Liquid-liquid phase separation (LLPS) has emerged in recent years as an important physicochemical process for organizing diverse processes within cells via the formation of membraneless organelles termed biomolecular condensates. Emerging evidence now suggests that the formation and regulation of biomolecular condensates are also intricately linked to cancer formation and progression. We review the most recent literature linking the existence and/or dissolution of biomolecular condensates to different hallmarks of cancer formation and progression. We then discuss the opportunities that this condensate perspective provides for cancer research and the development of novel therapeutic approaches, including the perturbation of condensates by small-molecule inhibitors.

Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing "Janelia Fluor" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator-Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

Deciphering the molecular basis of pluripotency is fundamental to our understanding of development and embryonic stem cell function. Here, we report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In this context, TAF3 is required for endoderm lineage differentiation and prevents premature specification of neuroectoderm and mesoderm. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to a subset of chromosomal regions bound by CTCF/cohesin that are selectively associated with genes upregulated by TAF3. Notably, CTCF directly recruits TAF3 to promoter distal sites and TAF3-dependent DNA looping is observed between the promoter distal sites and core promoters occupied by TAF3/CTCF/cohesin. Together, our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions that safeguard the finely-balanced transcriptional programs underlying pluripotency.

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline.

The inherent limitations of fluorescence microscopy, notably the restricted number of color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass the conventional color barrier. cycleHCR facilitates high-specificity, single-shot imaging per target for RNA and protein species within thick specimens, mitigating the molecular crowding issues encountered with other imaging-based spatial omics techniques. We demonstrate whole-mount transcriptomics imaging of 254 genes within an E6.5\~7.0 mouse embryo, achieving precise three-dimensional gene expression and cell fate mapping across a specimen depth of \~ 310 µm. Utilizing expansion microscopy alongside protein cycleHCR, we unveil the complex network of 10 subcellular structures in primary mouse embryonic fibroblasts. Furthermore, in mouse hippocampal slice, we image 8 protein targets and profile the transcriptome of 120 genes, uncovering complex gene expression gradients and cell-type specific nuclear structural variances. cycleHCR provides a unifying framework for multiplex RNA and protein imaging, offering a quantitative solution for elucidating spatial regulations in deep tissue contexts for research and potentially diagnostic applications.

Brain enriched voltage-gated sodium channel (VGSC) Na1.2 and Na1.6 are critical for electrical signaling in the central nervous system. Previous studies have extensively characterized cell-type specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, due to lack of reliable labeling and imaging methods, the sub-cellular localization and dynamics of these homologous Na1.2 and Na1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution and live-cell single molecule imaging to probe subcellular composition, relative abundances and trafficking dynamics of Na1.2 and Na1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Na1.2 to the distal axon of unmyelinated neurons. This pathway utilizes distinct signals residing in the intracellular loop 1 (ICL1) between transmembrane domain I and II to suppress the retention of Na1.2 in the axon initial segment (AIS) and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Na1.2 is gradually excluded from the distal axon as Na1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Na1.2 and Na1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.