Robert T. Doyle

Subcellular distributions and excited-state processes of hypericin in neurons.

Abstract— The photodynamic drug, hypericin, is studied in fetal rat neurons using fluorescence microscopy. Hypericin has an extremely high affinity for the cell membrane and is found to a smaller extent in the nucleus. Fluorescent excitation of hypericin is shown to cause irreversible damage to the cell membranes of living neurons. Fixed cells were used to make ultrafast time‐resolved measurements to avoid the deleterious effects of long‐term exposure to intense light and room temperatures. To our knowledge, these are the first ultrafast time‐resolved measurements of the fluorescence lifetime of hypericin in a subcellular environment. Nonexponential fluorescence decay is observed in hypericin in the neurons. This nonexponential decay is discussed in terms of other examples where non‐exponential decay is induced in hypericin upon its binding to biomolecules. The nonaradiative processes giving rise to the nonexponential hypericin decay are attributed to excited‐state electron transfer, excited‐state proton transfer or both.

Target-dependent induction of secretory capabilities in an identified motoneuron during synaptogenesis.

Cholinergic neurons isolated from the buccal ganglia of Helisoma were plated into cell culture with a variety of defined target cells to study the specificity of synaptogenesis. Motoneuron B19 selectively formed chemical connections with single dissociated muscle fibers derived from its appropriate target, the supralateral radular tensor (SLT) muscle. B19 did not form such connections with novel neuronal targets. In contrast to neuron B19, cholinergic neuron B5 nonselectively formed chemical connections with novel muscle and neuronal targets. Target cells were micromanipulated into contact with presynaptic neurons to examine the latent period until the onset of functional synaptic transmission. Neuron B5 formed chemical connections within the first minutes of contact with ACh-sensitive neurons and muscle while B19 required sustained periods of muscle-specific contact to induce the acquisition of a functional excitation-secretion coupling mechanism. These different latent periods from the onset of target contact suggest that neuron B5 acquires presynaptic secretory function before target contact, while B19 must receive a specific signal(s) from its appropriate target to induce the transformation of its terminal into a secretory state.

Target contact regulates the calcium responsiveness of the secretory machinery during synaptogenesis

Neuron B19 of Helisoma is selective in synaptogenesis. Presynaptic mechanisms underlying this selectivity were tested. Acetylcholine-sensitive assay cells were micromanipulated into contact with B19 somata to assess its secretory state. Prior to appropriate muscle target contact, spontaneous synaptic currents were detected; however, action potential-evoked release of neurotransmitter was detected only following hours of muscle contact. Photolysis of a calcium cage, DM-nitrophen, accelerated the frequency of synaptic currents in muscle-contacted, but not novel neuron-contacted, B19 somata. These studies demonstrate that contact with appropriate target muscle enhances the responsiveness of this neurons secretory machinery to internal calcium levels, thereby imparting the presynaptic cell with the ability to couple action potentials with neurotransmitter release.

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, alpha1B and beta3 subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional omega-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of alpha1B subunits was diffuse. The distribution of the alpha1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis.

Gamma–delta T cell subsets are differentially associated with granuloma development and organization in a bovine model of mycobacterial disease

The characteristic lesion in bovine tuberculosis is well‐organized respiratory granulomas. This is typically associated with a strong T‐helper 1 biased cell‐mediated immune response and eventual containment of the infection. In bovine paratuberculosis, the classic lesion is unorganized granulomatous intestinal inflammation. Clinical paratuberculosis is associated with a T‐helper 2 biased humoral immune response and eventual death because of inability of the host to contain the infection. Recent reports have suggested that gamma–delta (γδ) T cells play a significant role in granuloma development and/or maintenance during initial stages of infection and may influence the subsequent adaptive immune response. The objective of this study was to use an in vivo bovine model to evaluate γδ T cells during the early host immune response to mycobacterial infection. We used immunofluorescent staining, hyperspectral microscopy, and computerized assisted morphometry to evaluate staining and distribution of γδ T cells during development of organized and unorganized granulomas. Our data suggest that bovine γδ T cell subsets are differentially recruited to early infection sites, and may be instrumental during the initial antimycobacterial host immune response as well as for granuloma organization.

Dynamic imaging of purified individual synaptic vesicles.

The atomic force microscope (AFM) was used to directly image purified synaptic vesicles. Individual secretory vesicles (approximately 50 nm diameter) were resolved with the AFM when imaged either dry or in solution. Vesicles were observed repeatedly for periods of greater than 2 h. To ask whether the AFM can detect structural change of vesicles the osmolarity of the bathing medium was reduced from 330 to 110 mOsm. Hypo-osmotic treatment caused an expansion and flattening of the vesicles. Thus, using the AFM it is possible to resolve individual vesicles and follow changes in vesicular structure. This opens the possibility that the secretory event can be reconstituted and visualized in vitro in order to elucidate the roles of synaptic proteins in synaptic transmission.

Extraction of Near-Field Fluorescence from Composite Signals to Provide High Resolution Images of Glial Cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.

Glucose uptake in PC12 cells: GLUT3 vesicle trafficking and fusion as revealed with a novel GLUT3-GFP fusion protein

The distribution of glucose transporters at the cell surface has a major impact on cellular glucose uptake. In muscle cells and adipocytes, this distribution is under the control of insulin; however, neuronal glucose uptake is not acutely regulated by insulin. Factors that affect the translocation of the neuronal glucose transporter isoform GLUT3 vesicles to and their fusion with the plasma membrane are not well understood. We report that GLUT3 in PC12 cells colocalizes with SNARE complex proteins SNAP‐25 and syntaxin 1, suggesting that fusion of GLUT3‐containing vesicles with the plasma membrane is mediated by these proteins. In addition, it seems that GLUT3 vesicle fusion is regulated, as depolarization increases GLUT3 insertion into the plasma membrane. To study the dynamics of GLUT3 vesicle trafficking, we have created a GLUT3‐GFP fusion protein that is easily expressed in PC12 cells. Trafficking of GLUT3‐GFP seems normal, as 1) its distribution is similar to endogenous GLUT3, 2) GLUT3‐GFP containing vesicles fuse with the plasma membrane evidenced by labeling of the fusion protein with an antibody directed against the exofacial epitope of GLUT3, and 3) glucose uptake is similar to PC12 cells not transfected with GLUT3 fusion protein. These studies are the first to examine GLUT3 trafficking and fusion in PC12 cells, and establish a model system to study regulation of the neuronal glucose transporter.