Matthew C. Good

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Scaffold Proteins: Hubs for Controlling the Flow of Cellular Information

The spatial and temporal organization of molecules within a cell is critical for coordinating the many distinct activities carried out by the cell. In an increasing number of biological signaling processes, scaffold proteins have been found to play a central role in physically assembling the relevant molecular components. Although most scaffolds use a simple tethering mechanism to increase the efficiency of interaction between individual partner molecules, these proteins can also exert complex allosteric control over their partners and are themselves the target of regulation. Scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. As a result, scaffold proteins have been exploited by evolution, pathogens, and cellular engineers to reshape cellular behavior.

Deciphering Protein Kinase Specificity Through Large-Scale Analysis of Yeast Phosphorylation Site Motifs

A high-throughput peptide array approach reveals insight into kinase substrates and specificity. Exploring Kinase Selectivity Kinases are master regulators of cellular behavior. Because of the large number of kinases and the even larger number of substrates, approaches that permit global analysis are valuable tools for investigating kinase biology. Mok et al. identified the phosphorylation site selectivity for 61 of the 122 kinases in Saccharomyces cerevisiae by screening a miniaturized peptide library. By integrating these data with other data sets and structural information, they revealed information about the relationship between kinase catalytic residues and substrate selectivity. They also identified and experimentally verified substrates for kinases, including one for which limited functional information was previously available, showing the potential for this type of analysis as a launching point for the exploration of the biological functions of kinases. Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.

The Ste5 Scaffold Directs Mating Signaling by Catalytically Unlocking the Fus3 MAP Kinase for Activation

The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3s activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7.

Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

Scaling Spindle Size The difficulty of modulating cell size in vivo has made it hard to test hypotheses for organelle size scaling during development. To this end, Hazel et al. (p. 853) and Good et al. (p. 856) developed microfluidic systems in which cytoplasmic extracts are encapsulated in compartments with definable size. The size of mitotic spindles assembled within cell-free extracts scaled with the volume of the compartment within which the spindle assembled. The findings suggest that the diminished availability of cytoplasmic components, notably tubulin, concomitant with cell size reduction, prescribes a smaller spindle size. Microfluidic techniques reveal how mitotic spindle size is regulated by titratable cytosolic factors. Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures.

Preparation of Cellular Extracts from Xenopus Eggs and Embryos

Cell-free cytoplasmic extracts prepared from Xenopus eggs have been used extensively to recapitulate and characterize intracellular events in vitro. Egg extracts can be induced to transit the cell cycle and reconstitute assembly of dynamic structures including the interphase nucleus and the mitotic spindle. In this protocol, methods are described for preparing crude cytoplasmic extracts from Xenopus eggs and embryos that are arrested in metaphase of the cell cycle. The basic protocol uses unfertilized Xenopus laevis eggs, which are crushed by centrifugation in the presence of EGTA to preserve the natural cytostatic factor (CSF) activity that maintains high levels of Cdk1/cyclin B kinase and metaphase arrest. In the second method, the basic procedure is adapted for Xenopus tropicalis eggs with minor modifications to accommodate differences in frog size, timing of egg laying, and temperature and salt sensitivity. The third variation takes advantage of the synchronous divisions of fertilized X. laevis eggs to generate extracts from embryos, which are arrested in metaphase by the addition of nondegradable cyclin B and an inhibitor of the anaphase-promoting complex (APC) that together stabilize Cdk1/cyclin B kinase activity. Because they are obtained in much smaller amounts and their cell cycles are less perfectly synchronized, extracts prepared from embryos are less robust than egg extracts. X. laevis egg extracts have been used to study a wide range of cellular processes. In contrast, X. tropicalis egg extracts and X. laevis embryo extracts have been used primarily to characterize molecular mechanisms regulating spindle and nuclear size.

Encapsulation of Xenopus Egg and Embryo Extract Spindle Assembly Reactions in Synthetic Cell-Like Compartments with Tunable Size.

Methods are described for preparing Xenopus laevis egg and embryo cytoplasm and encapsulating extract spindle assembly reactions in cell-like compartments to investigate the effects of cell size on intracellular assembly. Cytoplasm prepared from the eggs or embryos of individual frogs is screened for the ability to form interphase nuclei and metaphase spindles, and subsequently packaged, along with DNA, into droplets of varying size using microfluidics. The dimensions of these cell-like droplets are specified to match the range of cell diameters present in early embryo development. The scaling relationship between droplets and spindles is quantified using live fluorescence imaging on a spinning-disk confocal microscope. By comparing the encapsulated assembly of spindles formed from cytoplasmic extracts prepared from embryos at distinct stages of Xenopus early development, the influence of cell composition and cell size on spindle scaling can be evaluated. Because the extract system is biochemically tractable, the function of individual proteins in spindle scaling can be evaluated by supplementing or depleting factors in the cytoplasm.

Controllable protein phase separation and modular recruitment to form responsive membraneless organelles

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.Designer organelles with new biochemical functionalities are of great interest in synthetic biology and cellular engineering. Here the authors present a single-protein-based platform for generating synthetic membraneless compartments that is capable of enzymatically-triggered alterations to phase behavior and of recruiting and concentrating cargo proteins.

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts

Cell‐free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell‐size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell‐free cytoplasm is confined using a two‐ or three‐dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell‐free cytoplasm and confinement technologies to generate synthetic cell‐like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.