TY  - INPR
A1  - Odenwald, Johanna
A1  - Gabiatti, Bernardo
A1  - Braune, Silke
A1  - Shen, Siqi
A1  - Zoltner, Martin
A1  - Kramer, Susanne
T1  - Beyond BioID: Streptavidin outcompetes antibody fluorescence signals in protein localization and readily visualises targets evading immunofluorescence detection
T2  - eLife
N2  - Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins.Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an “all in one” solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody-accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal a history of dynamic protein interaction.In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment.
KW  - BioID
KW  - Streptavidin
KW  - antibody fluorescence signals
KW  - protein localization
KW  - immunofluorescence detection
Y1  - 2024
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-360704
ER  - 
TY  - JOUR
A1  - Zoltner, Martin
A1  - Krienitz, Nina
A1  - Field, Mark C.
A1  - Kramer, Susanne
T1  - Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA
JF  - PLoS Neglected Tropical Diseases
N2  - Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
KW  - Trypanosoma
KW  - mRNA
KW  - T. brucei
KW  - PABPs
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177126
VL  - 12
IS  - 7
ER  -