Imaging Platforms for Measurement of Membrane Trafficking
Introduction
Membrane traffic enables cells to distribute proteins, lipids, and carbohydrates between membrane compartments and is thus vital for many cellular processes such as cell growth, homeostasis, and differentiation. Genetic and biochemical approaches have been very efficiently applied in the past to identify and characterize individual molecular components involved in the regulation of membrane traffic in the secretory and endocytic pathways. Central to many of these studies has been the reconstitution of the particular transport step of interest in vitro using purified components. Although this has led to an enormous body of information on how membrane traffic is organized at the molecular level, such simplified in vitro systems are lacking important regulatory elements relating to the spatial organization that occurs in living cells. More recently, systematic approaches, such as organelle proteomics or yeast two hybrid screening, have attempted to identify structural and regulatory components of membrane traffic with the goal of reaching a more complete description of its molecular regulation (see for example Bell 2001, Calero 2002, Monier 2002). However, despite their great potential, these techniques have limitations, not least of which is their lack of demonstrating a functional involvement of the molecules identified in the particular trafficking step under investigation.
Functional microscope‐based assays in intact living cells with the potential for large‐scale analyses have been recently developed and applied to problems in membrane trafficking (Ghosh et al., 2000; Liebel 2003, Pelkmans 2005, Starkuviene 2004). These techniques provide single cell or even subcellular resolution. They promise, in combination with genome‐wide RNA interference (RNAi, Elbashir et al., 2001) or over‐expression strategies (Starkuviene et al., 2004), to help to reveal comprehensively the regulatory networks underlying membrane traffic in intact cells. Here we describe such assays used to quantitatively monitor secretory transport to the plasma membrane and the subcellular kinetics of transport carriers in time‐lapse series.
Section snippets
Methodology
The problem of performing functional microscope‐based assays to address membrane trafficking on a large set of proteins in cells is presently still a challenge. It requires automation and coordination of various steps such as sample preparation, image acquisition, the handling and analysis of large sets of image data, and the integration of the results with existing knowledge provided by bioinformatic databases. A number of automated microscope‐based image acquisition systems are already
Comments
The stained samples are stored at 4° either embedded in Mowiol or in PBS solution containing 0.01% azide after a brief post‐staining fixation of the samples with 3% paraformaldehyde for 2 min. The initial cell density after plating of the cells on LabTek dishes is critical for the success of the experiments and needs to be adjusted when different cell types are used. If cell densities are too high, the efficiency of adenovirus transfection decreases considerably and the number of cells
Comments
It is important to test at the beginning of the image acquisition that images are not saturated as this distorts the quantification of fluorescence. Therefore, to adjust the systems parameters appropriately, the operator should take a few test images from different areas on the sample and check the images for saturation. Parameters should then be adjusted such that even the brightest signal can still be acquired at nonsaturating conditions.
Figure 2G shows the results of an example analysis of
Comments
The speed of image acquisition is a critical parameter for the success of the time‐space plot approach and needs to occur at a frequency high enough to result in a continuous trajectory in the projection image (refer to Step 4 in the protocol described earlier). The macro “read velocities from tsp” calculates a number of parameters such as, the entire length of the trajectory taken by the structure under view, average, and instantaneous speed. The speed is returned in units of pixels/frame,
Summary and Perspectives
We described two methods to quantify membrane transport in single cells using automated fluorescence microscopy and image analysis. The assay to measure the plasma membrane transport of ts‐O45‐G is robust and has already been used in systematic analyses to identify new proteins involved in the regulation of the secretory pathway (e.g., Starkuviene et al., 2004). Applying this approach to genome‐wide siRNA screens in intact cells, for example, may help to reveal the machinery and interaction
References (10)
- et al.
Proteomics characterization of abundant Golgi membrane proteins
J. Biol. Chem.
(2001) - et al.
Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors
FEBS Lett.
(2002) - et al.
A microscope‐based screening platform for large‐scale functional protein analysis in intact cells
FEBS Lett.
(2003) - et al.
Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein
Cell
(1980) - et al.
Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells
Nature
(2001)
Cited by (13)
Quantitative analysis of organelle abundance, morphology and dynamics
2011, Current Opinion in BiotechnologyCitation Excerpt :This approach can be used to monitor autophagic degradation of any organelle that is tagged with a GFP fusion protein. Pepperkok et al. [19] was one of the first who presented an example of a method to determine the kinetics of vesicular transport in mammalian cells. In this example, vesicle transport from the ER via the Golgi apparatus to the plasmamembrane was measured.
2D-GolgiTrack—a semi-automated tracking system to quantify morphological changes and dynamics of the Golgi apparatus and Golgi-derived membrane tubules
2022, Medical and Biological Engineering and ComputingCoordinated integrin activation by actin-dependent force during T-cell migration
2016, Nature Communications