Metabolic specialization and the assembly of microbial communities

Metabolic specialization is a general biological principle that shapes the assembly of microbial communities. Individual cell types rarely metabolize a wide range of substrates within their environment. Instead, different cell types often specialize at metabolizing only subsets of the available substrates. What is the advantage of metabolizing subsets of the available substrates rather than all of them? In this perspective piece, we argue that biochemical conflicts between different metabolic processes can promote metabolic specialization and that a better understanding of these conflicts is therefore important for revealing the general principles and rules that govern the assembly of microbial communities. We first discuss three types of biochemical conflicts that could promote metabolic specialization. Next, we demonstrate how knowledge about the consequences of biochemical conflicts can be used to predict whether different metabolic processes are likely to be performed by the same cell type or by different cell types. We then discuss the major challenges in identifying and assessing biochemical conflicts between different metabolic processes and propose several approaches for their measurement. Finally, we argue that a deeper understanding of the biochemical causes of metabolic specialization could serve as a foundation for the field of synthetic ecology, where the objective would be to rationally engineer the assembly of a microbial community to perform a desired biotransformation.     

Some Possible Dynamical Constraints for Life’s Origin

Oscillating biochemical reactions are common in cell dynamics and could be closely related to the emergence of the life phenomenon itself. In this work, we study the dynamical features of some classical chemical or biochemical oscillators where the effect of cell volume changes is explicitly considered. Such analysis enables us to find some general conditions about the cell membrane to preserve such oscillatory patterns, of possible relevance to hypothetical primitive cells in which these structures first appeared.     

On the impact on biochemical research of the discovery of stable isotopes: the outcome of the serendipic meeting of a refugee with the discoverer of heavy isotopes at Columbia University

As late as the 1930s, approaches to biochemical research not only were rather primitive, but a certain amount of mysticism still surrounded the biochemical events that occur in the living cell. To a great extent, this was due to the lack of techniques needed to uncover the subtle reactions in the living cell. In the early 1930s, an accidental meeting of two scientists revolutionized approaches in biochemical studies and led to the scientific explosion in molecular biology that has occurred during the last few decades. The dark political storm in Germany deposited Dr. Rudolf Schoenheimer on the New York shore, where he met Professor Urey, who recently had discovered "heavy" hydrogen. Schoenheimer suggested that biological compounds tagged with heavy atoms of hydrogen would enable investigators to follow their metabolic pathways. This intellectual leap revolutionized the thinking and design of experiments and made it possible to uncover the myriad reactions that occur in the living cell.     

Rac regulates endothelial morphogenesis and capillary assembly

Endothelial cells undergo branching morphogenesis to form capillary tubes. We have utilized an in vitro Matrigel overlay assay to analyze the role of the cytoskeleton and Rho GTPases during this process. The addition of matrix first induces changes in cell morphology characterized by the formation of dynamic cellular protrusions and the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, but without a recognizable lumen. This is followed by cell migration leading to the formation of a complex interconnecting network of capillary tubes with readily identifiable lumens. Inhibition of actin polymerization or actin-myosin contraction inhibits cell migration but has no effect on the initial changes in endothelial cell morphology. However, inhibition of microtubule dynamics prevents both the initial cell shape changes as well as cell migration. We find that the small GTPase Rac is essential for the matrix-induced changes in endothelial cell morphology, whereas p21-activated kinase, an effector of Rac, is required for cell motility. We conclude that Rac integrates signaling through both the actin and microtubule cytoskeletons to promote capillary tube assembly.     

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.     

Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.

We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.     

SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over Chromosomes in E. coli

Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

Evolution and mechanism of apolipoprotein B-containing lipoprotein assembly

PURPOSE OF REVIEW: Apolipoprotein B-containing lipoprotein assembly and secretion is critical for lipid absorption and triglyceride homeostasis, and plays a role in atherogenesis and the pathobiology of type 2 diabetes and obesity. This review highlights recent insights into the evolutionary, structural, and cell biology of hepatic and intestinal pathways for lipid mobilization, and the mechanisms and regulation of lipoprotein assembly and secretion. RECENT FINDINGS: Until recently it was assumed that microsomal triglyceride transfer protein-dependent apolipoprotein B-containing lipoprotein assembly was a unique adaptation associated with vertebrate lipid homeostasis. However, it is now clear that microsomal triglyceride transfer protein (MTP) exists in species whose last common ancestor diverged over 550 million years ago. In its long evolutionary history, the MTP gene has given rise to a series of paralogous lipid transport proteins, all of which require MTP for their biogenesis. During its evolution, MTP has acquired new functions, enabling it to participate in a disparate array of lipid mobilization and transport pathways, ranging from primitive lipoprotein assembly to antigenic lipid presentation. In addition to the complex and multifunctional role of MTP in apolipoprotein B assembly, other factors responsible for the generation of secretion-coupled lipids and the modulation of apolipoprotein B production are emerging. SUMMARY: The phylogenic dissection of MTP and apolipoprotein B function, coupled with ongoing structural and biochemical analyses, provide significant insights into the mechanisms of lipid mobilization and secretion. Some of these factors and processes may be targeted therapeutically to modulate the quantitative and qualitative aspects of apolipoprotein B production.     

Evolution of cell division in bacteria

Molecular evolution in bacteria is examined with an emphasis on cell division. For a bacterial cell to assemble and then divide required an immense amount of integrated cell and molecular biology structures/functions to be present, such as a stable cellular structure, enzyme catalysis, minimal genome, septum formation at mid-cell and mechanisms to take up nutrients and produce and use energy, as well as store it. The first bacterial cell(s) capable of division must have had complex cell and molecular biology functions. At this stage of evolution, they would not have been primitive cells but would have reached a threshold in evolution where cell division occurred in a regulated manner.     

Lipid protein interactions: the assembly of CD1d1 with cellular phospholipids occurs in the endoplasmic reticulum.

CD1d1 is a member of a family of lipid Ag-presenting molecules. The cellular ligands associated with CD1d1 were isolated and characterized by biochemical means as an approach to elucidate the mechanism by which CD1 molecules assemble in vivo. Natural ligands of mouse CD1d1 included cellular phosphatidylinositol and phosphatidylinositol-glycans that are synthesized in the endoplasmic reticulum. Further biochemical data revealed that the two CD1d1 mutants, one defective in recycling from-and-to the plasma membrane and the other in efficiently negotiating the secretory pathway, associated with phosphatidylinositol. Thus phosphatidylinositol associated with CD1d1 in the early secretory pathway. Phosphatidylinositol also associated with CD1d1 in Pig-A-deficient cells that are defective in the first glycosylation step of glycosylphosphatidylinositol biosynthesis. Moreover, cellular phosphatidylinositol-glycans are not Valpha14Jalpha15 natural T cell Ags. Therefore, we predict that cellular lipids occlude the hydrophobic Ag-binding groove of CD1 during assembly until they are exchanged for a glycolipid Ag(s) within the recycling compartment for display on the plasma membrane. In this manner, cellular lipids might play a chaperone-like role in the assembly of CD1d1 in vivo, akin to the function of invariant chain in MHC class II assembly.     

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.     

Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system

The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.     

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.     

Membrane Phospholipid Redistribution in Cytokinesis： A Theoretical Model

In cell mitosis, cytokinesis is a major deformation process, during which the site of the contractile ring is determined by the biochemical stimulus from asters of the mitotic apparatus, actin and myosin assembly is related to the motion of membrane phospholipids, and local distribution and arrangement of the microfilament cytoskeleton are different at different cytokinesis stages. Based on the Zinemanas-Nir model, a new model is proposed in this study to simulate the entire process by coupling the biochemical stimulus with the mechanical actions. There were three assumptions in this model： the movements of phospholipid proteins are driven by gradients of biochemical stimulus on the membrane surface; the local assembly of actin and myosin filament depends on the amount of phospholipid proteins at the same location; and the surface tension includes membrane tensions due to both the passive deformation of the membrane and the active contraction of actin filament, which is determined by microfilament redistribution and rearrangement. This model could explain the dynamic movement of microfilaments during cytokinesis and predict cell deformation. The calculated results from this model demonstrated that the reorientation of phospholipid proteins and the redistribution and reorientation of microfilaments may play a crucial role in cell division. This model may better represent the cytokinesis process by the introduction of biochemical stimulus.     

Epidermal Growth Factor–induced Contraction Regulates Paxillin Phosphorylation to Temporally Separate Traction Generation from De-adhesion

Directed cell migration is mediated by cycles of protrusion, adhesion, traction generation on the extracellular matrix and retraction. However, how the events after protrusion are timed, and what dictates their temporal order is completely unknown. We used acute epidermal growth factor (EGF) stimulation of epidermal keratinocytes to initiate the cell migration cycle to study the mechanism of the timing of adhesion, traction generation, and de-adhesion. Using microscopic and biochemical assays, we surprisingly found that at ∼2 min after EGF stimulation protrusion, activation of myosin-II, traction generation, adhesion assembly, and paxillin phosphorylation occurred nearly simultaneously, followed by a 10-min delay during which paxillin became dephosphorylated before cell retraction. Inhibition of myosin-II blocked both the EGF-stimulated paxillin phosphorylation and cell retraction, and a paxillin phosphomimic blocked retraction. These results suggest that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a cycle of paxillin phosphorylation/dephosphorylation that mediates a cycle of adhesion strengthening and weakening that delays cell retraction. Thus, we reveal for the first time a mechanism by which cells may temporally segregate protrusion, adhesion, and traction generation from retraction during EGF-stimulated cell migration.     

Macromolecules and the origin of life

From our knonwledge of present day organisms, it is hard to imagine a living assembly, even at its most primitive stage, without macromolecules.In order to look for the macromolecules which possibly participated in the assembly of the primitive organisms, the reaction and formation of polymers in HCN under irradiation of ultraviolet ray of 184.9 nm. was studied.As a example of a simple way of producing an assembly of macromolecules, the mechanism of coacervation was studied by using gelatin as the material.     

In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.