Biology subjects

Langdon, E. M.

Publications and source records attributed to Langdon, E. M..

3 recordsLinked to original sources

bioRxiv2019

3D neural network-based particle tracking reveals spatial heterogeneity of the cytosol

The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity, the lack of truly passive probes, and the need for probes of many sizes to accurately describe the state across scales. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). Using these probes to spatially resolve diffusivity of a cytoplasmic volume within a cell requires accurate and automated 3D tracking methods. We developed an image analysis pipeline for whole-cell imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We apply a neural network to track particles in 3D, generate surface meshes to project data on representations of the cell, and create dynamic visualizations of local diffusivities. Using this pipeline, we have found that there is remarkable variability in the properties of the cytosol both within a single cell and between cells. By analyzing the spatial diffusivity patterns, we saw an enrichment of low diffusivity zones at hyphal tips and near some nuclei. These results show that the physical state of the cytosol varies spatially within a single cell and exhibits significant cell-to-cell variability. Thus, molecular crowding contributes to heterogeneity within individual cells and across populations.

cell biology↗

bioRxiv2018

Regulation of the error-prone DNA polymerase polκ by oncogenic signaling and its contribution to drug resistance

Mutations in the proofreading domains of the replicative DNA polymerases pol{delta} and pol{varepsilon} are associated with elevated mutation rates in cancer, but the roles of other DNA polymerases in tumorigenesis remain poorly understood. One such polymerase is pol{kappa}, an enzyme that plays a key role in translesion synthesis. pol{kappa} contributes to cell survival in the face of DNA damage but can be highly mutagenic due to lack of a proofreading domain. Here we demonstrate that cancer cells under stress from oncogene inhibition upregulate pol{kappa} and shift its localization from the cytoplasm to the nucleus. This effect can be phenocopied by mTOR inhibition or glucose deprivation, analogous to stress-induced mutagenesis in E. coli whereby cell stress and nutrient deprivation can upregulate and activate DinB/pol IV (the bacterial orthologue of pol{kappa}). We find that cancer cells normally sequester pol{kappa} in the cytoplasm via exportin-1, likely to prevent excess mutagenesis from the error-prone nature of this polymerase. Subverting the normal nuclear-cytoplasmic shuttling by forced overexpression of nuclear pol{kappa} increases resistance of melanoma cells to the BRAFV600E inhibitor vemurafenib. This data suggests a mechanism by which cancer cells regulate the expression and localization of the error-prone polymerase pol{kappa}, abrogation of which can contribute to drug resistance.\n\nOne Sentence Summary: Cancer cells under stress from oncogene or mTOR inhibition dysregulate the error-prone DNA polymerase pol{kappa}, which contributes to drug resistance in melanoma cells.

cancer biology↗

bioRxiv2017

mRNA structure determines specificity of a polyQ-driven phase separation

RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.\n\nOne Sentence SummaryIdentity in cellular, phase-separated compartments arises from RNA-RNA complexes encoded by mRNA secondary structures.

cell biology↗