Biology subjects

Good, M. C.

Publications and source records attributed to Good, M. C..

7 recordsLinked to original sources

bioRxiv2025

53BP1 condensates function as bioreactors for NHEJ directed DNA repair and insulators to determine pathway choice

Genomic integrity requires efficient resolution of DNA damage. Non-homologous end joining (NHEJ) is the primary mechanism of DNA double strand break (DSB) repair in mammalian cells and is mediated by 53BP1, a tumor suppressor involved in preventing DSB end-resection and homologous recombination. NHEJ repair foci form following DSB formation, however how mesoscale assembly occurs and whether 53BP1 is the driver of this process are unknown. Also, despite knowledge of the identify of key pathway molecules, the specific functions of mesoscale repair condensates in DNA repair and pathway selectivity are unknown. To address these gaps, we determined the minimal domain of 53BP1 sufficient for phase separation in vitro and identified the key residues that governing its condensation. Utilizing a separation-of-function mutant, we demonstrate that 53BP1and its protein condensation is the core driver of NHEJ foci formation. 53BP1 condensates function as bioreactor compartments, increasing the effective concentration of substrates near double strand breaks and are essential for efficient DNA damage resolution. Additionally, we show that 53BP1 condensates function as insulators around DSB sites to prevent end-resection and direct repair pathway selectivity. Collectively our work reveals a specialized compartment for DNA repair through the spatial clustering of 53BP1 molecules into repair foci essential to maintain genome integrity.

cell biology↗

bioRxiv2024

Spatially ordered zygotic genome activation fulfills embryo quality control

Early embryo development features autonomous, maternally-driven cell divisions that self- organize the multicellular blastula or blastocyst tissue. Maternal control cedes to the zygote starting with the onset of widespread zygotic genome activation (ZGA), which is essential for subsequent cell fate determination and morphogenesis. Intriguingly, although the onset of ZGA is highly regulated at the level of an embryo, it can be non-homogenous and precisely patterned at the single-cell level. We previously demonstrated a stereotyped spatial and temporal ordering of ZGA in a model vertebrate embryo. Unknown, however, was whether this precise ZGA patterning was required for development. To address this fundamental question, we devised a strategy to spatially control cell divisions in the embryo that perturb blastula embryo organization. We demonstrate the feasibility of spatially inverting the cell size pattern of embryos and find that these inverted embryos undergo a flipped pattern of ZGA. Mispatterned ZGA along the animal-vegetal axis causes embryo apoptosis, revealing that gastrula embryos have a built-in quality control system to sense inappropriate ZGA patterning, including regional defects in transcriptional onset. The quality control response is non-autonomous which may depend on anti-apoptotic signals that repress cell death outside of the animal hemisphere. These results reveal the requirement of properly patterned ZGA for normal development and the existence of an embryo quality control response exquisitely tuned to the spatial and temporal ordering of genome activation and zygotic gene expression.

developmental biology↗

bioRxiv2024

Assembly of Hierarchical Multiphase Condensates Using Designer Surfactant Proteins

Biomolecular condensates are complex fluids formed by liquid-liquid phase separation of macromolecules. Similar to other types of soft matter, they feature a range of biophysical properties that distinguish them from the cellular milieu. As a separate phase, they have an identifiable interface that dictates their interaction with the cytoplasm and other membraneless organelles. In this work, we engineer the interface of condensates to build novel hierarchical mesoscale structures from two immiscible disordered proteins: the RGG domain of LAF-1, a RNA-processing protein involved in germ granule assembly and the low complexity domain (LC) of FUS, an RNA binding protein whose aggregation is implicated in age-related neurodegeneration. RGG and FUS LC do not co-partition with one another and instead form discrete protein-rich condensed phases. Despite their apparent immiscibility, we identified conditions that can promote hierarchical assembly, either kinetically trapping one phase in the other, or using a designer surfactant protein that reduces the interfacial tension between the two phases. In addition, we studied factors that impact condensate miscibility and structure formation, including surface properties and viscoelasticity. This study probes the principles that underlie formation and assembly of complex structures from biomolecular condensates and provides a strategy for designing synthetic multiphasic materials capable of spatial partitioning.

biophysics↗

bioRxiv2024

Delivery of Peptide Coacervates to Form Stable Interaction Hubs In Cells

Cells contain membrane-bound and membraneless organelles that operate as spatially distinct biochemical niches. However, these reaction centers lose fidelity due to aging or disease. A grand challenge for biomedicine is restoring or augmenting cellular functionalities. Although commonly tackled by gene replacement therapy, an exciting new strategy is the delivery of protein-based materials that directly interact with and alter intracellular pathways. In this study, we sought to develop long-lasting materials capable of cellular uptake, akin to artificial organelles or synthetic interaction hubs. We show efficient delivery of micron-size peptide-based compartments into a variety of cell types. By loading coacervates with nanobodies and bioPROTACs, we demonstrate successful native target sequestration, and hub bioreactor function to selectively degrade targets inside human cells. These results represent an important step toward the development of synthetic organelles that can be fabricated in vitro and taken up by cells for applications in cell engineering and regenerative medicine.

synthetic biology↗

bioRxiv2023

Determinants of Disordered Protein Co-Assembly Into Discrete Condensed Phases

Cells harbor numerous mesoscale membraneless compartments that house specific biochemical processes and perform distinct cellular functions. These protein and RNA-rich bodies are thought to form through multivalent interactions among proteins and nucleic acids resulting in demixing via liquid-liquid phase separation (LLPS). Proteins harboring intrinsically disordered regions (IDRs) predominate in membraneless organelles. However, it is not known whether IDR sequence alone can dictate the formation of distinct condensed phases. We identified a pair of IDRs capable of forming spatially distinct condensates when expressed in cells. When reconstituted in vitro, these model proteins do not co-partition, suggesting condensation specificity is encoded directly in the polypeptide sequences. Through computational modeling and mutagenesis, we identified the amino acids and chain properties governing homotypic and heterotypic interactions that direct selective condensation. These results form the basis of physicochemical principles that may direct subcellular organization of IDRs into specific condensates and reveal an IDR code that can guide construction of orthogonal membraneless compartments.

biophysics↗

bioRxiv2020

A C. elegans Zona Pellucida domain protein functions via its ZPc domain

Zona Pellucida domain (ZP) proteins are critical components of the bodys external-most protective layers, apical extracellular matrices (aECMs). Although their loss or dysfunction is associated with many diseases, it remains unclear how ZP proteins assemble in aECMs. Current models suggest that ZP proteins polymerize via their ZPn subdomains, while ZPc subdomains modulate ZPn behavior. Using the model organism C. elegans, we investigated the aECM assembly of one ZP protein, LET-653, which shapes several tubes. Contrary to prevailing models, we find that LET-653 localizes and functions via its ZPc domain. Furthermore, the ZPc domain is inhibited by the ZPn domain and cleavage of the LET-653 C-terminus relieves this inhibition. In vitro, the ZPc, but not ZPn, domain formed crystalline aggregates. These data offer a new model for ZP function whereby the ZPc domain is primarily responsible for matrix incorporation and tissue shaping.

genetics↗

bioRxiv2020

Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bio-inspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation. Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the proteins propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

biophysics↗