BJ Articles Ahead of Print

(Biophysical Journal 116, 1682–1691; May 7, 2019)

The nuclear environment is highly crowded by biological macromolecules, including chromatin and mobile proteins, which alter the kinetics and efficiency of transcriptional machinery. These alterations have been described, both theoretically and experimentally, for steady-state crowding densities; however, temporal changes in crowding density (“dynamic crowding”) have yet to be integrated with gene expression. Dynamic crowding is pertinent to nuclear biology, as processes such as chromatin translocation and protein diffusion lend to highly mobile biological crowders.

The electrical membrane potential (vm) is one of the components of the electrochemical potential of protons across the biological membrane (proton motive force), which powers many vital cellular processes. As vm also plays a role in signal transduction, measuring it is of great interest. Over the years a variety of techniques has been developed for the purpose. In bacteria, given their small size, Nernstian membrane voltage probes are arguably the favourite strategy, and their cytoplasmic accumulation depends on vm according to the Nernst equation.

We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGluh, iGlum and iGlul cover a broad range of Kd-s (5.8 μM, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP has a Kd of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerisation either of the apo or the ligand-bound binding protein.

Efficient engagement with the envelope glycoprotein (Env) membrane-proximal external region (MPER), results in robust blocking of viral infection by a class of a broadly neutralising antibodies (bnAbs) against HIV. Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralising potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arise only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids.

How chromatin is folded on the lengthscale of a gene is an open question. Recent experiments have suggested that, in vivo, chromatin is folded in an irregular manner and not as an ordered fiber with a width of 30 nm expected from theories of higher order packaging. Using computational methods, we examine how the interplay between DNA-bending non histone proteins, histone tails, intra-chromatin electrostatic and other interactions decide the nature of packaging of chromatin. We show that while the DNA-bending non histone proteins make the chromatin irregular, they may not alter the packing density and size of the fiber.

Cysteine palmitoylation, a form of S-acylation, is a key post-translational modification in cellular signaling. This type of reversible lipidation is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl-CoA from the lipid bilayer. The DHHC enzyme then becomes auto-acylated, at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering auto-acylation.

Knots in the human genome would greatly impact diverse cellular processes ranging from transcription to gene regulation. To date, it has not been possible to directly examine the genome in vivo for the presence of knots. Recently, methods for serial fluorescent in situ hybridization have made it possible to measure the 3d position of dozens of consecutive genomic loci, in vivo. However, the determination of whether genomic trajectories are knotted remains challenging, because small errors in the localization of a single locus can transform an unknotted trajectory into a highly-knotted trajectory, and vice versa.

The dynamic organization of chromatin inside the cell nucleus plays a key role in gene regulation, genome replication as well as maintaining genome integrity. While the static folded state of the genome has been extensively studied, dynamical signatures of processes such as transcription or DNA repair remain an open question. Here, we investigate the interphase chromatin dynamics in human cells in response to local DNA damage, specifically, DNA double strand breaks (DSBs). Using simultaneous two-color spinning disk confocal microscopy, we monitor the DSB dynamics and the compaction of the surrounding chromatin, visualized by fluorescently labeled 53BP1 and histone H2B, respectively.

The TRPV1 cation non-selective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature or by naturally occurring pungent molecules such as allicin, capsaicin or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior.

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins (MBP) and aggregation transitions between MBP substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates.

Bundles of actin filaments are central to a large variety of cellular structures, such as filopodia, stress fibers, cytokinetic rings or focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles’ rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this paper, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load.