Cellular logistics最新文献

There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies.

The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.

We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to "classify" entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.

Small GTPases of the Ras superfamily are important regulators of many cellular functions, including signal transduction, cytoskeleton assembly, metabolic regulation, organelle biogenesis and intracellular transport. Most GTPases act as binary switches, being "on" in the active, GTP-bound state and "off" in the inactive, GDP-bound state, and cycle between the two states with the aid of accessory proteins, referred to as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). This review will focus on the ADP-ribosylation factors (Arfs), a family of G-proteins that are essential regulators of carrier vesicle formation during vesicular transport. As for most other GTPases, the Arfs themselves are vastly outnumbered by the proteins that regulate them, and a major focus in the field has been to define the functional relationships between individual GEFs and GAPs and their substrates at the cellular level. Over the years, a variety of methods have been developed to measure GTPase activation in vitro and in vivo. In vitro analysis will be discussed in the accompanying article by Randazzo and colleagues. Here we will focus on cell- and tissue-based assays and their advantages/disadvantages relative to cell-free systems.

Cullin-RING-ligases (CRLs) comprise the largest class of multisubunit E3 ubiquitin ligases, which regulate a broad range of cellular processes. Cullin3 (Cul3) recently emerged as an important regulator of intracellular trafficking, in particular secretion and endosome maturation. Here we summarize and discuss possible functions and substrates of Cul3 in the endocytic system.

Retrograde trafficking mediates the transport of endocytic membranes from endosomes to the trans-Golgi network (TGN). Dysregulation of these pathways can result in multiple ailments, including late-onset Alzheimer disease. One of the key retrograde transport regulators, the retromer complex, is tightly controlled by many factors, including the C-terminal Eps15 homology domain (EHD) proteins. This mini-review focuses on recent findings and discusses the regulation of the retromer complex by EHD proteins and the novel EHD1 interaction partner, Rabankyrin-5 (Rank-5).

Exchange factors are enzymes that catalyze the exchange of GTP for GDP on guanine nucleotide binding proteins. Progress in understanding the molecular basis of action and the cellular functions of these enzymes has largely come from structural determinations (e.g., crystal structures) and studying effects on cells when expression levels of the exchange factors are perturbed or mutated exchange factors are expressed. Proportionally little effort has been expended on studying the kinetics of exchange; however, reaction rates are central to understanding enzymes. Here, we discuss the importance of kinetic analysis of exchange factors for guanine nucleotide binding proteins, with a focus on ADP-ribosylation factor (Arf) and heterotrimeric G proteins, for providing unique insights into molecular mechanisms and regulation as well as how kinetic analyses are used to complement other approaches.