Monitoring nucleolar-nucleoplasmic protein shuttling in living cells by high-content microscopy and automated image analysis.

The nucleolus has core functions in ribosome biosynthesis, but also acts as a regulatory hub in a plethora of non-canonical processes, including cellular stress. Upon DNA damage, several DNA repair factors shuttle between the nucleolus and the nucleoplasm. Yet, the molecular mechanisms underlying such spatio-temporal protein dynamics remain to be deciphered. Here, we present a novel imaging platform to investigate nucleolar-nucleoplasmic protein shuttling in living cells. For image acquisition, we used a commercially available automated fluorescence microscope and for image analysis, we developed a KNIME workflow with implementation of machine learning-based tools. We validated the method with different nucleolar proteins, i.e., PARP1, TARG1 and APE1, by monitoring their shuttling dynamics upon oxidative stress. As a paradigm, we analyzed PARP1 shuttling upon H2O2 treatment in combination with a range of pharmacological inhibitors in a novel reporter cell line. These experiments revealed that inhibition of SIRT7 results in a loss of nucleolar PARP1 localization. Finally, we unraveled specific differences in PARP1 shuttling dynamics after co-treatment with H2O2 and different clinical PARP inhibitors. Collectively, this work delineates a highly sensitive and versatile bioimaging platform to investigate swift nucleolar-nucleoplasmic protein shuttling in living cells, which can be employed for pharmacological screening and in-depth mechanistic analyses.

The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles.

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions.

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.