A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1.

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

Peptide-Tag Specific Nanobodies for Studying Proteins in Live Cells.

Single-domain antibodies such as nanobodies (Nbs) have substantially expanded the possibilities of advanced cellular imaging. In comparison to conventional antibodies, Nbs are characterized by small size, high stability, and solubility in many environments, including the cytoplasm. Nbs can be efficiently functionalized or modified according to the needs of the imaging approach. Target-specific Nbs can be easily converted into genetically encoded fluorescently labeled intrabodies, also known as chromobodies (CBs), which represent powerful tools to study the dynamics of different proteins of interest within living cells. In this context, CBs specific for a short peptide epitope provide a versatile alternative to bypass the limitations observed with larger fluorescent protein fusions and can be readily used to visualize and monitor peptide-tagged proteins for which specific Nbs are not available. Here, we present our novel detection system comprising a 15 amino acid peptide-tag (PepTag) in combination with a peptide-tag specific CB (PepCB). We provide protocols for adding the PepTag to different proteins of interest, reformatting the peptide-specific Nb (PepNb) into a CB for expression in mammalian cells, and establishment of stable cell lines expressing the PepCB for protein interaction assays and compound screenings.

A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies.

Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.