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Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.
The immune system makes use of major histocompatibility complex class I (MHC I) molecules to present peptides to other immune cells, which can evoke an immune response. Within this process of antigen presentation, the MHC I peptide loading complex, consisting of a transporter associated with antigen processing TAP, MHC I, and chaperones, is key to the initiation of immune response by shuttling peptides from the cytosol into the ER lumen. However, it is still enigmatic how the flux of antigens is precisely coordinated in time and space, limiting our understanding of antigen presentation pathways. Here, we report on the development of a synthetic viral TAP inhibitor that can be cleaved by light. This photo-conditional inhibitor shows temporal blockade of TAP-mediated antigen translocation, which is unleashed upon illumination. The recovery of TAP activity was monitored at single-cell resolution both in human immune cell lines and primary cells. The development of a photo-conditional TAP inhibitor thus expands the repertoire of chemical intervention tools for immunological processes.
Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.