Springer protocols feed by Cell Biology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Application of RNAi Technology and Fluorescent Protein Markers to Study Membrane Traffic in C. elegans
RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenome...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Chemical Genomics: Characterizing Target Pathways for Bioactive Compounds Using the Endomembrane Trafficking Network
The plant endomembrane trafficking system is a highly complex set of processes. This complexity presents a challenge for its study. Classical plant genetics often struggles with loss-of-function lethality and gene redundancy. Chemical genomics allows overcoming many of these issues by using small molecules of natural or synthetic origin to inhibit specific trafficking proteins thereby affecting the processes in a tunable and reversible manner. Bioactive chemicals identified by high-throughput phenotype screens must be characterized in detail starting with understanding of the specific trafficking pathways affected. Here, w...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Use of Kaede and Kikume Green–Red Fusions for Live Cell Imaging of G Protein-Coupled Receptors
The fusion of fluorescent proteins to G protein-coupled receptors (GPCRs) is an important tool to study, e.g., trafficking and protein interactions of these important drug targets. In the past, the green fluorescent protein and its derivatives have been widely used as fluorescent tags. More recently, it was reported that photoconvertible fluorescent proteins (PCFPs) such as Kaede or Kikume green–red protein could also be used as fluorescent tags for GPCRs. These proteins have the obvious advantage that their fluorescence can be switched once the GPCR of interest has reached a specific subcellular compartment. Here, w...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Probabilistic Density Maps to Study the Spatial Organization of Endocytosis
Despite a large body of publications on endocytosis, only a few studies have focused on its spatial organization. To study how endocytosis is related to distinct cellular sites, we combine cell normalization by the “micropatterning technique” with the quantification of spatial organization by “probabilistic density mapping.” Micropatterns of extracellular matrix proteins impose adhesive and non-adhesive areas to cultured cells and allow the control of adhesion geometry, shape, and cell organization. Probabilistic density maps provide a visual summary for 3D localization of the structures of interest...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
VIS2FIX: Rapid Chemical Fixation of Vitreous Sections for Immuno-Electron Microscopy
Immuno-electron microscopy uniquely allows high-resolution localization of proteins in their cellular context. Usually, affinity labeling with an electron-dense marker, e.g., small gold particles, is performed on sections of chemically fixed cells or tissues. In this chapter, we describe two novel protocols, the VIS2FIX methods, for chemical fixation of sections of cryo-immobilized biological samples. This method involves production of thin sections of high-pressure frozen cells that are statically adhered to a TEM grid. Subsequent steps involve chemical fixation of the samples by either the VIS2FIXH (“H” for &...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
A Novel Permeabilization Protocol to Obtain Intracellular 3D Immunolabeling for Electron Tomography
Electron tomography (ET) is a very important high-resolution tool for 3D imaging in cell biology. By combining the technique with immunolabeling, ET can provide essential insights into both cellular architecture and dynamics. We recently developed a protocol to achieve 3D immunolabeling of intracellular antigens without the need for uncontrolled permeabilization steps that cause random, extensive cell membrane disruption. Here we describe this novel method based on well-controlled permeabilization by targeted laser cell perforation. Mechanical permeabilization of the plasma membrane can be applied at specific sites without...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Nanocones to Study Initial Steps of Endocytosis
Vesicle endocytosis at the plasma membrane is associated with a precise temporal choreography in the recruitment of cytosolic proteins that sense, generate, or stabilize locally curved membrane regions. To dissect the role of membrane curvature sensing from other co-occurring events during the initial steps of endocytosis, we developed a method to artificially induce nanoscale deformations of the PM in living cells that is based on cone-shaped nanostructures (i.e., Nanocones). When cultured on Nanocones, cells create stable inward plasma membrane deformations to which curvature-sensing proteins are recruited. Here, we prov...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Real-Time Investigation of Plasma Membrane Deformation and Fusion Pore Expansion Using Polarized Total Internal Reflection Fluorescence Microscopy
Polarized Total Internal Reflection Fluorescence Microscopy (pTIRFM) allows for real-time observation of plasma membrane deformations. The technique provides insights into the dynamics of biological processes requiring rapid and localized changes in membrane shape. Such processes include exocytosis, endocytosis, cytokinesis, and cell motility. In this chapter, we describe how to implement a polarization-based TIRF imaging system to monitor exocytosis in adrenal chromaffin cells. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
A Novel Pair of Split Venus Fragments to Detect Protein–Protein Interactions by In Vitro and In Vivo Bimolecular Fluorescence Complementation Assays
Protein–protein interactions are critical components of almost every cellular process. The bimolecular fluorescence complementation (BiFC) method has been used to detect protein–protein interactions in both living cells and cell-free systems. The BiFC method is based on the principle that a fluorescent protein is reassembled from its two complementary non-fluorescent fragments when an interaction occurs between two proteins, each one fused to each fragment. In vivo and in vitro BiFC assays, which use a new pair of split Venus fragments composed of VN210 (amino acids 1–210) and VC210 (amino acids 210&ndash...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
A Method to Rapidly Induce Organelle-Specific Molecular Activities and Membrane Tethering
In this chapter we describe a technique for rapid protein targeting to individual intracellular organelles. This method enables a real-time imaging-based study of cellular behavior in response to controlled induction of signaling events in a specifically targeted cellular compartment. We provide rationales and a step-by-step protocol for probe design and imaging of protein targeting along with two different applications of this technique. One application involves organelle-specific activation of small GTPases, while the other application involves membrane tethering of two different organelles. In the former case, we activa...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Use of Transmembrane FRET to Investigate the Internalization of Glycosylated Proteins
The importance of glycans in various cellular events, especially intracellular and intercellular trafficking of proteins, has been reported in numerous studies. Here, we present a novel method to monitor endocytosis of proteins of interest bearing a specific glycan modification. Using a fluorescence resonance energy transfer technique, we investigated the role of glycan structure on the internalization of insulin-responsive glucose transporter GLUT4. We found that sialylated glycoforms of GFP-tagged GLUT4 appear to be internalized more slowly than non-sialylated GLUT4 upon insulin removal. This novel glycan imaging tool al...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Synchronization of Secretory Cargos Trafficking in Populations of Cells
In mammalian cells, secretory proteins are transported to their destination compartment via the secretory pathway. Cargos start their journey in the endoplasmic reticulum and then reach the Golgi complex where they are processed and sorted to be delivered to their target intracellular compartment. To analyze and visualize this flow of proteins, one needs to synchronize their transport. We recently developed the retention using selective hooks (RUSH) system enabling simultaneous and synchronous release of secretory cargos from the endoplasmic reticulum in a population of cells. Here, we describe how to subclone the gene cod...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Analysis of Protein Dynamics with Tandem Fluorescent Protein Timers
Fluorescent timers (FTs) are fluorescent proteins that change color with time. FTs can be used as tags to follow protein dynamics in living cells. Recently we described a novel class of FTs called tandem fluorescent protein timers (tFTs). Each tFT is a tandem fusion of two different conventional fluorescent proteins having distinct kinetics of fluorophore maturation. tFTs suitable for studying protein dynamics on different scales can be generated from a broad range of commonly used fluorescent proteins. Here we describe how to establish new tFTs and consider potential pitfalls. We detail a protocol for quantitative fluores...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
FlAsH-PALM: Super-resolution Pointillist Imaging with FlAsH-Tetracysteine Labeling
Super-resolution light microscopy including pointillist methods based on single molecule localization (e.g., PALM/STORM) allow to image protein structures much smaller than the diffraction limit (200–300 nm). However, commonly used labeling strategies such as antibodies or protein fusions have several important drawbacks, including the risk to alter the function or distribution of the imaged proteins. We recently demonstrated that pointillist imaging can be performed using the alternative labeling technique known as FlAsH, which better preserves protein function, is compatible with live cell imaging, and may help rea...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
SNAP-Tag to Monitor Trafficking of Membrane Proteins in Polarized Epithelial Cells
In order to understand the mechanisms through which apical and basolateral membrane proteins achieve their subcellular distributions in polarized epithelial cells, it is critical to develop techniques that permit the selective observation of newly synthesized populations of these proteins. The SNAP tag system permits the detection and visualization of distinct spatially and temporally defined cohorts of tagged proteins. Thus, this technique is especially well suited to studying the trafficking routes pursued by newly synthesized proteins. The SNAP tag can be applied in the setting of fixed or live cell fluorescence microsc...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
