Whole genome sequencing of CRISPR/Cas9-engineered NF-κB reporter mice for validation and variant discovery.

Targeted knockout, mutations, or knock-in of genomic DNA fragments in model organisms have been used widely for functional and cell-tracking studies. The desired genetic perturbation is often accomplished by recombination-based or CRISPR/Cas9-based genome engineering. For validating the intended genetic modification, a local region surrounding the targeted locus is typically examined based on enzymatic cleavage and consequent length patterns, e.

Biomolecular Condensates as Novel Antiviral Targets.

Viral infections pose a significant health risk worldwide. There is a pressing need for more effective antiviral drugs to combat emerging novel viruses and the reemergence of previously controlled viruses. Biomolecular condensates are crucial for viral replication and are promising targets for novel antiviral therapies.

Double knockin mice show NF-κB trajectories in immune signaling and aging.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites.

Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers.

A multi-step nucleation process determines the kinetics of prion-like domain phase separation.

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters.

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.

Liquid-liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1.

Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit.

The condensation of biomolecules into biomolecular condensates via liquid-liquid phase separation (LLPS) is a ubiquitous mechanism that drives cellular organization. To enable these functions, biomolecules have evolved to drive LLPS and facilitate partitioning into biomolecular condensates. Determining the molecular features of proteins that encode LLPS will provide critical insights into a plethora of biological processes.

Intrinsically disordered protein regions and phase separation: sequence determinants of assembly or lack thereof.

Intrinsically disordered protein regions (IDRs) - regions that do not fold into a fixed three-dimensional structure but instead exist in a heterogeneous ensemble of conformations - have recently entered mainstream cell biology in the context of liquid-liquid phase separation (LLPS). IDRs are frequently found to be enriched in phase-separated compartments. Due to this observation, the presence of an IDR in a protein is frequently assumed to be diagnostic of its ability to phase separate.

Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture.

Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment.

G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network.

Integrative analysis suggests cell type-specific decoding of NF-κB dynamics.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts.

Valence and patterning of aromatic residues determine the phase behavior of prion-like domains.

Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions.

Protein Network Structure Enables Switching between Liquid and Gel States.

Biomolecular condensates are emerging as an important organizational principle within living cells. These condensed states are formed by phase separation, yet little is known about how material properties are encoded within the constituent molecules and how the specificity for being in different phases is established. Here we use analytic theory to explain the phase behavior of the cancer-related protein SPOP and its substrate DAXX.

Assaying Homodimers of NF-κB in Live Single Cells.

NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as "NF-κB" in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types.

Correction to: PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

The affiliation of Erik W. Martin is corrected in this paper.

PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis.

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood.

Challenges of Decoding Transcription Factor Dynamics in Terms of Gene Regulation.

Technological advances are continually improving our ability to obtain more accurate views about the inner workings of biological systems. One such rapidly evolving area is single cell biology, and in particular gene expression and its regulation by transcription factors in response to intrinsic and extrinsic factors. Regarding the study of transcription factors, we discuss some of the promises and pitfalls associated with investigating how individual cells regulate gene expression through modulation of transcription factor activities.

Relationship of Sequence and Phase Separation in Protein Low-Complexity Regions.

Liquid-liquid phase separation seems to play critical roles in the compartmentalization of cells through the formation of biomolecular condensates. Many proteins with low-complexity regions are found in these condensates, and they can undergo phase separation in vitro in response to changes in temperature, pH, and ion concentration. Low-complexity regions are thus likely important players in mediating compartmentalization in response to stress.

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing.

TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) , fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity.

A two-helix motif positions the lysophosphatidic acid acyltransferase active site for catalysis within the membrane bilayer.

Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga maritima, revealing an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ-domain that contains the catalytic HXD motif.