Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.

Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations.

Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

To a large extent functional diversity in cells is achieved by the expansion of molecular complexity beyond that of the coding genome. Various processes create multiple distinct but related proteins per coding gene - so-called proteoforms - that expand the functional capacity of a cell. Evaluating proteoforms from classical bottom-up proteomics datasets, where peptides instead of intact proteoforms are measured, has remained difficult. Here we present COPF, a tool for COrrelation-based functional ProteoForm assessment in bottom-up proteomics data. It leverages the concept of peptide correlation analysis to systematically assign peptides to co-varying proteoform groups. We show applications of COPF to protein complex co-fractionation data as well as to more typical protein abundance vs. sample data matrices, demonstrating the systematic detection of assembly- and tissue-specific proteoform groups, respectively, in either dataset. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from bottom-up proteomic datasets.

Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.