Person:

Drokhlyansky, Eugene

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.

Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

The brain has a tightly regulated environment that protects non-regenerating post-mitotic neurons and limits inflammation, which led to its description as a site of ‘immune privilege’. For example, viral and bacterial stimuli elicit a weaker immune response within the brain than following systemic application; injection of pathogenic stimuli into the brain leads to significantly less, if any, monocyte recruitment, T-cell priming and B-cell antibody production when compared to systemic applications over the same time period. However, the difference between the immune response of the brain and systemic circulation is not absolute, but rather relative, and applies to both the innate and adaptive immune systems.

Innate immunity provides a rapid response to infections, which is often referred to as a first line of host defense, and it also enhances adaptive immune responses. Innate immune pathways are activated in the brain by local infections, during neurodegeneration, as part of neuropsychiatric disorders, and following systemic pathogenic stimuli. However, the types of innate immune pathways that are activated in the brain are unclear, including the types of cells that mount an innate immune response, and the ways in which particular cell types respond to the different innate immunity signaling molecules are also not well defined. Perhaps of most importance, however, is whether these responses are functionally effective and can limit virus spread in the brain.

The brain is structurally complex, harboring extensive interactions with both the lymphatic and vascular architecture. Within the brain, the parenchyma, which includes the white and grey matter, is the area of immune privilege. We sought to test if the brain parenchyma has the cellular and molecular elements to initiate an innate immune response capable of limiting the spread of directly delivered virus. We injected VSV, a well-characterized transsynaptic tracer, or VSV-derived defective interfering particles, into the caudate-putamen and scored for an innate immune response and inhibition of virus spread. We report that the brain parenchyma has a functional type I interferon response that can limit VSV spread at both the inoculation site and between neurons. Furthermore, we characterize the response of the microglia, which are believed to be the brain’s immune cells, to VSV infection and demonstrate that infected microglia produce type I interferon and that the innate immune response is induced in un-infected microglia following infection.