Edited by Alex TokerSpatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) and are either activated or inactivated by local PI(4,5)P 2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P 2sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P 2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P 2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P 2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P 2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P 2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P 2 with neomycin or disrupting PI(4,5)P 2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.

Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the α- and β-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic β-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the β-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb β-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division.

Deciphering chromatin regulation at the molecular level is of fundamental importance for an understanding of cellular physiological and pathological processes. Chromatin is an extremely complex system due to its molecular organization, heterogeneous structure and multiscale dynamics induced by post translational modifications on chromatin itself and other regulatory effectors including transcription factors (TFs). One key class of chromatin interacting proteins are pioneer transcription factors. The main characteristic that distinguishes pioneer transcription factors from other TFs is their ability to specifically recognize their target DNA sequences in compacted chromatin and consequently to trigger chromatin opening, thus enabling the cellular machinery to locally access the DNA. In the context of cell fate reprogramming, this pioneer action is crucial, but its molecular mechanism is poorly understood. Known to be an essential protein involved in multiple steps of DNA regulation, Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is the pioneer transcription factor inspiring us to explore its pioneer role in such complex and dynamic system as chromatin. We recently established a single-molecule Förster resonance energy transfer (FRET) method, using micromirror total internal reflection fluorescence (mmTIRF) microscopy, dedicated to investigate structural dynamics of chromatin fibers. Combining single-molecule FRET and chemically defined synthetic chromatin segments, we uncovered the interconversion kinetics of discrete tetranucleosome units and the impact of the post-translational modifications by ubiquitylation on chromatin structure. Here, we demonstrate that Rap1 invades compacted chromatin and exerts chromatin remodeling. These studies yield fundamental insights into the molecular mechanisms of gene regulation by molecular interactions. 200-Plat Single Molecule Measurements Reveal Conformational TransitionsDuring DNA Clamp Loading and Unloading SeungWon Lee, Eunjin Ryu, Sukhyun Kang, Hajin Kim. UNIST, Ulsan, Republic of Korea. Proliferation cell nuclear antigen (PCNA) is a DNA clamp, playing an important role of providing a ''platform'' for various enzymes during DNA replication. The loading of the closed trimeric ring of PCNA into duplex DNA requires the ATP-dependent activity of replication factor C (RFC) complex. The unloading of PCNA from chromatin is crucial for the regulation of replication process and maintaining genomic stability and it was recently found that ATAD5 protein is complexed with RFC-like complex (RLC) to get involved in the unloading of PCNA. However, the molecular mechanisms of PCNA loading and unloading processes have remained poorly understood. Here, we report direct observation of the loading and unloading dynamics of human PCNA driven by RFC and ATAD5-RLC complexes, respectively, by single molecule fluorescence resonance energy transfer measurements. Distinct conformational stages during PCNA loading were clearly detected that represent open and closed conformations of P...

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In this work we are presenting the coupling of the Dry Martini CG model for lipids with the lattice Boltzmann molecular dynamics technique that allows to include hydrodynamic interactions in implicit solvent CG simulations. We present the coupling of this force field with the OPEP CG model for proteins. These advances allow us to investigate systems and biophysical processes where the fluid environment and motion is key: for instance from vesicular and membrane fusion, shear effects, fluid transport across the membrane to protein aggregation in a membrane environment. We will showcase not only the basic coupling but also simulations of challenging systems, like a nanoreactor where enzymes, substrates and crowding proteins are confined by a lipid vesicle.