Molecular cloning offers an opportunity for designing constructs for expressing chimeric proteins containing incorporated reporter molecules. In this approach, molecules are identified based upon reporter molecules which are expressed within cells as a result of transfection, instead of marking them through immunolabeling, in situ hybridization, or derivative incorporation. This approach is particularly useful for studies involving integrated microscopy. Integrated microscopy allows us to assemble images of the same cell obtained with different microscopes into one comprehensive message concerning cellular functions. Integration of fluorescence and electron spectroscopic imaging is particularly promising. The main advantage of this approach relies in overcoming limitations of each type of microscopy alone i.e. in studies on living cells limitations in spatial resolution of light microscopy and in analysis of supramolecular organization, limitation of electron microscopy to frozen or fixed cells. For this purpose, special reporter molecules suitable for selected modes of microscopy have to be used.

In situ detection of specific antigens and nucleic acid sequences is possible with a variety of immunohistochemical (IHC) and in situ hybridization (ISH) methods. The drawback of conventional methodologies is their relatively low detection sensitivity. Improved detection efficiency is required in preparations containing only minute amounts of detectable antigens or only few copies of specific DNA and/or RNA sequences, which is often the case in routine paraffin cytoand histopathologic material. The use of gold as the label, followed by silver amplification (autometallography) avoids hazardous reagents and also gives an improved resolution compared to „standard” methods. Sensitivity problems may now be overcome by our specific combinations of recent technologies, utilitizing reporter molecule amplification with labeled tyramides (CARD, catalyzed reporter deposition, or tyramide signal amplification), followed by streptavidin bound to a new gold label, Nanogold™ (Nanoprobes, Stony Brook, NY), and silver acetate autometallography.

Colloidal gold is routinely used conjugated to ligands or antibodies as a label for the detection of cell structures by transmission (TEM) and scanning (SEM) electron microscopy. Silver enhancement, a procedure that amplifies the size of gold spheres, is used for light microscopic (LM) observation of colloidal gold. These techniques are valuable in localization studies but it is difficult to quantitate the level of labeling in larger volumes of tissue solely by microscopic means.

Instrumental Neutron Activation Analysis (INAA) can be used to provide a quantitative index of gold accumulation at the organ and tissue levels. Given that gold is not naturally found in the body in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be determined at the organ and tissue levels in whole animal systems.

The green fluorescent protein (GFP) has great potential as a tool for biologists because it can be used as an in vivo real time reporter of protein localization and gene expression in a variety of experimental systems. Wild type GFP, however, has several undesirable properties including low brightness, a significant lag in the development of fluorescence, complex photoisomerization, inefficient protein folding at 37°C, and poor expression in several species. To improve upon these qualities, we have combined an ultra-bright variant of GFP, GFPmutl, with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yields an enhanced variant (EGFP) (Table 1) that greatly increases the sensitivity of this reporter.

The natural green emission of GFP can conveniently be monitored by optics designed to detect fluorescein. There are, however, many potential applications for GFP that require additional emission colors.

The emission spectrum from bioluminescent bacteria has been observed to depend on the type of bacteria. Photobacterium phosphoreum species usually show bioluminescence maxima around 472 nm and Photobacterium leiognathi species to slightly longer wavelength. A certain strain (Yl) of Vibrio fischeri, has a yellow bioluminescence with maximum at 542 nm. These differences have been explained as due to the bioluminescence originating from the fluorescence transition of an “antenna” protein, participating in the bioluminescence reaction along with the enzyme bacterial luciferase. The bioluminescence from a number of coelenterates involves a similar participation of an antenna protein, the famous “Green-Fluorescent Protein” being the origin of the bioluminescence emission from these organisms.

The cloning and expression of GFP in heterologous systems introduced a fantastic tool for studying specific gene expression and protein localization inside living cells. However, one aspect of GFP that has not been exploited to its full potential is its use as a quantitative imaging tool. To determine its quantitative usefulness, we have addressed five points that are important in GFP imaging: detectable signal over background, photostability, pH stability of the molecule, temperature dependence of chromophore formation, and estimation and normalization of GFP levels.

To determine the quantitative limits of GFP in cells, several GFP versions (wtGFP, αGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F)) were compared by imaging of GFP expressing cells or by spectroscopic measurements of purified proteins. When imaged, the GFP signals are contaminated by the naturally occurring background autofluorescence, but improved detection can be achieved for each green GFP by combination of confocal microscopy using 488 nm excitation, a rapid cut-on dichroic mirror, and a narrow bandpass emission filter (Figure l).

Green fluorescent protein (GFP) was evaluated as a non-invasive probe to quantify the rheological properties and pH of intracellular aqueous compartments. Spectroscopic studies were done on several bacterially-expressed and purified GFP mutants. GFP-S65T was brightly fluorescent in solution (ƛex 492 nm, ƛem 509 nm) with lifetime 2.9 ns and rotational correlation time ( tc ) ∼20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 ± 2 ms (5 μm diameter spot) giving a diffusion coefficient of 8.7 x 10-7 cm2/s. The t1/2 was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein, GFP photobleaching efficiency was not affected by solution O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), independent of spot diameter, and not affected by O2 or quenchers.

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.

We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

The cytoskeleton plays an important role in cell structure, polarity, locomotion and division. Individual elements of the cytoskeleton are composed of subunit proteins which assemble and disassemble in specific places and times within the cell. Knowledge of the temporal and spatial regulation of subunit assembly and disassembly is essential to understanding how the cytoskeleton contributes to cellular activities. The assembly and dynamics of two cytoskeletal structures, namely adhesion plaques (APs) and intermediate filaments (IFs), have been difficult to study by traditional methods. We have generated GFP-chimeras to label these structures and to study their dynamics in motile fibroblasts.

To study the dynamics of APs, we prepared stable 3T3 cell lines expressing a GFP-β1 integrin chimera. The chimera was prepared by fusing GFP to the transmembrane and cytoplasmic portions of β1 intergrin, since previous studies had shown that the cytoplasmic tail of β integrins is sufficient to direct integrins to APs.

A variety of extracellular signals, including hormones, neurotransmitters, growth factors and cytokines, are capable of activating vascular smooth muscle cells (VSMC). These can modulate the VSMC phenotype, which underlies the progression of vascular diseases. We study gene expression control responses in VSMC that are regulated by different classes of cell surface receptors. Taken as simpler model systems for the pleiotropic sensitivity of VSMC to extracellular stimuli, we focus on understanding the elements of intracellular signal transduction pathways involved in controlling gene expression. In particular, we are interested in identifying signaling elements that can be activated by multiple classes of cell surface receptors.

Previous studies had supported both a role of protein kinase A (PKA) activity and transcriptional induction of an unknown factor in control of ATI angiotensin receptor gene expression in VSMC (1). The mRNA from this gene is down-regulated by a variety of extracellular stimuli, and we hypothesized that stimulation of PKA by several classes of receptors might explain the apparently pleiotropic nature of ATI-receptor down regulation.

We have investigated the translocation of different GFP-tagged protein domains in response to various receptor stimuli. Confocal fluorescent imaging was used to study the localization of the GFP-tagged protein domain before, during and after the receptor stimuli was applied. A minimal cysteine-rich domain from protein kinase C was shown to be a useful tool to study localized diacylglycerol changes. Furthermore, the calcium-induced plasma membrane translocation of the GFP-tagged C2-domain from PKC could be used to study receptor-mediated changes in near plasma membrane calcium concentration. In addition, different pleckstrin homology domains (PH-domains) could be employed to measure changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI4.5P2) as well as of P13,4P2and PO,4,5P3. In combination, these fluorescent translocation probes provide a new means to investigate the local and temporal control of cell signaling processes in individual living cells.

Choristoneura fumiferana nucleopolyhedrovirus(CfMNPV) is a baculovirus that infects the spruce budworm, Choristoneura fumiferana(Cf), and a few other related species. To study the infection process of this virus within the spruce budworm, we have constructed a recombinant virus (CfMNPV-GFP) expressing the green fluorescence protein(GFP) under the control of the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Each larva was fed 10,000 CfMNPV-GFP occlusion bodies(OB) applied to diet plugs and various tissues were dissected from the larvae at 24 hr intervals and examined for the presence of the OB and/or GFP using a fluorescence microscope. Larvae were also fixed and embedded for light and electron microscopy.

GFP-specific fluorescence was first detected as a few spots at the posterior end of the midgut at 72 hr post feeding(PF). By 96 hr PF, the fluorescence had expanded to include adjacent cells and some hemocytes.

The cell surface mediates the flow of information and metabolites between a cell and its environment. We are interested in understanding the lateral organization of the surface, as part of our program in understanding its function. The model of lateral organization of cell surface membranes is evolving from one which emphasizes mobility and autonomy of membrane constituent molecules, to another which emphasizes the lateral concentration of membrane proteins and lipids into patches and membrane microdomains. Indirect evidence suggests that diameters of these patches and microdomains are often ≤ a wavelength of visible light, and so cannot be readily resolved by conventional light microscopy.

We have developed two complementary techniques for detecting patches and microdomains on the nm scale: image fluorescence resonance energy transfer, FRET, and near-field scanning optical microscopy, NSOM. The first of these techniques detects proximity of membrane proteins and lipids on a scale of a few nm in terms of the interactions of donor and acceptor fluorophores.

The nervous system has evolved to make use of a variety of mechanisms that allow information to flow and be processed among a large collection of individual cells. The communication between individual brain cells occurs largely at chemical synapses. In these compartments, chemical messengers are packaged into small vesicles that fuse with the cell membrane upon stimulation, releasing neurotransmitter.. The average total number of synaptic vesicles in a typical central nervous system synapse is only a few hundred and as a result an efficient local recycling mechanism operates in order to replenish this pool during periods of even modest neuronal activity. Without this membrane recycling, synapses quickly become depleted of vesicles, and soon fail to communicate information between cells.

We make use of optical techniques to follow the trafficking of synaptic vesicles at synapses formed between hippocampal neurons grown in culture. Recycling synaptic vesicles can be readily labeled using the fluorescent amphipathic membrane dye FM 1-43.

Dynamin, a 100 kDa GTPase, is essential for receptor mediated endocytosis and synaptic vesicle recycling; however its mechanism of action is unknown. The requirement for dynamin was first elucidated by the discovery that the shibire gene product in Drosophila melanogaster was homologous to mammalian dynamin-1 (1,2). The shibire flies exhibit a depletion of synaptic vesicles and an accumulation of collared clathrin-coated pits at the plasma membrane of their nerve termini (3). It was later demonstrated that endocytosis was inhibited by the overexpression of dominant negative mutants of dynamin (4,5), and that purified dynamin can self-associate to form spirals which resemble the collars of shibire and structures seen in synaptosomes treated with GTPγS (6,7). These observations led to the speculation that dynamin pinches the clathrin-coated bud from the plasma membrane. In support of this hypothesis, we show that purified recombinant dynamin can bind to a lipid bilayer in a regular and repeating pattern to form helical tubes which vesiculate upon the addition of GTP.

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.

We have used green fluorescent protein (GFP) chimeras to examine the dynamics of the early secretory pathway and the role of the peripheral coat protein COP I. To describe the overall properties of ER to Golgi transport we used the temperature sensitive viral glycoprotein, ts045 VSVG, tagged with GFP at its cytoplasmic tail. VSVG-GFP retained the temperature sensitive phenotype of its parent: it reversibly misfolded and was retained in the ER at 40°C. Upon shift to 32°C it was rapidly exported from the ER, moving as a synchronous pool into the Golgi complex and then to the cell surface. Using time-lapse imaging of living cells expressing VSVG-GFP we found that the carriers for ER to Golgi traffic are tubulovesicular pre-Golgi intermediates that move centrosomally to the Golgi at speeds of>1 μM2/sec and then fuse with the cis face of the Golgi complex. These movements are dependant on microtubules and the dynein/dynactin complex.

The origin and role of confronting cisternae (CC) have been clouded in mystery since their discovery in 1955. Early investigations suggested that CC arose from either nuclear envelope (NE) or by stacking of endoplasmic reticulum (ER) cisternae. Electron microscopic studies of mitotic HeLa cells suggest that CC are fragments of NE (Fig. 1) that arise as portions of the NE fold back on one another. Because lamin B is a NE specific protein, a monoclonal antibody to lamin B was used to probe the origin of CC. The monoclonal antibody to lamin B was then detected by utilizing a secondary antibody conjugated to the fluorochrome rhodamine which fluoresces red when exposed to UV light. Fluorescent microscopy of interphase HeLa cells demonstrates that only the NE fluoresces red when the cells are probed with a monoclonal antibody to lamin B (Fig. 2). There is no label localized in the cytoplasm which supports the evidence that lamin B is only localized to the NE in interphase cells.

We have previously described a cryohomogenization method for making in vitropreparations of rough endoplasmic reticulum (RER) and other organelles. The goal of that approach was to minimize the extensive vesicular fragmentation of the endoplasmic reticulum that occurs during homogenization for conventional cell fractionation. The microsome fraction of cell fractionation consists primarily of small vesicles derived from rough and smooth endoplasmic reticulum, and has been of great value in biochemical studies of protein synthesis and secretion. However, the small size of the microsome vesicles has made them less useful for in vitro studies of bound polysomes by electron microscopy. As a further development of our cryohomogenization approach, we here describe a method for removing larger particles and debris from the cryohomogenate by filtration, and the application of in vitro RER and other organelles to EM grid membranes for negative staining and viewing by electron microscopy.

Chlamydia trachomatis is responsible for several significant human diseases including trachoma, the primary source of preventable blindness in developing countries, and is the most common cause of sexually transmitted disease. C. trachomatis is an obligate intracellular prokaryote (ICP) relying on eukaryotic host cells for growth and replication. Typically, microorganisms engulfed by host cells, are trafficked through maturing endosomes to the lysosomal pathway and ultimately destroyed. Survival in a host cell requires the invading organism to either adapt or modify their host environment to avoid fusion with lysosomal vesicles. Organisms such as Mycobacterium tuberculosis have evolved mechanisms to arrest maturation of the endosomes, such that they avoid lysosomal fusion.3 C trachomatis has developed alternative strategies for successful intracellular survival and growth.

C. trachomatis exists in two morphologically and functionally distinct forms which multiply in vacuoles termed inclusions. A small dense form known as the elementary body (EB), is the stable extracellular stage of the life cycle capable of attachment and entry into host cells.