Crowding increases the tendency of macromolecules to aggregate and phase separate, and high crowding can induce glass-like states of cytoplasm. To explore the effect of crowding in a well-characterized model cytoplasm we developed methods to selectively concentrate components larger than 25 kDa from Xenopus egg extracts. When crowding was increased 1.4x, the egg cytoplasm demixed into two liquid phases of approximately equal volume. One of the phases was highly enriched in glycogen while the other had a higher protein concentration. Glycogen hydrolysis blocked or reversed demixing. Quantitative proteomics showed that the glycogen phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are in complexes with especially high native molecular weight. The glycogen phase was depleted of ribosomes, ER and mitochondria. These results inform on the physical nature of a glycogen-rich cytoplasm and suggest a role of demixing in the localization of glycogen particles in tissue cells.

Drugs that modulate microtubule (MT) dynamics are well-characterized at the molecular level, yet the mechanisms linking these molecular effects to their distinct clinical outcomes remain unclear. Several MT-destabilizing drugs, including vinblastine, combretastatin A4, and plinabulin, are widely used, or are under evaluation for cancer treatment. Although all three depolymerize MTs, they do so through distinct biochemical mechanisms. Furthermore, their clinical profiles and therapeutic uses differ considerably. To investigate whether differential modulation of molecular pathways might account for clinical differences, we compared gene expression and signaling pathway responses in human pulmonary microvascular endothelial cells (HPMECs), alongside the MT-stabilizing drug docetaxel and the pro-inflammatory cytokine TNF-α. RNA-sequencing and phosphoproteomics revealed that all three MT destabilizers triggered equivalent molecular responses. The substantial changes in gene expression caused by MT destabilization were completely dependent on Rho family GTPase activation. These findings suggest that the distinct clinical profiles of the destabilizing drugs depend on differences in pharmacokinetics (PK) and tissue distribution rather than molecular actions. The washout rate of the three drugs differed, which likely translates to PK differences. Our data provide insights into how MT destabilization triggers signaling changes, potentially explaining how these drugs induce cell cycle re-entry in quiescent cells and how plinabulin ameliorates chemotherapy-induced neutropenia.

Assembly of cells into epithelial layers marks the early steps of tissue organization and embryonic development in most animals. Although several conserved proteins are known to be essential for epithelialization in bilaterians, it is unclear whether these are sufficient to drive epithelial organization in all metazoans. Using proteomics and knockdown approaches in embryos of the early-branching sea anemone Nematostella vectensis, we identified the neural cell adhesion molecule NCAM2 to be essential for organization of the primary epithelial layer. NCAM2 belongs to the immunoglobulin family of cell adhesion molecules with roles in inter-cellular adhesion and signaling. In this work, we show that NCAM2 is enriched at the apical cell junctions and is required for Nematostella epithelial cell identity. Importantly, embryos depleted for NCAM2 fail to gastrulate. Both whole-embryo germ layer patterning and tissue changes required for gastrulation are impaired. Together, our data show that in Nematostella NCAM2 is required for both epithelial and germ layer organization.

Loss of the tubulin-binding protein STMN2 is implicated in amyotrophic lateral sclerosis (ALS) but how it protects neurons is not known. STMN2 is known to turn over rapidly and accumulate at axotomy sites. We confirmed fast turnover of STMN2 in U2OS cells and iPSC-derived neurons and showed that degradation occurs mainly by the ubiquitin-proteasome system. The membrane targeting N-terminal domain of STMN2 promoted fast turnover, whereas its tubulin binding stathmin-like domain (SLD) promoted stabilization. Proximity labeling and imaging showed that STMN2 localizes to trans-Golgi network membranes and that tubulin binding reduces this localization. Pull-down assays showed that tubulin prefers to bind to soluble over membrane-bound STMN2. Our data suggest that STMN2 interconverts between a soluble form that is rapidly degraded unless bound to tubulin and a membrane-bound form that does not bind tubulin. We propose that STMN2 is sequestered and stabilized by tubulin binding, while its neuroprotective function depends on an unknown molecular activity of its membrane-bound form.

The small, tubulin-binding protein STMN2 is highly expressed in neurons and is implicated in amyotrophic lateral sclerosis. STMN2 degrades rapidly and accumulates at axotomy sites, suggesting fast turnover is crucial for its neuroprotective function. We show that STMN2 was primarily degraded by the ubiquitin-proteasome system. Its membrane-targeting N-terminal domain promoted fast turnover, whereas its tubulin-binding domain promoted stabilization. Proximity labeling and imaging showed that tubulin binding reduced STMN2 targeting to trans-Golgi network membranes. Pull-down assays showed that tubulin binds preferentially to soluble over membrane-bound STMN2. Our observations suggest that STMN2 interconverts between a soluble, tubulin-bound form and a membrane-bound, tubulin-free form, and is rapidly degraded when released from both membranes and tubulin. We propose that tubulin binding sequesters and stabilizes STMN2, while its neuroprotective function involves an unknown membrane activity.

Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 h post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.

Microtubules physically organize eukaryotic cells by serving as structural elements and polarized transport tracks. This article advances the hypothesis that dynamic microtubules also serve as sensors of cell shape and cytoplasmic state, building on ideas proposed for higher plant cells. Microtubule polymerization dynamics and lattice structure are sensitive to mechanical, chemical, and signaling inputs that alter the balance between microtubules and soluble tubulin and regulate MAP binding affinity. These changes are detected by transducers, which include the GTP exchange factor GEF-H1 (ARHGEF2) and MARK family kinases. The resulting signals regulate cytoplasmic behavior, gene expression, and tissue physiology. The microtubule-destabilizing drugs colchicine and plinabulin may mimic sensing of pathophysiological cues by microtubules, leading to activation of gene expression programs that promote cell survival, growth, and repair, which account for the therapeutic actions of the drugs. In tissue cells with stable morphologies, the sensory functions of microtubules may be as or more important than their architectural functions. This reframing of microtubule biology suggests new directions for mechanistic inquiry and drug discovery.

Eukaryotic cells direct toxic misfolded proteins to various quality control pathways based on their chemical properties and aggregation status. Aggregated proteins are targeted to selective autophagy or specifically sequestered into the "aggresome", a perinuclear inclusion at the microtubule-organizing center (MTOC). However, the mechanism for selective aggresome recruitment remains unclear. To investigate this process, here we reconstitute MTOC-directed aggregate transport in Xenopus laevis egg extract using AgDD, a chemically inducible aggregation system. High-resolution single-particle tracking reveals that dynein-mediated aggregate transport is highly episodic, with average velocity positively correlating with aggregate size. Mechanistic modeling suggests that recurrent formation of the dynein transport complex biases larger aggregates towards active transport, compensating for the slowdown due to viscosity. Both episodic transport and positive size selectivity are conferred by aggresome-specific dynein adapters. Coupling an aggresome adapter to polystyrene beads recapitulates positive size selectivity in transport, while recruiting conventional dynein adapters to protein aggregates perturbs aggresome formation and reverses the size selectivity.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

Cells are essential to understanding health and disease, yet traditional models fall short of modeling and simulating their function and behavior. Advances in AI and omics offer groundbreaking opportunities to create an AI virtual cell (AIVC), a multi-scale, multi-modal large-neural-network-based model that can represent and simulate the behavior of molecules, cells, and tissues across diverse states. This Perspective provides a vision on their design and how collaborative efforts to build AIVCs will transform biological research by allowing high-fidelity simulations, accelerating discoveries, and guiding experimental studies, offering new opportunities for understanding cellular functions and fostering interdisciplinary collaborations in open science.

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the non-structural viral membrane protein nsp4 is the key DMV pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, with broad spectrum anti-coronaviral activity. ### Competing Interest Statement The authors have declared no competing interest.

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining. Nsp4 interacts with nsp3 on the cytoplasmic side and with the viral replicase inside the DMV. Newly synthesized mRNAs exit the DMV into the cytoplasm, passing through a narrow ring of conserved nsp4 residues. Steric constraints imposed by the ring predict that modified nucleobases block mRNA transit, resulting in broad-spectrum anticoronaviral activity.

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al ., Cell Rep. 33 , 108234 (2020); L. Miorin et al ., Proc. Natl. Acad. Sci. U.S.A. 117 , 28344–28354 (2020); and M. Frieman et al ., J. Virol. 81 , 9812–9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98–RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98–RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Supplementary Figure 6a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Video 1 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by <i>In Vivo</i> Microscopy and Single-Cell Pharmacodynamics

Keratins are the most diverse family of intermediate filaments and are expressed in most epithelial tissues and malignancies. They form highly stable polymers that need to be cut through during cytokinesis. Previous work suggested a role of phosphorylation, but keratin regulation during cell division is not understood in detail. Depletion of Keratin 8 in an epithelial cancer cell line (HeLa) caused chromosome segregation and cytokinesis defects. Aurora B kinase localization to midzones and cleavage furrows was reduced in Keratin 8 knockouts, suggesting that Keratin 8 helps scaffold Aurora B during cytokinesis. We mapped eleven Aurora B kinase sites in Keratin 8 that were associated with cell division. Keratin 8 S34 phosphorylation occurred specifically at the cleavage furrow and persisted at the midzone until the end of cytokinesis. Inhibition of Aurora B kinase or non-phosphorylatable Keratin 8 mutant prevented the disassembly of keratin bundles at the cleavage furrow, which blocked furrow ingression. Our data reveal a functional inter-dependency between Keratin 8 and Aurora B kinase in epithelial cells. Keratin 8 promotes the translocation of Aurora B to the midzone. Aurora B kinase then generates a local zone of Keratin phosphorylation which promotes keratin filament disassembly and allows the cleavage furrow to cut through the keratin network.

Supplementary Figure 4a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Figure 5a from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

Supplementary Table S3 from Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Supplementary Video 6 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by <i>In Vivo</i> Microscopy and Single-Cell Pharmacodynamics