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.

Serosal ADH stimulation enhances water flow under an imposed osmotic gradient through insertion of water channels (aggrephores) into the mucosal plasma membrane of toad urinary bladder sacs. Following cessation of ADH actions, water channels are retrieved as endosomes that can be visualized by mucosal inclusion of horseradish peroxidase (HRP) into round vesicles, long tubules and multivesicular bodies within the cytosol (1,2,3). Endosomes also occur adjacent to golgi bodies or lysosomes (1,2,3). However, true nature of endosomes including their formation at the mucosal surface and their shuttling in granular cells is still unclear (4,5). Current studies were undertaken to understand the role of endosomes in water channel cycling in this renal membrane model.

Urinary bladder sacs removed surgically from doubly-pithed toads, were suspended at ends of glass tubes. Control (no hormone) and experimental bladder sacs were exposed to ADH for 10 min in the absence of osmotic gradient.

Several clinically important anti-cancer agents exert their effects on tumor cells through interference with the function of microtubules. In addition to the Vinca alkaloids, such as vinblastine and vincristine, the taxanes, such as paclitaxel (Trade Name: Taxol), kill tumor cells through a microtubular target. Treatment with taxol leads to the inability of microtubules to depolymerize, leading to the formation of large intracellular microtubular bundles. In tumor cells that progress through the cell cycle, this leads to the inability of these cells to disassembly interphase microtubule networks and a failure to form functional mitotic spindles. These cells arrest in M phase, from which they eventually progress, either by the induction of apoptotic cell death, or by micronucleation and the formation of tetraploid cells. There is also the possibility that taxol has other effects on the regulation of genes or other systems to enhance cell killing, perhaps through lowering the threshold of cells to the induction of apoptotic cell death.

The microtubule (MT) cytoskeleton plays a crucial role in a number of cellular functions including cytoplasmic organization, intracellular transport, cellular polarity, and cell division. Microtubules are linear polymeric structures composed of heterodimers of α- and β-tubulin with a class of specialized proteins, the microtubule-associated proteins, typically being present. Microtubules are dynamic structures that undergo repeated rounds of assembly/disassembly. The half-life of MTs, an index of their dynamics (i.e., assembly-disassembly), has been estimated from studies of single cells in culture and typically ranges from ≈ 5 - 20 minutes depending on the cell type. These dynamic properties of MTs have been explained in terms of the “dynamic instability” model. Five parameters have been used to describe dynamic instability of MTs: (1) elongation velocity; (2) shortening velocity; (3) nucleation frequency; (4) catastrophe frequency; and (5) rescue frequency. Catastrophe is the transition from elongation to shortening while rescue is the transition from shortening to elongation.

Following intracellular uptake, taxanes bind to β tubulin and suppress microtubular dynamics, resulting in stable microtubule bundles and mitotic arrest of cancer cells. This proximal taxane- β tubulin interaction depends on the amount of intracellular accumulation of taxanes. Since taxanes are substrates for the membrane transporter P-glycoprotein (PGP) but not the MRP, overexpression of PGP but not MRP reduces the intracellular accumulation and cytotoxicity of taxanes. Recent reports have also suggested that alterations in the levels of specific β tubulin isotypes can reduce the effects of taxanes on microtubule dynamics, thereby inhibiting the mitotic effects and resulting cytotoxicity of taxanes.6 For example, increased βIV and (βIII tubulin levels may confer resistance against microtubule and cytostatic effects of taxanes. Taxane-induced G2/M arrest is preceded by the activation of cell-cycle mitotic kinase CDK1, which promotes the G2/M transition. Recent studies have indicated that the loss of cell-cycle Gl checkpoint due to p53 mutations may enhance taxane-induced mitotic arrest and cytotoxicity.

Taxol, a plant-derived antimicrotubule agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. This naturally occurring antineoplastic drug has been demonstrated to possess broad activity against a variety of human solid tumors, particularly in drug-refractory ovarian cancer and metastatic breast cancer (1). However, the exact mechanism of taxol's cytotoxicity against tumor cells is not entirely clear. Recent studies have demonstrated that taxol, besides causing microtubule bundling and mitotic arrest, is able to induce internucleosomal DNA fragmentation and typical morphological features of apoptosis in a number of solid tumor cells. These results clearly indicate that taxol, in addition to its classical activity against microtubules and cell cycle arrest, also possesses significant cell-killing activity by induction of apoptosis.

Although it is well recognized that taxol can cause both mitotic arrest and apoptotic cell death, it remains unclear whether taxol-induced cell death is a secondary event resulting from mitotic arrest or represents a novel mechanism of taxol against tumor cells.

Correlative microscopy is employed in a great variety of settings by both diagnostic and investigative pathologists. Combinations of conventional light microscopy (LM), immunohistology, and electron microscopy (EM) are used in a wide range of diagnostic settings, including the analysis of tumors, autoimmune disorders, and infectious diseases. Valuable diagnostic information is also frequently obtained by simultaneous or sequential examination of exfoliated or aspirated cell suspensions (cytopathology) and tissue sections (histopathology) from the same lesion. An even wider range of correlative microscopic methods is employed by pathologists in basic and clinical research. The rationales for using correlative techniques are many and varied, but in most cases fall within a limited number of categories.

Pathologists frequently use a second microscopic or preparative technique to improve on the resolution afforded by an initial technique. Electron microscopy is often used to refine the analysis of features initially detected by routine LM or immunohistology.

Molecular pathology represents a distinctive new discipline in medicine with many potential applications that have been heretofore untapped. This discipline is introducing important changes in the concepts of disease causation and in the classification of diseases. Transfer of molecular genetic technology and knowledge to the pathology laboratory is giving the pathologist new and powerful tools to diagnose, classify, and monitor disease. Methodologies such as in situ hybridization (ISH), immunohistochemistry (IHC), and the polymerase chain reaction (PCR) will continue to allow more systematic and accurate classification of human diseases. IHC and ISH allow the identification and localization of cells containing specific antigens or nucleic acid sequences. The main advantage of these methods is their ability to localize targets and define their morphologic features in various tissues, allowing correlations with histopathologic changes.