Current Protocols in Cell Biology最新文献

Mass spectrometry-based proteomic technology experienced remarkable advancement in the past decades. However, their application was still hampered by the complexity of sample preparation. Conventional strategies for sample preparation incorporate multiple time-consuming steps, including cell lysis, protein extraction, protease cleavage, and desalting. Thus, we explored a simplified method (the cell-absorb method) during which living cells were absorbed into vacuum-dried polyacrylamide gel and directly digested in gel into peptides for subsequent LC-MS/MS analysis. As a consequence, both of the steps for cell lysis and protein extraction involved in traditional protocol were skipped. In addition to the decrease in time, more proteins were identified. Indeed, 3022 proteins were identified by the cell-absorb method. Meanwhile, only 2642 and 2420 proteins were identified by the classical SDS-PAGE based method and the reported gel absorption-based method, respectively. The cell-absorb method exhibited apparent advantage in terms of the depth of proteome coverage. Furthermore, the number of proteins identified show excellent reproducibility with a CV (coefficient of variation) of 0.03 among three replicates using the cell-absorb method. These advantages suggest that cell-absorb method is a promising choice for mapping the whole proteome of cells. © 2018 by John Wiley & Sons, Inc.

This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described. Curr. Protoc. Cell Biol. 60:21.7.1-21.7.29. © 2013 by John Wiley & Sons, Inc.

Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure sequesters a portion of the cytoplasm by the formation of a double-membrane compartment called the autophagosome. The autophagosome then delivers the cargo to the lysosome to complete degradation. The molecular mechanism accounting for the generation of the autophagic membrane is a longstanding question. Here, a cell-free approach that has been established to understand the mechanism of early autophagic membrane generation is described. This system has provided insight into the membrane source of the autophagosome, the early protein-membrane associations, and the membrane remodeling that generates the autophagosomal precursors. The cell-free assay, in combination with other established approaches (e.g., cell imaging), will facilitate a deeper understanding of the mechanism of autophagy. © 2017 by John Wiley & Sons, Inc.

Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF-based single-filament imaging techniques have proven instrumental in uncovering mechanisms of actin regulation. In this unit, novel single-filament approaches using microfluidics-assisted TIRF imaging are described. These methods can be used to grow anchored actin filaments aligned in a flow, thus making the analysis much easier as compared to open flow cell approaches. The microfluidic nature of the system also enables rapid change of biochemical conditions and allows simultaneous imaging of a large number of actin filaments. Support protocols for preparing microfluidic chambers and purifying spectrin-actin seeds used for nucleating anchored filaments are also described. © 2017 by John Wiley & Sons, Inc.

Almost all types of cells secrete extracellular vesicles (EVs), including exosomes and microvesicles. EVs carry various proteins, lipids, mRNAs, and microRNAs, and may participate in many aspects of physiological and pathophysiological processes. Various studies are currently being conducted to develop therapeutic and diagnostic methods targeting or utilizing EVs. Therefore, developing ideal methods for isolating and quantifying EVs is an active area of research. EVs express phosphatidylserine on their outer lipid bilayer. This unit describes an affinity-based method for isolating EVs using TIM4, which binds phosphatidylserine in a specific and calcium-dependent manner. EVs captured by TIM4 can be easily released by addition of a chelating agent, or can be retained for quantification by ELISA or flow cytometry. These methods enable the isolation of highly purified EVs and the sensitive quantification of EVs, which will accelerate EV research beyond what has been achievable with conventional methods. © 2017 by John Wiley & Sons, Inc.

The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene expression responses in individual cells. For this reason, it is desirable to simultaneously image signal transduction and gene expression events in the same cells. Here, we describe a method that combines fluorescence and bioluminescence microscopy to image protein kinase C (PKC) translocation from the cytosol to the plasma membrane and the expression of nuclear factor kappa-light polypeptide B (NF-κB)-regulated genes. © 2017 by John Wiley & Sons, Inc.

Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments. Apoptosis of breast cancer cells in 2D culture, spheroids, and in vivo murine breast tumor xenografts in response to a variety of genetic and pharmacologic methods implicated in apoptosis of cancer cells was studied. This approach for quantifying apoptosis of cancer cells is based on caspase-3 activity at single-cell resolution using FLIM. © 2017 by John Wiley & Sons, Inc.

This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein–coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases: (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live-cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc.