Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Nanoscopic structure of DNA condensed for gene delivery.

Scanning force microscopy was used to examine DNA condensates prepared with varying stoichiometries of lipospermine or polyethylenimine in physiological solution. For the first time, individual DNA strands were clearly visualized in incomplete condensates without drying. Using lipospermine at sub-saturating concentrations, discrete nuclei of condensation were observed often surrounded by folded loops of DNA. Similar packing of DNA loops occurred for polyethylenimine-induced condensation. Increasing the amount of the condensing agent led to the progressive coalescence or aggregation of initial condensation nuclei through folding rather than winding the DNA. At over-saturating charge ratios of the cationic lipid or polymer to DNA, condensates had sizes smaller than or equal to those measured previously in electron micrographs. Polyethylenimine condensates were more compact than lipospermine condensates and both produced more homogeneously compacted plasmids when used in a 2-4-fold charge excess. The size and morphology of the condensates may affect their efficiency in transfection.

Masking generates contiguous segments of metal-coated and bare DNA for scanning tunneling microscope imaging.

To date, no microscopic methods are available to confirm scanning tunneling microscope (STM) images of DNA. The difficulties encountered in repeating these images may be attributed to inadequate distribution of molecules on the substrate, poor adhesion to the substrate, or the low conductivity of the molecules. However, these factors are difficult to assess in an STM experiment where they may act simultaneously. A method to isolate these factors involves partly masking the deposited molecules before coating them with a conductive film to produce adjacent segments of coated and bare DNA after the mask is removed. The coated DNA segments are conductive and mechanically stable to allow easy identification of DNA by the STM. Furthermore, the path of a molecule can be traced from a coated to an uncoated region to test STM imaging of bare DNA. Masked preparations of DNA deposited on platinum/carbon-coated mica and highly oriented pyrolytic graphite were examined with a tunneling current 1000 times lower than the usual nanoamps. The tip apparently displaces molecules adsorbed to graphite to preclude imaging whereas more stably bound DNA on platinum/carbon-coated mica appears in reversed contrast.

Images of single-stranded nucleic acids by scanning tunnelling microscopy.

The scanning tunnelling microscope has the potential to resolve the structure of biological molecules with atomic detail. Progress has been made in the imaging of dried, unshadowed double helices of DNA4-7 and in recording images of DNA under water. Also, images of unshadowed complexes of DNA with the RecA protein from Escherichia coli indicate that this technique may not be restricted to thin biological samples. Here we present images of polydeoxyadenylate molecules aligned in parallel, with their bases lying flat on a surface of highly oriented pyrolytic graphite and with their charged phosphodiester backbones protruding upwards. Based on these images, a molecular model has been built which suggests the presence of a hydrogen bond that could stabilize the parallel alignment. Our micrographs demonstrate the potential application of scanning tunnelling microscopy in structural studies of nucleic acids and provide evidence that it could be used to sequence DNA.