Relocation sensors to quantify signaling dynamics in live single cells

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Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.     

A sensitive switch for visualizing natural gene silencing in single cells

RNA interference is a natural gene expression silencing system that appears throughout the tree of life. As the list of cellular processes linked to RNAi grows, so does the demand for tools to accurately measure RNAi dynamics in living cells. We engineered a synthetic RNAi sensor that converts this negative regulatory signal into a positive output in living mammalian cells thereby allowing increased sensitivity and activation. Furthermore, the circuit's modular design allows potentially any microRNA of interest to be detected. We demonstrated that the circuit responds to an artificial microRNA and becomes activated when the RNAi target is replaced by a natural microRNA target (miR-34) in U2OS osteosarcoma cells. Our studies extend the application of rationally designed synthetic switches to RNAi, providing a sensitive way to visualize the dynamics of RNAi activity rather than just the presence of miRNA molecules.     

Protein kinase activities in the growth cycle of suspension cultured plant cells

In batch suspension cultures of Nicotiana tabacum and Datura innoxia protein kinase activity extracted from the whole cells and assayed with casein as substrate was followed over the growth cycle. In one case (N. tabacum) the activity was also determined in the nuclei preparation obtained from the suspension cultured cells. Immediately at the onset of the growth curve the protein kinase level increases strongly and reaches a maximum value at the early phase of proliferation; the enzyme level from the nuclei is slightly delayed. A comparison with protein synthesis shows that protein kinases are among the first proteins synthesized in the growth cycle. Chromatographic separation of the enzymes contributing to the total activity revealed that both in the extract of whole cells and in the nuclei two enzyme species are present. Their time course is similar to that of the total protein kinase level, although the activity corresponding to the enzyme with the higher molecular weight in the case of the whole cell extract is slightly delayed. The possible significance of protein phosphorylation in the growth cycle is discussed.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

DNA markers in plant improvement: an overview

The progress made in DNA marker technology has been tremendous and exciting. DNA markers have provided valuable tools in various analyses ranging from phylogenetic analysis to the positional cloning of genes. The development of high-density molecular maps which has been facilitated by PCR-based markers, have made the mapping and tagging of almost any trait possible. Marker-assisted selection has the potential to deploy favorable gene combinations for disease control. Comparative studies between incompatible species using these markers has resulted in synteny maps which are useful not only in predicting genome organization and evolution but also have practical application in plant breeding. DNA marker technology has found application in fingerprinting genotypes, in determining seed purity, in systematic sampling of germplasm, and in phylogenetic analysis. This review discusses the use of this technology for the genetic improvement of plants.     

Protein dynamics in living cells

A protein's structure is most often used to explain its function, but function also depends on dynamics. To date, protein dynamics have been studied only in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein dynamics? The answer will help in assessing the biological significance of the NMR-derived dynamics data collected to date. We investigated fast protein dynamics inside living Escherichia coli by using in-cell NMR. The backbone dynamics of apocytochrome b5 were quantified using {1H}-15N nuclear Overhauser effect (nOe) measurements, which characterize motions on the pico- to nanosecond time scale. The overall trend of backbone dynamics remains the same in cells. Some of the nOe values differ, but most of the differences track the increased intracellular viscosity rather than a change in dynamics. Therefore, it appears that dilute solution steady-state {1H}-15N nOe measurements provide biologically relevant information about pico- to nanosecond backbone motion in proteins.     

Protein kinase CK2: a new view of an old molecular complex

Protein kinase CK2 (formerly known as casein kinase II) has been viewed traditionally as a stable heterotetrameric complex, but new analytical techniques are bringing a different picture into focus. The transient nature of this complex has been highlighted by the elucidation of its structure. Furthermore, analysis of the spatiotemporal organization of individual CK2 subunits in living cells has shown that they are dynamic and that they integrate into different multimolecular assemblies. These new studies give an additional dimension to the challenge of determining the cellular regulation of this protein kinase.     

Protein trafficking in plant cells

The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.     

Media for cultivation of animal cells: an overview

The increasing interest in products from animal cells has caused an extensive research effort towards development of media for cell cultivation.The basic components in the media used for cultivation of animal cells vary depending upon the characters of the cells and the cultivation method. Basic components consist of an energy source, nitrogen source, vitamins, fats and fatty soluble components, inorganic salts, nucleic acid precursors, antibiotics, oxygen, pH buffering systems, hormones, growth factors and serum. Extensive efforts are directed towards developing serum-free or chemically defined media. Among the serum substitutes is a long list of hormones and growth factors.     

Mobile factories: Golgi dynamics in plant cells

The plant Golgi apparatus plays a central role in the synthesis of cell wall material and the modification and sorting of proteins destined for the cell surface and vacuoles. Earlier perceptions of this organelle were shaped by static transmission electron micrographs and by its biosynthetic functions. However, it has become increasingly clear that many Golgi activities can only be understood in the context of its dynamic organization. Significant new insights have been gained recently into the molecules that mediate this dynamic behavior, and how this machinery differs between plants and animals or yeast. Most notable is the discovery that plant Golgi stacks can actively move through the cytoplasm along actin filaments, an observation that has major implications for trafficking to, through and from this organelle.     

The biophysical design of plant cuticles: an overview

The outer surfaces of epidermal cell walls are impregnated with an extracellular matrix called the cuticle. This composite matrix provides several functions at the interface level that enable plants to thrive in different habitats and withstand adverse environmental conditions. The lipid polymer cutin, which is the main constituent of the plant cuticle, has some unique biophysical properties resulting from its composition and structure. This review summarizes the progress made towards understanding the biophysical significance of this biopolymer with special focus on its structural, thermal, biomechanical, and hydric properties and relationships. The physiological relevance of such biophysical properties is discussed in light of existing knowledge on the plant cuticle.     

Protein kinase resource: an integrated environment for phosphorylation research

The protein kinase superfamily is an important group of enzymes controlling cellular signaling cascades. The increasing amount of available experimental data provides a foundation for deeper understanding of details of signaling systems and the underlying cellular processes. Here, we describe the Protein Kinase Resource, an integrated online service that provides access to information relevant to cell signaling and enables kinase researchers to visualize and analyze the data directly in an online environment. The data set is synchronized with Uniprot and Protein Data Bank (PDB) databases and is regularly updated and verified. Additional annotation includes interactive display of domain composition, cross-references between orthologs and functional mapping to OMIM records. The Protein Kinase Resource provides an integrated view of the protein kinase superfamily by linking data with their visual representation. Thus, human kinases can be mapped onto the human kinome tree via an interactive display. Sequence and structure data can be easily displayed using applications developed for the PKR and integrated with the website and the underlying database. Advanced search mechanisms, such as multiparameter lookup, sequence pattern, and blast search, enable fast access to the desired information, while statistics tools provide the ability to analyze the relationships among the kinases under study. The integration of data presentation and visualization implemented in the Protein Kinase Resource can be adapted by other online providers of scientific data and should become an effective way to access available experimental information.