Biology subjects

McLaughlin, G. A.

Publications and source records attributed to McLaughlin, G. A..

3 recordsLinked to original sources

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2025

A phase oscillator model of cell cycles reveals nuclear density control in a branched fungal network

Multinucleate cells are widespread in biology, from human skeletal muscle and placenta to many filamentous fungi, yet their basic cell biology remains poorly understood. Maintaining an appropriate nuclear-to-cytoplasmic ratio is essential across cell types for physiological function, and mechanisms of size control have been extensively studied in mononucleate cells. Much less is known about how comparable control is achieved in cells where many nuclei share a common cytoplasm. The filamentous fungus Ashbya gossypii forms a branching mycelial network in which individual nuclei divide asynchronously, while the number of nuclei per cell volume (the nuclear density) is tightly controlled. How global regulation of nuclear density coexists with local cell cycle asynchrony remains unclear. We address this by combining a mathematical model of nuclear division in a growing and branching cell with live-cell microscopy. We model nuclei as a dividing population of phase oscillators within a branching cell network and parameterize the model with measurements from Ashbya cells. The model demonstrates that asynchrony is required to prevent large density fluctuations that would result from synchronous division, and that introducing a nuclear-density checkpoint to the cell cycles leads to synchrony if it is the only mechanism of density control. We find that coupling branch formation to nuclear density both stabilizes nuclear density and prevents the emergence of synchronous cycles. Our results indicate that asynchronous nuclear cycles together with density-responsive branching maintain a constant nuclear density, revealing a strategy for regulating the nuclear-to-cytoplasmic ratio in large multinucleate cells. SignificanceMultinucleate cells appear in diverse biological contexts, from human tissues to filamentous fungi, yet many fundamental aspects of their cell biology are still unclear. Regulating the nuclear-to-cytoplasmic ratio is important across cell types, and little is known about how this is achieved in large, multinucleate fungal cells. Using mathematical modeling and live-cell imaging, we identify how the density of nuclei is controlled in a growing and branching fungal mycelial network. We find that asynchronous nuclear cycles together with density-responsive branching can stabilize the nuclear-to-cytoplasmic ratio within a growing fungal network. This illustrates how large multinucleate cells can control nuclear density even as their morphology becomes increasingly complex.

cell biology↗

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↗