Springer protocols feed by Biochemistry 
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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.Determining the Role of Rab7 in Constitutive and Ligand-Mediated Epidermal Growth Factor Receptor Endocytic Trafficking Using Single Cell Assays
RAB proteins are essential for the proper membrane trafficking of a number of proteins. Each of the 60+ RABs that have been identified has a discrete role in coordinating the movement from one subcellular compartment to another. Early attempts at deciphering the roles of individual RAB proteins relied heavily on the use of activating and/or dominant negative mutants (Ceresa, Histol Histopathol 21:987–993, 2006). However, overexpression of mutant proteins can lead to misleading information; high levels of expression can drive low affinity (and possibly, nonphysiological) interactions as well as cause mislocalization. ...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Differential Effects of Overexpression of Rab5 and Rab22 on Autophagy in PC12 Cells with or without NGF
Macroautophagy selectively recycles damaged or unneeded proteins and organelles by degradation via targeting to the autophagosome. The following method seeks to identify candidate Rab GTPases that likely modulate autophagy in PC12 cells during nerve growth factor (NGF) starvation. This microscopy-based assay is a single cell-based quantification of the presence of autophagosomes by fluorescently labeled markers in response to the overexpression of Rabs and mutants in the presence or absence of NGF. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Role of the Rab5 Guanine Nucleotide Exchange Factor, Rme-6, in the Regulation of Clathrin-Coated Vesicle Uncoating
Clathrin-coated pits are major ports of entry into eukaryotic cells. Following scission of clathrin-coated pits to form coated vesicles, the peripheral membrane proteins that form the coat need to be removed. Here we describe an assay that provides a measure of the extent of clathrin coat uncoating in intact cells. This assay has been used to investigate rab5-dependent modulation of uncoating. Specifically, it has been used to identify functional differences between rab5 guanine nucleotide exchange factors. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Identification of the Rab5 Binding Site in p11β: Assays for PI3Kβ Binding to Rab5
Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110β, and p110δ), p110β has unique properties. Unlike the other family members, p110β directly binds to Gβγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110β binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for ...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Determination of Rab5 Activity in the Cell by Effector Pull-Down Assay
Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 tha...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Methods for Analysis of AP-3/Rabin4’ in Regulation of Lysosome Distribution
The position of lysosomes in the cytoplasm is important for their ability to fuse with the plasma membrane and release of proteases that are involved in tissue remodeling. Motor-directed bidirectional transport along microtubules is a critical process determining the distribution of lysosomes. How lysosomes are tethered to microtubules is incompletely understood, but a role for small GTPases of rab and arl families has been documented. We recently found that the rab5 and rab4 effector rabip4′ interacts with the adaptor complex AP-3 in a rab4-dependent manner on tubular endosomes. We here describe the assays that led ...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Assay of Rab17 and Its Guanine Nucleotide Exchange Factor Rabex-5 in the Dendrites of Hippocampal Neurons
Neurons are functionally and morphologically compartmentalized into axons and dendrites, and the localization of specific proteins within these compartments is critical to the proper formation of neuronal networks, which includes neurite morphogenesis and synapse formation. The small GTPase Rab17 is specifically localized in dendrites and is not found in axons, and it regulates the dendrite morphogenesis and postsynaptic development of mouse hippocampal neurons. However, the spatiotemporal regulation of Rab17 is poorly understood. We recently identified Rabex-5, originally described as a Rab5-guanine nucleotide exchange fa...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Rab Family of GTPases
Rab proteins represent the largest branch of the Ras-like small GTPase superfamily and there are 66 Rab genes in the human genome. They alternate between GTP- and GDP-bound states, which are facilitated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and function as molecular switches in regulation of intracellular membrane trafficking in all eukaryotic cells. Each Rab targets to an organelle and specify a transport step along exocytic, endocytic, and recycling pathways as well as the crosstalk between these pathways. Through interactions with multiple effectors temporally, a Rab can co...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Analysis of Connecdenn 1–3 (DENN1A-C) GEF Activity for Rab35
Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that direct...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Measurement of Rab35 Activity with the GTP-Rab35 Trapper RBD35
Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 t...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Characterization of the Role Rab25 in Energy Metabolism and Cancer Using Extracellular Flux Analysis and Material Balance
Rab25, by altering trafficking of critical cellular resources, influences cell metabolism and survival during stress conditions. Overall, perturbations in the vesicular trafficking machinery change cellular bioenergetics that can be directly measured in real time as Oxygen Consumption Rate, OCR (mitochondrial respiration) and Extracellular Acidification Rate, ECAR (glycolysis) by an extracellular flux analyzer (XF96, Seahorse Biosciences, MA). Additionally, overall turnover of glucose, lactate, as well as glutamine and glutamate can be measured biochemically using the YSI2900 Biochemistry Analyzer (YSI Incorporated, Life S...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
In Vitro and In Vivo Characterization of the Rab11-GAP Activity of Drosophila Evi5
Small GTPases of the Rab family are master regulators of vesicular trafficking. As such, they control the spatial distribution of various proteins, including proteins involved in cell signaling and the regulation of cell polarity. Misregulation of Rab proteins is associated with a large array of diseases. Surprisingly, the target of some key regulators of Rab proteins, including many GTPase-activating protein (GAP) is still unknown. Identifying the target of a specific GAP requires the combination of both in vitro and in vivo experiments to avoid any misinterpretation. Here is described the methodology we used to character...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
3D Time-Lapse Analysis of Rab11/FIP5 Complex: Spatiotemporal Dynamics During Apical Lumen Formation
Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells. (Sour...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Selective Visualization of GLUT4 Storage Vesicles and Associated Rab Proteins Using IRAP-pHluorin
Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
Rab Antibody Characterization: Comparison of Rab14 Antibodies
Rab14 functions in the endocytic recycling pathway, having been implicated in the trafficking of the ADAM10 protease, GLUT4, and components of cell–cell junctions to the plasma membrane. It localizes predominantly to endocytic membranes with a pool also found on trans-Golgi network (TGN) membranes, and is most closely related to the Rab11 subfamily of GTPases. Certain intracellular bacteria such as Legionella pneumophila, Chlamydia trachomatis, and Salmonella enterica utilize Rab14 to promote their maturation and replication. Furthermore, the HIV envelope glycoprotein complex subverts the function of Rab14, and its e...
Source: Springer protocols feed by Biochemistry - March 26, 2015 Category: Biochemistry Source Type: news
