The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.