Holt Liam J.

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Holt
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Liam J.
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  • Article
    Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization
    (Elsevier, 2022-02-28) Molines, Arthur T. ; Lemière, Joë ; Gazzola, Morgan ; Steinmark, Ida Emilie ; Edrington, Claire H. ; Hsu, Chieh-Ting ; Real-Calderon, Paula ; Suhling, Klaus ; Goshima, Gohta ; Holt, Liam J. ; Thery, Manuel ; Brouhard, Gary J. ; Chang, Fred
    The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.
  • Article
    Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking
    (American Society for Cell Biology, 2020-06-29) McLaughlin, Grace A. ; Langdon, Erin M. ; Crutchley, John M. ; Holt, Liam J. ; Forest, M. Gregory ; Newby, Jay M. ; Gladfelter, Amy S.
    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. 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). We developed an image analysis pipeline for 3D 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 applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.