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mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding

Delarue, M; Brittingham, G P; Pfeffer, S; Surovtsev, I V; Pinglay, S; Kennedy, K J; Schaffer, M; Gutierrez, J I; Sang, D; Poterewicz, G; Chung, J K; Plitzko, J M; Groves, J T; Jacobs-Wagner, C; Engel, B D; Holt, L J
Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles >=20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules <=5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.
PMID: 29937223
ISSN: 1097-4172
CID: 3161522

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence

Neurohr, Gabriel E; Terry, Rachel L; Lengefeld, Jette; Bonney, Megan; Brittingham, Gregory P; Moretto, Fabien; Miettinen, Teemu P; Vaites, Laura Pontano; Soares, Luis M; Paulo, Joao A; Harper, J Wade; Buratowski, Stephen; Manalis, Scott; van Werven, Folkert J; Holt, Liam J; Amon, Angelika
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.
PMID: 30739799
ISSN: 1097-4172
CID: 3655982

Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly

Cuylen-Haering, Sara; Petrovic, Mina; Hernandez-Armendariz, Alberto; Schneider, Maximilian W G; Samwer, Matthias; Blaukopf, Claudia; Holt, Liam J; Gerlich, Daniel W
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 nanometres4-7, 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.
PMID: 32879492
ISSN: 1476-4687
CID: 4615422

The Genome Project-Write

Boeke, Jef D; Church, George; Hessel, Andrew; Kelley, Nancy J; Arkin, Adam; Cai, Yizhi; Carlson, Rob; Chakravarti, Aravinda; Cornish, Virginia W; Holt, Liam; Isaacs, Farren J; Kuiken, Todd; Lajoie, Marc; Lessor, Tracy; Lunshof, Jeantine; Maurano, Matthew T; Mitchell, Leslie A; Rine, Jasper; Rosser, Susan; Sanjana, Neville E; Silver, Pamela A; Valle, David; Wang, Harris; Way, Jeffrey C; Yang, Luhan
PMID: 27256881
ISSN: 1095-9203
CID: 2126732

Microtubules Enhance Mesoscale Effective Diffusivity in the Crowded Metaphase Cytoplasm

Carlini, Lina; Brittingham, Gregory P; Holt, Liam J; Kapoor, Tarun M
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.
PMID: 32818469
ISSN: 1878-1551
CID: 4578222

Ancestral reconstruction reveals mechanisms of ERK regulatory evolution

Sang, Dajun; Pinglay, Sudarshan; Wiewiora, Rafal P; Selvan, Myvizhi E; Lou, Hua Jane; Chodera, John D; Turk, Benjamin E; Gümüş, Zeynep H; Holt, Liam J
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.
PMID: 31407663
ISSN: 2050-084x
CID: 4043262

Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution

Holt, Liam J; Tuch, Brian B; Villen, Judit; Johnson, Alexander D; Gygi, Steven P; Morgan, David O
To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.
PMID: 19779198
ISSN: 1095-9203
CID: 1876032

Positive feedback sharpens the anaphase switch

Holt, Liam J; Krutchinsky, Andrew N; Morgan, David O
At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behaviour are unclear. Anaphase is initiated when a ubiquitin ligase, the anaphase-promoting complex (APC), triggers the destruction of securin, thereby allowing separase, a protease, to disrupt sister-chromatid cohesion. Here we demonstrate that the cyclin-dependent kinase 1 (Cdk1)-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 (refs 5 and 6), our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.
PMID: 18552837
ISSN: 1476-4687
CID: 1876062

Control of nuclear size by osmotic forces in Schizosaccharomyces pombe

Lemière, Joël; Real-Calderon, Paula; Holt, Liam J; Fai, Thomas G; Chang, Fred
The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.
PMID: 35856499
ISSN: 2050-084x
CID: 5279082

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation

Pinglay, Sudarshan; Bulajić, Milica; Rahe, Dylan P; Huang, Emily; Brosh, Ran; Mamrak, Nicholas E; King, Benjamin R; German, Sergei; Cadley, John A; Rieber, Lila; Easo, Nicole; Lionnet, Timothée; Mahony, Shaun; Maurano, Matthew T; Holt, Liam J; Mazzoni, Esteban O; Boeke, Jef D
Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.
PMID: 35771912
ISSN: 1095-9203
CID: 5268842