GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Promiscuity and specificity of eukaryotic glycosyltransferases.

Glycosyltransferases are a large family of enzymes responsible for covalently linking sugar monosaccharides to a variety of organic substrates. These enzymes drive the synthesis of complex oligosaccharides known as glycans, which play key roles in inter-cellular interactions across all the kingdoms of life; they also catalyze sugar attachment during the synthesis of small-molecule metabolites such as plant flavonoids. A given glycosyltransferase enzyme is typically responsible for attaching a specific donor monosaccharide, via a specific glycosidic linkage, to a specific moiety on the acceptor substrate. However these enzymes are often promiscuous, able catalyze linkages between a variety of donors and acceptors. In this review we discuss distinct classes of glycosyltransferase promiscuity, each illustrated by enzymatic examples from small-molecule or glycan synthesis. We highlight the physical causes of promiscuity, and its biochemical consequences. Structural studies of glycosyltransferases involved in glycan synthesis show that they make specific contacts with 'recognition motifs' that are much smaller than the full oligosaccharide substrate. There is a wide range in the sizes of glycosyltransferase recognition motifs: highly promiscuous enzymes recognize monosaccharide or disaccharide motifs across multiple oligosaccharides, while highly specific enzymes recognize large, complex motifs found on few oligosaccharides. In eukaryotes, the localization of glycosyltransferases within compartments of the Golgi apparatus may play a role in mitigating the glycan variability caused by enzyme promiscuity.

Ancient dynamin segments capture early stages of host-mitochondrial integration.

Eukaryotic cells use dynamins-mechano-chemical GTPases--to drive the division of endosymbiotic organelles. Here we probe early steps of mitochondrial and chloroplast endosymbiosis by tracing the evolution of dynamins. We develop a parsimony-based phylogenetic method for protein sequence reconstruction, with deep time resolution. Using this, we demonstrate that dynamins diversify through the punctuated transformation of sequence segments on the scale of secondary-structural elements. We find examples of segments that have remained essentially unchanged from the 1.8-billion-y-old last eukaryotic common ancestor to the present day. Stitching these together, we reconstruct three ancestral dynamins: The first is nearly identical to the ubiquitous mitochondrial division dynamins of extant eukaryotes, the second is partially preserved in the myxovirus-resistance--like dynamins of metazoans, and the third gives rise to the cytokinetic dynamins of amoebozoans and plants and to chloroplast division dynamins. The reconstructed sequences, combined with evolutionary models and published functional data, suggest that the ancestral mitochondrial division dynamin also mediated vesicle scission. This bifunctional protein duplicated into specialized mitochondrial and vesicle variants at least three independent times--in alveolates, green algae, and the ancestor of fungi and metazoans-accompanied by the loss of the ancient prokaryotic mitochondrial division protein FtsZ. Remarkably, many extant species that retain FtsZ also retain the predicted ancestral bifunctional dynamin. The mitochondrial division apparatus of such organisms, including amoebozoans, red algae, and stramenopiles, seems preserved in a near-primordial form.

Organelle acidification: an ancient cellular leak detector.

Intracellular membrane-bounded organelles of eukaryotic cells transiently contact the extracellular environment during endocytosis and secretion. Such contacts must be precisely timed to prevent leakage of cargo. I argue that early eukaryotes evolved organelle acidification as a way to detect and prevent leakage.

Using stochastic cell division and death to probe minimal units of cellular replication.

The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in Escherichia coli populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term 'widgets' reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget's molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

Universal Poisson Statistics of mRNAs with Complex Decay Pathways.

Messenger RNA (mRNA) dynamics in single cells are often modeled as a memoryless birth-death process with a constant probability per unit time that an mRNA molecule is synthesized or degraded. This predicts a Poisson steady-state distribution of mRNA number, in close agreement with experiments. This is surprising, since mRNA decay is known to be a complex process. The paradox is resolved by realizing that the Poisson steady state generalizes to arbitrary mRNA lifetime distributions. A mapping between mRNA dynamics and queueing theory highlights an identifiability problem: a measured Poisson steady state is consistent with a large variety of microscopic models. Here, I provide a rigorous and intuitive explanation for the universality of the Poisson steady state. I show that the mRNA birth-death process and its complex decay variants all take the form of the familiar Poisson law of rare events, under a nonlinear rescaling of time. As a corollary, not only steady-states but also transients are Poisson distributed. Deviations from the Poisson form occur only under two conditions, promoter fluctuations leading to transcriptional bursts or nonindependent degradation of mRNA molecules. These results place severe limits on the power of single-cell experiments to probe microscopic mechanisms, and they highlight the need for single-molecule measurements.

Current approaches in evolution: from molecules to cells and organisms.

This is an exciting time to be an evolutionary biologist. Indeed, it is difficult to keep up with all the studies that fall under the broad category of "Evolution" since they span species, traits, and scales of organization. This special issue gives a flavor of exciting new approaches in evolutionary biology, but also emphasizes universal themes. The reviews contained here discuss important aspects of molecular evolution at multiple scales, from individual proteins to complex regulatory networks, as well as from unicellular organisms to macroscopic traits in animals. Though the model systems are diverse, the issues addressed are fundamental: the origin of evolutionary novelties, and the forces that drive them to fixation.

Wisdom of (molecular) crowds: How a snake's temperaturesensing superpower separates information from misinformation.

The namesake pit organ of pit vipers and other temperature-sensing snakes is a remarkable biological thermometer, one that converts infrared light into an electrical signal (Bullock and Diecke 1956). The organ is arranged like a pinhole camera, with a small outward-facing opening covering a pit membrane dense with neuronal projections. This geometry ensures that only light from a narrow angular cone lands on the membrane. By reorienting its head to scan its surroundings, the snake can precisely detect and localise warm-blooded prey even in complete darkness.

New organelles by gene duplication in a biophysical model of eukaryote endomembrane evolution.

Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification.

Prediction by promoter logic in bacterial quorum sensing.

Quorum-sensing systems mediate chemical communication between bacterial cells, coordinating cell-density-dependent processes like biofilm formation and virulence-factor expression. In the proteobacterial LuxI/LuxR quorum sensing paradigm, a signaling molecule generated by an enzyme (LuxI) diffuses between cells and allosterically stimulates a transcriptional regulator (LuxR) to activate its cognate promoter (pR). By expressing either LuxI or LuxR in positive feedback from pR, these versatile systems can generate smooth (monostable) or abrupt (bistable) density-dependent responses to suit the ecological context. Here we combine theory and experiment to demonstrate that the promoter logic of pR - its measured activity as a function of LuxI and LuxR levels - contains all the biochemical information required to quantitatively predict the responses of such feedback loops. The interplay of promoter logic with feedback topology underlies the versatility of the LuxI/LuxR paradigm: LuxR and LuxI positive-feedback systems show dramatically different responses, while a dual positive/negative-feedback system displays synchronized oscillations. These results highlight the dual utility of promoter logic: to probe microscopic parameters and predict macroscopic phenotype.

Regulation of noise in the expression of a single gene.

Stochastic mechanisms are ubiquitous in biological systems. Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations. This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-lambda, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals. We sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations within single cells (biochemical noise) could explain the variation of gene expression levels between cells in a genetically identical population (phenotypic noise). We have isolated the biochemical contribution to phenotypic noise from that of other noise sources by carrying out a series of differential measurements. We varied independently the rates of transcription and translation of a single fluorescent reporter gene in the chromosome of Bacillus subtilis, and we quantitatively measured the resulting changes in the phenotypic noise characteristics. We report that of these two parameters, increased translational efficiency is the predominant source of increased phenotypic noise. This effect is consistent with a stochastic model of gene expression in which proteins are produced in random and sharp bursts. Our results thus provide the first direct experimental evidence of the biochemical origin of phenotypic noise, demonstrating that the level of phenotypic variation in an isogenic population can be regulated by genetic parameters.

Molecular and cellular constraints on vesicle traffic evolution.

In eukaryotic cells, the budding and fusion of intracellular transport vesicles is carefully orchestrated in space and time. Locally, a vesicle's source compartment, its cargo, and its destination compartment are controlled by dynamic multi-protein specificity modules. Globally, vesicle constituents must be recycled to ensure homeostasis of compartment compositions. The emergence of a novel vesicle pathway therefore requires new specificity modules as well as new recycling routes. Here, we review recent research on local (molecular) constraints on gene module duplication and global (cellular) constraints on intracellular recycling. By studying the evolution of vesicle traffic, we may discover general principles of how complex traits arise via multiple intermediate steps.

Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2.

PURPOSE: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. METHODS: We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. RESULTS: We observed a 75.4% agreement between saliva and N/OPS-VTM, that increased drastically to 83% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5% test sensitivity. Our simulations suggest that a test with 75% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. CONCLUSION: These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of saliva-based screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.

Emergent gene order in a model of modular polyketide synthases.

Polyketides are a class of biologically active heteropolymers produced by assembly line-like multiprotein complexes of modular polyketide synthases (PKS). The polyketide product is encoded in the order of the PKS proteins in the assembly line, suggesting that polyketide diversity derives from combinatorial rearrangement of these PKS complexes. Remarkably, the order of PKS genes on the chromosome follows the order of PKS proteins in the assembly line: This fact is commonly referred to as "collinearity". Here we propose an evolutionary origin for collinearity and demonstrate the mechanism by using a computational model of PKS evolution in a population. Assuming continuous evolutionary pressure for novel polyketides, and that new polyketide pathways are formed by horizontal transfer/recombination of PKS-encoding DNA, we demonstrate the existence of a broad range of parameters for which collinearity emerges spontaneously. Collinearity confers no fitness advantage in our model; it is established and maintained through a "secondary selection" mechanism, as a trait which increases the probability of forming long, novel PKS complexes through recombination. Consequently, collinearity hitchhikes on the successful genotypes which periodically sweep through the evolving population. In addition to computer simulation of a simplified model of PKS evolution, we provide a mathematical framework describing the secondary selection mechanism, which generalizes beyond the context of the present model.

Genome doubling enabled the expansion of yeast vesicle traffic pathways.

Vesicle budding and fusion in eukaryotes depend on a suite of protein types, such as Arfs, Rabs, coats and SNAREs. Distinct paralogs of these proteins act at distinct intracellular locations, suggesting a link between gene duplication and the expansion of vesicle traffic pathways. Genome doubling, a common source of paralogous genes in fungi, provides an ideal setting in which to explore this link. Here we trace the fates of paralog doublets derived from the 100-Ma-old hybridization event that gave rise to the whole genome duplication clade of budding yeast. We find that paralog doublets involved in specific vesicle traffic functions and pathways are convergently retained across the entire clade. Vesicle coats and adaptors involved in secretory and early-endocytic pathways are retained as doublets, at rates several-fold higher than expected by chance. Proteins involved in later endocytic steps and intra-Golgi traffic, including the entire set of multi-subunit and coiled-coil tethers, have reverted to singletons. These patterns demonstrate that selection has acted to expand and diversify the yeast vesicle traffic apparatus, across species and time.

Graph-theoretic constraints on vesicle traffic networks.

Eukaryotic cells use small membrane-enclosed vesicles to transport molecular cargo between intracellular compartments. Interactions between molecules on vesicles and compartments determine the source and target compartment of each vesicle type. The set of compartment and vesicle types in a cell define the nodes and edges of a transport graph known as the vesicle traffic network. The transmembrane SNARE proteins that regulate vesicle fusion to target compartments travel in cycles through the transport graph, but the paths they follow must be tightly regulated to avoid aberrant vesicle fusion. Here we use graph-theoretic ideas to understand how such molecular constraints place constraints on the structure of the transport graph. We identify edge connectivity (the minimum number of edges that must be removed to disconnect a graph) as a key determinant that separates allowed and disallowed types of transport graphs. As we increase the flexibility of molecular regulation, the required edge connectivity decreases, so more types of vesicle transport graphs are allowed. These results can be used to aid the discovery of new modes of molecular regulation and new vesicle traffic pathways.

Multistability in the lactose utilization network of Escherichia coli.

Multistability, the capacity to achieve multiple internal states in response to a single set of external inputs, is the defining characteristic of a switch. Biological switches are essential for the determination of cell fate in multicellular organisms, the regulation of cell-cycle oscillations during mitosis and the maintenance of epigenetic traits in microbes. The multistability of several natural and synthetic systems has been attributed to positive feedback loops in their regulatory networks. However, feedback alone does not guarantee multistability. The phase diagram of a multistable system, a concise description of internal states as key parameters are varied, reveals the conditions required to produce a functional switch. Here we present the phase diagram of the bistable lactose utilization network of Escherichia coli. We use this phase diagram, coupled with a mathematical model of the network, to quantitatively investigate processes such as sugar uptake and transcriptional regulation in vivo. We then show how the hysteretic response of the wild-type system can be converted to an ultrasensitive graded response. The phase diagram thus serves as a sensitive probe of molecular interactions and as a powerful tool for rational network design.

Golgi compartments enable controlled biomolecular assembly using promiscuous enzymes.

The synthesis of eukaryotic glycans - branched sugar oligomers attached to cell-surface proteins and lipids - is organized like a factory assembly line. Specific enzymes within successive compartments of the Golgi apparatus determine where new monomer building blocks are linked to the growing oligomer. These enzymes act promiscuously and stochastically, causing microheterogeneity (molecule-to-molecule variability) in the final oligomer products. However, this variability is tightly controlled: a given eukaryotic protein type is typically associated with a narrow, specific glycan oligomer profile. Here, we use ideas from the mathematical theory of self-assembly to enumerate the enzymatic causes of oligomer variability and show how to eliminate each cause. We rigorously demonstrate that cells can specifically synthesize a larger repertoire of glycan oligomers by partitioning promiscuous enzymes across multiple Golgi compartments. This places limits on biomolecular assembly: glycan microheterogeneity becomes unavoidable when the number of compartments is limited, or enzymes are excessively promiscuous.

How contraction has shaped evolution.

Two unicellular relatives of animals reveal that coordinated contractions of groups of cells using actomyosin predated animal multicellularity during evolution.

Frustration and fidelity in influenza genome assembly.

The genome of the influenza virus consists of eight distinct single-stranded RNA segments, each encoding proteins essential for the viral life cycle. When the virus infects a host cell, these segments must be replicated and packaged into new budding virions. The viral genome is assembled with remarkably high fidelity: experiments reveal that most virions contain precisely one copy of each of the eight RNA segments. Cell-biological studies suggest that genome assembly is mediated by specific reversible and irreversible interactions between the RNA segments and their associated proteins. However, the precise inter-segment interaction network remains unresolved. Here, we computationally predict that tree-like irreversible interaction networks guarantee high-fidelity genome assembly, while cyclic interaction networks lead to futile or frustrated off-pathway products. We test our prediction against multiple experimental datasets. We find that tree-like networks capture the nearest-neighbour statistics of RNA segments in packaged virions, as observed by electron tomography. Just eight tree-like networks (of a possible 262 144) optimally capture both the nearest-neighbour data and independently measured RNA-RNA binding and co-localization propensities. These eight do not include the previously proposed hub-and-spoke and linear networks. Rather, each predicted network combines hub-like and linear features, consistent with evolutionary models of interaction gain and loss.