Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.
Background: Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues. These procedures are often time consuming, require substantial amounts of plant material, have low yield or do not result in pure organelle fractions. Moreover, barely a protocol allows rapid and flexible isolation of different subcellular compartments. The recently published SpySystem enables the in vitro and in vivo covalent linkage between proteins and protein complexes. Here we describe the use of this system to tag and purify plant organelles. Results: We developed a simple and specific method to in vivo tag and visualize, as well as isolate organelles of interest from crude plant extracts. This was achieved by expressing the covalent split-isopeptide interaction system, consisting of SpyTag and SpyCatcher, in Nicotiana benthamiana leaves. The functionality of the SpySystem in planta, combined with downstream applications, was proven. Using organelle-specific membrane anchor sequences to program the sub-cellular localization of the SpyTag peptide, we could tag the outer envelope of chloroplasts and mitochondria. By co-expression of a cytosolic, soluble eGFP-SpyCatcher fusion protein, we could demonstrate intermolecular isopeptide formation in planta and proper organelle targeting of the SpyTag peptides to the respective organelles. For one-step organelle purification, recombinantly expressed SpyCatcher protein was immobilized on magnetic microbeads via covalent thiol-etherification. To isolate tagged organelles, crude plant filtrates were mixed with SpyCatcher-coated beads which allowed isolation of SpyTag-labelled chloroplasts and mitochondria. The isolated organelles were intact, showed high yield and hardly contaminants and can be subsequently used for further molecular or biochemical analysis. Conclusion: The SpySystem can be used to in planta label subcellular structures, which enables the one-step purification of organelles from crude plant extracts. The beauty of the system is that it works as a covalent toolbox. Labeling of different organelles with individual tags under control of cell-specific and/or inducible promoter sequences will allow the rapid organelle and cell-type specific purification. Simultaneous labeling of different organelles with specific Tag/Catcher combinations will enable simultaneous isolation of different organelles from one plant extract in future experiments.
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