Dr. Ivana Nikić-Spiegel
Talk - Genetic code expansion-based live-cell protein labelling and its applications for super-resolution microscopy and neurobiology
Genetic code expansion (GCE) is emerging as an important technology for in vitro and in vivo protein manipulation and labelling. In combination with click-chemistry it allows site-specific labelling of proteins with small organic dyes. This is achieved by co-translational incorporation of unnatural amino acids (UAAs) in target proteins by using tRNA/amino-acyl tRNA synthetase pairs orthogonal to the host translational machinery. In a subsequent step, unique functional groups of UAAs are labelled with functionalized dyes in ultrafast and biocompatible click-chemistry reactions. The fact that any dye can be directly attached to the target protein in a minimally invasive way is of particular importance for single molecule science and super-resolution microscopy (SRM). I previously used this technology for dual-colour live-cell labelling and SRM of distinct populations of membrane proteins in mammalian cells, as well as for the intracellular labelling of low-copy number proteins inside the cell nucleus. In my laboratory we are currently using this technique to address some of the challenges in understanding pathogenesis of neuroinflammatory diseases, such as multiple sclerosis.
Molecular Mechanisms of Axonal Injury
Axons transmit electrical impulses and transport cargos between neuronal cell body and synapses. Due to their unique roles and architecture, they can cover remarkable distances and are highly prone to injury.
In neuroinflammatory diseases, such as multiple sclerosis (MS), axons are damaged by infiltrating immune cells. MS is one of the most common neuroinflammatory diseases of the human CNS and a leading cause of non-traumatic neurological disability in young adults. Since the amount of axonal injury determines the extent of irreversible clinical deficits that patients suffering from this incurable disease develop, understanding how axons are damaged in MS is crucial for development of new therapeutic approaches. Despite its importance, molecular mechanisms underlying neuroinflammatory axonal injury are poorly understood.
Previous work (Nikić et al., Nature Medicine 2011) identified a novel form of neuroinflammatory axon loss - focal axonal degeneration (FAD). The main feature of FAD is that early stages of axonal injury, induced by oxidative stress, are reversible. In order to understand what determines this reversibility and define new neuroprotective strategies, our aim is to further understand mechanisms underlying neuroinflammatory axonal injury at a molecular level.
We study mechanisms of neuroinflammatory axonal injury in mouse and in vitro models. In addition to more standard microscopy techniques (live cell and intravital widefield imaging, confocal microscopy), we use modern super-resolution microscopy techniques, such as stochastic optical reconstruction microscopy (STORM), to obtain molecular-scale information. We are also using and developing new cutting-edge protein engineering tools based on selective incorporation of unnatural amino acids (Nikić et al., Angewandte 2014; Nikić, Kang, Girona et al., Nature Protocols 2015; Nikić & Lemke, Current Opinion in Chemical Biology 2016). Unnatural amino acids give us a unique opportunity to introduce new properties/functional groups, such as dyes, affinity tags for proteomics, post-translation modifications, cross-linkers, optogenetic tools, etc. into proteins at a single cell and even whole organism level.