In vitro synthesis and assembly of picornaviral capsid intermediate structures.

Cell-free translation of encephalomyocarditis RNA in extracts of rabbit reticulocytes results in the synthesis of viral proteins indistinguishable from those produced during virus infection of cells. The viral capsid proteins are produced in an active form capable of assembly into viral capsid intermediate structures. Protomers (5S), pentamers (14S), and shell-like structures (75 to 85S) can be detected after prolonged incubation in the extracts. Proteolytic cleavage of capsid precursor proteins appears to be a prerequisite for assembly, in apparent contrast to cell-associated assembly. Assembly of pentamers is also preceded by conversion of protein epsilon 1 to epsilon in a step which may reflect an amino-terminal blocking reaction.     

Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules

Turbidity measurements have been used to study the in vitro assembly and disassembly of porcine neurotubules. All measurements were carried out with tubulin with a purity higher than 80%. Tubules formed by in vitro assembly of this protein are so long that the turbidity is insensitive to length and is a function only of the total mass of high molecular weight material. Porcine tubulin shows a critical concentration for assembly of about 0.2 mg/ml under optimal conditions, pH 6.6, 0.1m-2-(N-morpholino)ethane sulfonic acid, 26 to 37 °C. Under these conditions assembly and disassembly are essentially fully reversible in the presence of excess GTP. The kinetics of assembly show an initial lag and initial rates which are strongly temperature dependent. Our samples show a concentration dependence of no more than second order. The apparent activation enthalpy of assembly is 25 kcal/mol; the apparent reaction enthalpy of assembly for the chain propagation step is 21 kcal/mol. Disassembly kinetics show an apparent negative activation enthalpy of ?28 kcal/mol. They are independent of tubule length implying a slow activation step followed by rapid depolymerization. At 20 °C, cycles of polymerization and depolymerization show hysteresis effects in the assembly kinetics though not in disassembly rates or final states. This is most easily explained by postulating a slow reversible inactivation at 4 °C of the initiation complex for tubule assembly. Conditions are reported for producing tubulin in a state which cannot assemble in aqueous buffer unless nucleotides are added. GTP, ATP and ADP, but not GDP, are effective in promoting tubule assembly. An adenylate kinase impurity in our preparation may be the cause of this unusual effect. Whether or not it is actually associated with tubulin or tubules is unknown.     

A reassessment of the factors affecting microtubule assembly and disassembly in vitro

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.     

Effects of spirogermanium on the in vitro assembly and disassembly of brain microtubules

The inhibition of microtubule proteins (MTP) assembly by Spirogermanium (SP, 1.25-100 microM) has been studied. Assembly at 37 degrees C was monitored by turbidity measurements and electron microscopy. For SP in 1:1 protein-drug ratio the inhibition of assembly was 50%. Addition of 12.5 microM SP to microtubules induced spontaneous disassembly. SP had less effect on the assembly of pure tubulin (tubulin 6S). Complete inhibition of assembly induced by glycerol and Mg2+ was found with 250 microM and the ratio of SP to tubulin to obtain 50% inhibition was higher than with MTP.     

Mechanisms of synapse assembly and disassembly

The mechanisms that govern synapse formation and elimination are fundamental to our understanding of neural development and plasticity. The wiring of neural circuitry requires that vast numbers of synapses be formed in a relatively short time. The subsequent refinement of neural circuitry involves the formation of additional synapses coincident with the disassembly of previously functional synapses. There is increasing evidence that activity-dependent plasticity also involves the formation and disassembly of synapses. While we are gaining insight into the mechanisms of both synapse assembly and disassembly, we understand very little about how these phenomena are related to each other and how they might be coordinately controlled to achieve the precise patterns of synaptic connectivity in the nervous system. Here, we review our current understanding of both synapse assembly and disassembly in an effort to unravel the relationship between these fundamental developmental processes.     

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Assembly of bacteriophage T4 head-related strucutres. II. In vitro assembly of prehead-like structures

A protein mixture which is derived from bacteriophage T4 preheads formed in vivo contains all the important prehead proteins: i.e. protein P23, which forms the icosahedral prehead shell; the core proteins P22 and internal protein III; and two quantitatively minor proteins, P24 and P20. Conditions are described under which these proteins assemble in vitro into structures that (1) resemble preheads when visualized by electron microscopy, (2) contain all prehead proteins, and (3) have a similar length and diameter as preheads formed in vivo. It is concluded that prehead-like structures can be assembled in vitro, and that the mechanism that determines the length and diameter of the T4 prehead is active in our in vitro system. Evidence is presented that the core proteins play an important role in specifying the prehead diameter. The result of assembly experiments after partial fractionation of the protein mixture by gel filtration suggests that P20 plays a key role in the assembly of prehead-like structures in vitro, whereas P24 is not required. A possible mechanism by which P20 governs tha assembly of P23 and the core proteins is discussed.     

The assembly and disassembly of ecological networks

Global change has created a severe biodiversity crisis. Species are driven extinct at an increasing rate, and this has the potential to cause further coextinction cascades. The rate and shape of these coextinction cascades depend very much on the structure of the networks of interactions across species. Understanding network structure and how it relates to network disassembly, therefore, is a priority for system-level conservation biology. This process of network collapse may indeed be related to the process of network build-up, although very little is known about both processes and even less about their relationship. Here we review recent work that provides some preliminary answers to these questions. First, we focus on network assembly by emphasizing temporal processes at the species level, as well as the structural building blocks of complex ecological networks. Second, we focus on network disassembly as a consequence of species extinctions or habitat loss. We conclude by emphasizing some general rules of thumb that can help in building a comprehensive framework to understand the responses of ecological networks to global change.     

In vitro assembly of U1 snRNPs.

An efficient system for the in vitro assembly of U1 snRNPs is described. RNA-protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP-specific proteins 70K and A require only the 5'-most stem-loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.     

In vitro assembly of novel cholera toxin-like complexes

Cholera toxin (CT) is responsible for the major pathological features of cholera, but in addition to its cytotoxic properties, CT is a potent mucosal adjuvant when coadministered with antigens at mucosal sites. Discovery of CT adjuvanticity has prompted the generation of CT chimeras with reduced toxicity and improved efficiency for antigen presentation at mucosal sites. To date, chimeric forms of CT have been produced in bacterial strains by coexpressing the CT B subunit and a chimeric form of the CT A subunit consisting of a target protein antigen fused with the A2 polypeptide of CT. In this study, a chimeric protein consisting of green fluorescent protein (GFP) fused with polypeptide A2 was generated to investigate the feasibility of assembling CT holotoxin-like complexes in vitro. The assembly of such holotoxin-like complexes would expand the variety of antigenic compounds that could be incorporated into CT-based vaccines. In this study, GFP-A2/CTB complexes could be generated in vitro using a stepwise denaturation-renaturation process. These findings suggest that it is possible to generate novel mucosal vaccines consisting of macromolecules that are chemically coupled to polypeptide A2 and reconstituted into CT-like complexes in vitro.     

In vitro generation of three-dimensional renal structures

End-stage renal disease is currently being treated effectively by transplantation. However, increasing demand and donor shortage make this treatment challenging. Recent advances in cell-based therapies have provided potential opportunities to alleviate the current challenges of donor shortage. In this study we developed a system to generate renal structures in vitro using primary kidney cells. This system involves the cultivation of expanded primary renal cells in a three-dimensional collagen-based culture system. After one week of growth, individual renal cells began to form renal structures resembling tubules and glomeruli. Histologically, these structures show phenotypic resemblance to native kidney structures. The reconstituted tubules stained positively for Tamm-Horsfall protein, which is expressed in the thick ascending limb of Henle's Loop and distal convoluted tubules. These results show that renal structures can be reconstituted in a three-dimensional culture system, which may eventually be used for renal cell therapy applications.     

In vitro collagen fibril assembly: thermodynamic studies

The in vitro fibril assembly of calf skin collagen was examined as a function of ionic strength and temperature. In a 0.03 M NaPi, pH 7.0, buffer, fibril assembly required a minimum critical concentration of collagen. At nearly physiological ionic strengths and temperatures, the critical concentration was less than 1 microgram/mL and required a very sensitive method for measurement. Raising the ionic strength of the buffer resulted first in higher and then lower critical concentrations. Raising the temperature led to lower critical concentrations. A van't Hoff plot of the fibril growth constant calculated from the critical concentration gave positive enthalpy changes and positive heat capacity changes which indicate that the fibril growth is driven by both hydrophobic and ionic inter-collagen interactions. Sedimentation equilibrium studies showed the collagen to be monomeric at subcritical concentrations. Differential scanning microcalorimetric studies showed only one very sharp heat absorption peak for the fibril assembly which coincided with the appearance of solution turbidity. Within experimental error, the enthalpy changes of the fibril assembly measured with the microcalorimeter were of the same magnitude as the van't Hoff enthalpy changes. These results are discussed in light of a cooperative nucleation-growth mechanism of collagen fibril assembly proposed earlier.     

In vitro matrix assembly induced by critical assembly concentration (CAC).

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.     

Regulation of microtubule assembly and disassembly by nucleotides

The role of nucleotides in microtubular assembly and disassembly has been reviewed. Two possible functions of GTP hydrolysis during assembly are discussed: (1) hydrolysis renders sensitivity to factor(s) regulating microtubule depolymerization; (2) the energy of GTP hydrolysis is utilized for the subunit flow from one end of the microtubule to the other. In the second part of the review, experiments are considered showing that microtubular disassembly takes place in the cells only in the presence of ATP, and, therefore, this process is regulated via some ATP-dependent mechanism (most probably, phosphorylation of microtubule-associated proteins).     

Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.     

Evolutionary assembly and disassembly of the mammalian sternum

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