotemporal characterization of proteins in endosomes and lysosomes (LYSs) from microscopic images. SpatTrack collects a wide range of analysis methods, and is tested on synthetic data and on LYSs containing fluorescent Niemann Pick C2 protein in disease fibroblasts.

Targeting of Viral Capsids to Nuclear Pores in a Cell-Free Reconstitution System
Fenja Anderson, Anca F. Savulescu, Kathrin Rudolph, Julia Schipke, Ilana Cohen, Iosune Ibiricu, Asaf Rotem, Kay Grünewald, Beate Sodeik, and Amnon Harel
Traffic, Volume 15, Issue 11: 1266-1281, Abstract
A novel assay based on nuclei reconstituted in vitro from Xenopus egg extracts was used for studying the interactions between viruses and nuclear pore complexes (NPCs). The targeting of GFP-tagged herpes simplex virus capsids to NPCs was followed by fluorescence and scanning electron microscopy. Surface features of different capsid types were analyzed by electron cryo tomography and compared with their targeting efficiency in the in vitro assay. Capsids exposing outer capsid and inner tegument proteins are targeted preferentially to NPCs.

Immuno- and Correlative Light Microscopy-Electron Tomography Methods for 3D Protein Localization in Yeast
Muriel Mari, Willie J.C. Geerts, and Fulvio Reggiori
Traffic, Volume 15, Issue 10: 1164-1178, Abstract
Three-dimensional reconstructions with nanoscale resolution in combination with protein subcellular localization are a key investigation tool for the molecular dissection of cellular processes. Here we present two complementary methods optimized for the study of yeast ultrastructure. The first is an immuno-electron tomography (IET) procedure, which allows determining protein distribution patterns on reconstructed organelles. The second is a correlative light microscopy-electron tomography method where compartments containing a specific protein that has been localized through a fluorescent signal are resolved in 3D.

Isolation of the Lateral Border Recycling Compartment Using a Diaminobenzidine-Induced Density Shift
David P. Sullivan, Claas Rüffer, and William A. Muller
Traffic, Volume 15, Issue 9: 1016-1029, Abstract
The lateral border recycling compartment (LBRC) is a novel endothelial cell compartment that appears as a multivesicular invagination of the plasma membrane. The LBRC is critical for the movement of leukocytes across the endothelium. Here, we detail the use of a diaminobenzidine-induced density shift for the purification of the LBRC for proteomic analysis. Vimentin and IQGAP1 were identified from LBRC-enriched fractions.

The CryoCapsule: Simplifying Correlative Light to Electron Microscopy
Xavier Heiligenstein, Jérôme Heiligenstein, Cédric Delevoye, Ilse Hurbain, Sabine Bardin, Perrine Paul-Gilloteaux, Lucie Sengmanivong, Gilles Régnier, Jean Salamero, Claude Antony, and Graca Raposo
Traffic, Volume 15, Issue 6: 700-716, Abstract
Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy.

High-Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry
Pei Zhi Cheryl Chia, Yasmin M. Ramdzan, Fiona J. Houghton, Danny M. Hatters, and Paul A. Gleeson
Traffic, Volume 15, Issue 5: 572-582, Abstract
Standard approaches for analysing the intracellular location of membrane cargo involve microscopy-based methods, which have limitations in throughput capacity and acquisition speed. We describe a flow cytometry-based method, which allows 1000s of cells to be analysed per second. We demonstrate that fluorescence pulse-width, a standard parameter of flow cytometers, can be used to determine the location of membrane proteins, to monitor their trafficking and can be exploited to sort cells based on differences in the intracellular localization of membrane proteins.

DropArray™, a Wall-Less 96-Well Plate for Uptake and Immunofluorescence Microscopy, Confirms CD22 Recycles
Gladys S. Ingle, and Suzie J. Scales
Traffic, Volume 15, Issue 3: 255-272, Abstract
Internalization and immunofluorescence in suspension cells is time-consuming compared with that in adherent cells. Here, we demonstrate the utility of the DropArray™ platform, consisting of 96-‘wall-less well’ plates and a gentle plate washer, for internalization, staining and imaging in B-cell lymphomas. We demonstrate that five different anti-CD22 antibodies all colocalize with transferrin at all time-points of internalization in eight B-cell lines, indicative of trafficking via the recycling pathway. Our results confirm recent biochemical data rather than earlier suggestions of lysosomal trafficking.

Dissecting Functions of the Conserved Oligomeric Golgi Tethering Complex Using a Cell-Free Assay
Nathanael P. Cottam, Katherine M. Wilson, Bobby G. Ng, Christian Körner, Hudson H. Freeze, and Daniel Ungar
Traffic, Volume 15, Issue 1: 12-21, Abstract
Intra-Golgi vesicle transport was reconstituted in vitro using fluorescently marked galactosyltransferase as a marker. Microscopic analysis of transport reactions provides the readout, and shows physiological properties of the reconstitution, including protein, energy and temperature dependence. The new assay was used to show antagonistic functions of the two halves of the conserved oligomeric Golgi complex during trans-Golgi vesicle tethering. The assay will be particularly useful to study molecular determinants of tethering, and human congenital glycosylation disorders that affect Golgi trafficking.

Simple and Direct Assembly of Kymographs from Movies Using KYMOMAKER
Kyoko Chiba, Yuki Shimada, Masataka Kinjo, Toshiharu Suzuki, and Seiich Uchida
Traffic, Volume 15, Issue 1: 1-11, Abstract
KYMOMAKER is an open access program that allows cell biologists to easily and directly generate kymographs from movies. This software can automatically extract faint traces; therefore, it is particularly useful for analyzing low levels of fluorescently labeled vesicular cargos in axons. Filters can be applied to remove Brownian motion. Color kymographs can be generated that include the position of the cargo perpendicular to the long axis of the axon, information that is lost in traditional kymographs.

Automated Quantification of the Subcellular Localization of Multicompartment Proteins via Q-SCAn
Nicholas C. Bauer, Anita H. Corbett, and Paul W. Doetsch
Traffic, Volume 14, Issue 12: 1200-1208, Abstract
In eukaryotic cells, proteins can occupy multiple intracellular compartments and move between compartments to fulfill critical biological functions. Unfortunately, no methods have been developed to robustly measure the distribution of a protein among compartments. To address this need, we have developed an automated method termed quantitative subcellular compartmentalization analysis (Q-SCAn). Q-SCAn is a quantitative analytical tool for providing broader and more detailed analysis of the localization of multicompartment proteins as compared to the currently available approaches.

Cryo-Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates
Thomas Schmiedinger, Georg F. Vogel, Oliver Eiter, Kristian Pfaller, Walter A. Kaufmann, Angelika Flörl, Karin Gutleben, Sabine Schönherr, Barbara Witting, Thomas W. Lechleitner, Hannes-L. Ebner, Thomas Seppi, and Michael W. Hess
Traffic, Volume 14, Issue 8: 886–894, Abstract
Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling ...

A Novel Assay for Measurement of Membrane-Protein Surface Expression using a β-lactamase Reporter
Vincent M. Lam, Pieter Beerepoot, Stephane Angers, and Ali Salahpour
Traffic, Volume 14, Issue 7: 778–784, Abstract
The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one-step and potentially high-throughput assay, using the β-lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins ...

Analysis of ER Resident Proteins in Saccharomyces cerevisiae: Implementation of H/KDEL Retrieval Sequences
Carissa L. Young, David L. Raden, and Anne S. Robinson
Traffic, Volume 14, Issue 4: 365–381, Abstract
An elaborate quality control system regulates endoplasmic reticulum (ER) homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high-throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of polymerase chain reaction (PCR)-based modules including codon-optimized epitopes and fluorescent protein (FP) variants complete with C-terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins ...

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response
James G. Burchfield, Jinling Lu, Daniel J. Fazakerley, Shi-Xiong Tan, Yvonne Ng, Katarina Mele, Michael J. Buckley, Weiping Han, William E. Hughes, and David E. James
Traffic, Volume 14, Issue 3: 259–273, Abstract
Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles ...

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.