Regulatable assembly of synthetic microtubule architectures using engineered microtubule-associated protein-IDR condensates.

Microtubule filaments are assembled into higher-order structures using microtubule-associated proteins. However, synthetic MAPs that direct the formation of new structures are challenging to design, as nanoscale biochemical activities must be organized across micron length-scales. Here, we develop modular MAP-IDR condensates (synMAPs) that enable inducible assembly of higher-order microtubule structures for synthetic exploration in vitro and in mammalian cells.

A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design.

Cells self-organize molecules in space and time to generate complex behaviors, but we lack synthetic strategies for engineering spatiotemporal signaling. We present a programmable reaction-diffusion platform for designing protein oscillations, patterns, and circuits in mammalian cells using two bacterial proteins, MinD and MinE (MinDE). MinDE circuits act like "single-cell radios," emitting frequency-barcoded fluorescence signals that can be spectrally isolated and analyzed using digital signal processing tools.

Regulatable assembly of synthetic microtubule architectures using engineered MAP-IDR condensates.

Microtubules filaments are assembled into higher-order structures and machines critical for cellular processes using microtubule-associated proteins (MAPs). However, the design of synthetic MAPs that direct the formation of new structures in cells is challenging, as nanoscale biochemical activities must be organized across micron length-scales. Here we develop synthetic MAP-IDR condensates (synMAPs) that provide tunable and regulatable assembly of higher-order microtubule structures and in mammalian cells.

Comparative profiling of cellular gait on adhesive micropatterns defines statistical patterns of activity that underlie native and cancerous cell dynamics.

Cell dynamics are powered by patterns of activity, but it is not straightforward to quantify these patterns or compare them across different environmental conditions or cell-types. Here we digitize the long-term shape fluctuations of metazoan cells grown on micropatterned fibronectin islands to define and extract statistical features of cell dynamics without the need for genetic modification or fluorescence imaging. These shape fluctuations generate single-cell morphological signals that can be decomposed into two major components: a continuous, slow-timescale meandering of morphology about an average steady-state shape; and short-lived "events" of rapid morphology change that sporadically occur throughout the timecourse.

Ciliate behavior: blueprints for dynamic cell biology and microscale robotics.

Place a drop of pond water under the microscope, and you will likely find an ocean of extraordinary and diverse single-celled organisms called . This remarkable group of single-celled organisms wield microtubules, active systems, electrical signaling, and chemical sensors to build intricate geometrical structures and perform complex behaviors that can appear indistinguishable from those of macroscopic animals. Advances in computer vision and machine learning are making it possible to completely digitize and track the dynamics of complex ciliates and mine these data for the hidden structure, patterns, and motifs that are responsible for their behaviors.

Coupled Active Systems Encode an Emergent Hunting Behavior in the Unicellular Predator Lacrymaria olor.

Many single-celled protists use rapid morphology changes to perform fast animal-like behaviors. To understand how such behaviors are encoded, we analyzed the hunting dynamics of the predatory ciliate Lacrymaria olor, which locates and captures prey using the tip of a slender "neck" that can rapidly extend more than seven times its body length (500 μm from its body) and retract in seconds. By tracking single cells in real-time over hours and analyzing millions of sub-cellular postures, we find that these fast extension-contraction cycles underlie an emergent hunting behavior that comprehensively samples a broad area within the cell's reach.

Reverse engineering GTPase programming languages with reconstituted signaling networks.

The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors.

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.

The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems.

Specificity in endoplasmic reticulum-stress signaling in yeast entails a step-wise engagement of HAC1 mRNA to clusters of the stress sensor Ire1.

Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing.

Product binding enforces the genomic specificity of a yeast polycomb repressive complex.

We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute.

Exploitation of latent allostery enables the evolution of new modes of MAP kinase regulation.

Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets coevolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages.

Starvation-associated genome restructuring can lead to reproductive isolation in yeast.

Knowledge of the mechanisms that lead to reproductive isolation is essential for understanding population structure and speciation. While several models have been advanced to explain post-mating reproductive isolation, experimental data supporting most are indirect. Laboratory investigations of this phenomenon are typically carried out under benign conditions, which result in low rates of genetic change unlikely to initiate reproductive isolation.

Conformational control of the Ste5 scaffold protein insulates against MAP kinase misactivation.

Cells reuse signaling proteins in multiple pathways, raising the potential for improper cross talk. Scaffold proteins are thought to insulate against such miscommunication by sequestering proteins into distinct physical complexes. We show that the scaffold protein Ste5, which organizes the yeast mating mitogen-activated protein kinase (MAPK) pathway, does not use sequestration to prevent misactivation of the mating response.

Ers1 links HP1 to RNAi.

Pericentromeric heterochromatin formation is mediated by repressive histone H3 lysine 9 methylation (H3K9Me) and its recognition by HP1 proteins. Intriguingly, in many organisms, RNAi is coupled to this process through poorly understood mechanisms. In Schizosaccharomyces pombe, the H3-K9 methyltransferase Clr4 and the heterochromatin protein 1 (HP1) ortholog Swi6 are critical for RNAi, whereas RNAi stimulates H3K9Me.

Coupled 5' nucleotide recognition and processivity in Xrn1-mediated mRNA decay.

Messenger RNA decay plays a central role in the regulation and surveillance of eukaryotic gene expression. The conserved multidomain exoribonuclease Xrn1 targets cytoplasmic RNA substrates marked by a 5' monophosphate for processive 5'-to-3' degradation by an unknown mechanism. Here, we report the crystal structure of an Xrn1-substrate complex.

Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity.

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity.

Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation.

GW182-family proteins are essential for microRNA-mediated translational repression and deadenylation in animal cells. Here we show that a conserved motif in the human GW182 paralog TNRC6C interacts with the C-terminal domain of polyadenylate binding protein 1 (PABC) and present the crystal structure of the complex. Mutations at the complex interface impair mRNA deadenylation in mammalian cell extracts, suggesting that the GW182-PABC interaction contributes to microRNA-mediated gene silencing.

Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense.

Acquired immunity in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into clustered regularly interspaced short palindromic repeats (CRISPRs). This nucleic acid-based immune system is mediated by a variable cassette of up to 45 protein families that represent distinct immune system subtypes. CRISPR-associated gene 1 (cas1) encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown.

Direct link between RACK1 function and localization at the ribosome in vivo.

The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-A crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function.

Starvation induces genomic rearrangements and starvation-resilient phenotypes in yeast.

Evolution has shaped a wide variety of genomes across eukaryotic taxa. However, the forces that shape the genomes are generally unknown. Because organisms in nature commonly experience prolonged periods of nutrient depletion, we posit that diverse demographic, physiological, and genomic responses to starvation can occur.