Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

University of Kentucky

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in . In previous studies, we created a mutant AcrB, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB in  cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB-YPet in live  cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter.

This paper presented the dataset of correction parameters used in the determination of the energy transfer efficiencies from the spectrum-based fluorescence resonance energy transfer (FRET) measurement in a trimeric membrane protein AcrB. The cyan fluorescent protein (CFP) and yellow fluorescent protein (YPet) were used as the donor and acceptor, respectively. Two AcrB fusion proteins were constructed, AcrB-CFP and AcrB-YPet. The proteins were co-expressed in  cells, and energy transfer efficiency were determined in live cells. To obtain reliable energy transfer data, a complete set of correction parameters need to be first determined to accommodate for factors such as background fluorescence and spectra overlap. This paper described the methodology and determination of the correction factors, which are useful data and reference points for researchers working on fluorescence measurement of membrane protein complexes in live bacteria cells. Further interpretation and discussion of these data can be found in "Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter" (Wang et al., in press).

Data on spectrum-based fluorescence resonance energy transfer measurement of  multidrug transporter AcrB.

The plant endomembrane system facilitates the transport of polysaccharides, associated enzymes, and glycoproteins through its dynamic pathways. Although enzymes involved in cell wall biosynthesis have been identified, little is known about the endomembrane-based transport of glycan components. This is partially attributed to technical challenges in biochemically determining polysaccharide cargo in specific vesicles. Here, we introduce a hybrid approach addressing this limitation. By combining vesicle isolation with a large-scale carbohydrate antibody arraying technique, we charted an initial large-scale map describing the glycome profile of the SYNTAXIN OF PLANTS61 (SYP61) -Golgi network compartment in Arabidopsis (). A library of antibodies recognizing specific noncellulosic carbohydrate epitopes allowed us to identify a range of diverse glycans, including pectins, xyloglucans (XyGs), and arabinogalactan proteins in isolated vesicles. Changes in XyG- and pectin-specific epitopes in the cell wall of an Arabidopsis  mutant corroborate our findings. Our data provide evidence that SYP61 vesicles are involved in the transport and deposition of structural polysaccharides and glycoproteins. Adaptation of our methodology can enable studies characterizing the glycome profiles of various vesicle populations in plant and animal systems and their respective roles in glycan transport defined by subcellular markers, developmental stages, or environmental stimuli.

A Hybrid Approach Enabling Large-Scale Glycomic Analysis of Post-Golgi Vesicles Reveals a Transport Route for Polysaccharides.

The dynamic -Golgi network/early endosome (TGN/EE) facilitates cargo sorting and trafficking and plays a vital role in plant development and environmental response. Transport protein particles (TRAPPs) are multi-protein complexes acting as guanine nucleotide exchange factors and possibly as tethers, regulating intracellular trafficking. TRAPPs are essential in all eukaryotic cells and are implicated in a number of human diseases. It has been proposed that they also play crucial roles in plants; however, our current knowledge about the structure and function of plant TRAPPs is very limited. Here, we identified and characterized AtTRAPPC11/RESPONSE TO OLIGOGALACTURONIDE2 (AtTRAPPC11/ROG2), a TGN/EE-associated, evolutionarily conserved TRAPP protein in Arabidopsis (). AtTRAPPC11/ROG2 regulates TGN integrity, as evidenced by altered TGN/EE association of several residents, including SYNTAXIN OF PLANTS61, and altered vesicle morphology in  mutants. Furthermore, endocytic traffic and brefeldin A body formation are perturbed in , suggesting a role for AtTRAPPC11/ROG2 in regulation of endosomal function. Proteomic analysis showed that AtTRAPPC11/ROG2 defines a hitherto uncharacterized TRAPPIII complex in plants. In addition,  mutants are hypersensitive to salinity, indicating an undescribed role of TRAPPs in stress responses. Overall, our study illustrates the plasticity of the endomembrane system through TRAPP protein functions and opens new avenues to explore this dynamic network.

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant -Golgi Network/Early Endosome Organization and Function.

We present the application of multiphoton  fluorescence correlation spectroscopy (FCS) of fluorescent nanoparticles for the measurement of cerebral blood flow with excellent spatial and temporal resolution. Through the detection of single nanoparticles within the complex vessel architecture of a live mouse, this new approach enables the quantification of nanoparticle dynamics occurring within the vasculature along with simultaneous measurements of blood flow properties in the brain. In addition to providing high resolution blood flow measurements, this approach enables real-time quantification of nanoparticle concentration, degradation, and transport. This method is capable of quantifying flow rates at each pixel with submicron resolution to enable monitoring of dynamic changes in flow rates in response to changes in the animal's physiological condition. Scanning the excitation beam using FCS provides pixel by pixel mapping of flow rates with subvessel resolution across capillaries 300 μm deep in the brains of mice.

 Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow.

The dynamic endomembrane system facilitates sorting and transport of diverse cargo. Therefore, it is crucial for plant growth and development. Vesicle proteomic studies have made substantial progress in recent years. In contrast, much less is known about the identity of vesicle compartments that mediate the transport of polysaccharides to and from the plasma membrane and the types of sugars they selectively transport. In this chapter, we provide a detailed description of the protocol used for the elucidation of the SYP61 vesicle population glycome. Our methodology can be easily adapted to perform glycomic studies of a broad variety of plant cell vesicle populations defined via subcellular markers or different treatments.

Thomas Wilkop

Department of Physiology,
UK Light Microscopy Core

University of Kentucky

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Thomas Wilkop

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology,UK Light Microscopy Core

University of Kentucky

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Department of Physiology,
UK Light Microscopy Core