Faculty Bibliography

Metabolism and mechanics are intrinsically intertwined. External forces, sensed through the cytoskeleton or distortion of the cell and organelles, induce metabolic changes in the cell. The resulting changes in metabolism, in turn, feed back to regulate every level of cell biology, including the mechanical properties of cells and tissues. Here we examine the links between metabolism and mechanics, highlighting signalling pathways involved in the regulation and response to cellular mechanosensing. We consider how forces and metabolism regulate one another through nanoscale molecular sensors, micrometre-scale cytoskeletal networks, organelles and dynamic biomolecular condensates. Understanding this cross-talk will create diagnostic and therapeutic opportunities for metabolic disorders such as cancer, cardiovascular pathologies and obesity.

Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation¹. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores². During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix³. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores². These pores have a size limit of 39 nanometres^4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis⁸, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.

Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40 nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by ~30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments.

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. [Media: see text] [Media: see text] [Media: see text].

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1- encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy and paste" mechanism. ORF1p is an RNA-binding protein and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p, only 16% of trialanine mutants retained near-wild-type activity. All ORF1p mutants formed near-wild-type levels of mRNA transcripts and seventy-five percent formed near-wild-type levels of protein. Two ORF1p mutants present a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis, but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins and target DNA throughout the L1 life cycle.

Protein kinases are crucial to coordinate cellular decisions and therefore their activities are strictly regulated. Previously we used ancestral reconstruction to determine how CMGC group kinase specificity evolved (Howard et al., 2014). In the present study, we reconstructed ancestral kinases to study the evolution of regulation, from the inferred ancestor of CDKs and MAPKs, to modern ERKs. Kinases switched from high to low autophosphorylation activity at the transition to the inferred ancestor of ERKs 1 and 2. Two synergistic amino acid changes were sufficient to induce this change: shortening of the β3-αC loop and mutation of the gatekeeper residue. Restoring these two mutations to their inferred ancestral state led to a loss of dependence of modern ERKs 1 and 2 on the upstream activating kinase MEK in human cells. Our results shed light on the evolutionary mechanisms that led to the tight regulation of a kinase that is central in development and disease.

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae. This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.