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PubMed1982

Particle assemblies in astrocytic plasma membranes are rearranged by various agents in vitro and cold injury in vivo.

Distinct aggregates of small intramembranous particles and assemblies characterize the P-face of freeze-fractured astrocytic membranes. To test the lability of the assemblies, astrocytes were treated in vitro with different chemical agents and in vivo by cold injury. The assemblies appeared either to contain or be associated with protein because exposure to medium containing cycloheximide, an inhibitor of protein synthesis, led to a sharp decrease in assemblies, down to 1% of the control levels within three hours. To ascertain whether the assemblies were tethered to the cytoskeleton, the cells were treated in vitro with disruptors of microtubules (colchicine) or microfilaments (cytochalasins); the assemblies became consistently rearranged. Protein denaturants, urea and guanidine HCl, brought about a selective aggregation of assembly with assembly. The lectin, concanavalin A, did not alter the distribution of the assemblies within the plane of the membrane fracture. Surface replicas of in vitro, non-fractured, astrocytes revealed surface particles which did not resemble assemblies. In vivo, the plasma membranes of astrocytes were altered within minutes of cold injury to the brain surface. In the centre of the lesions, damaged astrocytes had assemblies that were clumped like those of in vitro astrocytes exposed to denaturants. In the periphery of the lesions, however, the assemblies did not aggregate but increased in number. These results provide indirect evidence that assemblies may consist of protein, that the recognizable particle constituent of the assembly is confined to the interior of the membrane and is not present on the uncleaved cell surface, and that assemblies are connected with the cytoskeleton. Therefore, certain changes in the environment of the astrocyte caused by injury in vivo or addition of chemical agents in vitro alter the distribution of assemblies in the astrocytic plasma membrane either by a direct effect on the assemblies or indirectly by an alteration of the cytoplasmic proteins.

Animals↗
PubMed2004

Identifying the task variables that influence perceived object assembly complexity.

There is a general lack of understanding as to what issues affect assembly task performance when using diagrammatic instructions because few of the task variables contributing to assembly complexity have been identified. Using a task analysis of a range of self-assembly products, seven task variables hypothesized to predict assembly complexity were identified and studied in the instruction comprehension phase of assembly. Experiment 1 took nine real world assembly instructions and described each in terms of the seven task variables. Seventy-two participants gave a subjective rating of assembly difficulty for each assembly, showing a clear relationship between the task variables and perceived assembly difficulty. As real world assemblies provide little control a second experiment used an orthogonal design to systematically vary the values of each of the assembly task variables in 16 abstract assemblies. Forty-two participants compared the 16 assembly instructions to a final assembly. There was a clear relationship between the task variables and the time taken to view the instructions. Further, it was found that it is possible to predict the complexity of assembly tasks based upon the levels of the task variables identified. The task variables identified are a significant step towards identifying the factors that influence assembly complexity, together with providing progress towards a tool for predicting assembly complexity.

Audiovisual Aids↗
PubMed2006

Identifying the task variables that predict object assembly difficulty.

OBJECTIVE: We investigated the physical attributes of an object that influence the difficulty of its assembly. Identifying attributes that contribute to assembly difficulty will provide a method for predicting assembly complexity. BACKGROUND: Despite object assembly being a widespread task, there has been insufficient research into information processing and cognition during assembly. The lack of research means that the variables that affect the performance of procedural assembly tasks with illustration-only instructions are unknown. METHOD: In Experiment 1, seven physical characteristics (task variables) of assembly objects were systematically varied in a balanced fractional factorial and orthogonal design to create 16 abstract assemblies, which were assembled by 12 participants (6 men and 6 women aged 18-56). A second experiment (20 participants, 8 men and 12 women aged 18 to 52) involved scaled-down models of 8 real-world assemblies. RESULTS: A clear relationship between the task variables and assembly difficulty was found in both studies, and the regression model from the first experiment was able to predict the assembly difficulty timings in Experiment 2. CONCLUSION: The proposed task variables are associated with assembly difficulty, and the regression analysis has shown four of the task variables to be significant predictors of difficulty. APPLICATION: Applications of this research include the use of the regression model as a tool to evaluate the difficulty of assemblies or assembly steps defined by instructions. The task variables can also be used to produce guidelines to ensure that assemblies or assembly steps are manageable.

Adolescent↗
PubMed1996

Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA.

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.

Animals↗
PubMed2002

The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag.

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.

Animals↗
PubMed2000

Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA.

In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIA(F46) and MIIB(alpha)(F47)) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIB(alpha)(F47) [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(F47) mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.

Actin Cytoskeleton↗
PubMed1989

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues.

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.

Adenosine Triphosphate↗
PubMed1991

Chaperone-assisted assembly and molecular architecture of adhesive pili.

The assembly of bacterial pili as exemplified here by P and type 1 pili of E. coli is a complex process involving specific molecular interactions between structural and chaperone proteins. The assembly process occurs postsecretionally, i.e. after the subunits are translocated across the cytoplasmic membrane. In a single cell, hundreds of thousands of interactive subunits are typically surface localized and assembled into pili. Periplasmic chaperones are generally required to bind to the interactive subunits and partition them into assembly-competent complexes. The binding of the chaperone to the subunits apparently protects the interactive surfaces and prevents them from aggregating at the wrong time and place within the cell. Pili are most likely assembled into linear polymers that package into right-handed helices after their translocation through specific outer-membrane channels. Each pilus filament is a quaternary assembly of the structural subunit and several minor subunits including the adhesin moiety. Although the assembly and organization of P and type 1 pili are very similar, there are some notable differences. For example, the P pilus adhesin is located exclusively at the tips of the pilus filament and forms part of a morphologically distinct structure. In contrast, the adhesion moiety of type 1 pili is inserted into the pilus filament at intervals, but only the adhesin molecule exposed at the pilus tip is functional. The variability in isoreceptor recognition amongst P pili has been solely ascribed to structural differences in the respective adhesin molecules, whereas in type 1 pili, variability in binding specificity has been attributed to the pilus filament that influences the conformation of the adhesin moiety. Less is known about the structure or assembly of type 4 pili, which are a unique class of pili expressed by several different species of gram-negative bacteria. The phase variation of the pilC assembly gene in N. gonorrheae to the off state results in the accumulation of unassembled subunits toxic to the cells. This process exerts a strong selection pressure on the cells that triggers alterations in the pilin structural gene. Thus, antigenic variation of pili in this organism may be regulated at the level of assembly. Finally, the concept of periplasmic chaperones in postsecretional assembly is most likely a general phenomenon in the biology of gram-negative bacteria. The investigations of pilus assembly will continue to provide insight into the details of how macromolecular assembly reactions are coordinated in the bacterial cell and how the regulation of assembly genes can profoundly affect biological processes.

Adhesins, Escherichia coli↗
PubMed1999

The effects of various GTP analogues on microtubule assembly.

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.

Adenine Nucleotides↗
PubMed2001

Signposts in the assembly of chylomicrons.

Intestinal cells synthesize and secrete chylomicrons in the postprandial state. Synthesis of these particles is defective in abetalipoproteinemia and chylomicron retention disease. Chylomicrons are very large, heterogeneous, lipid-rich particles ranging in diameters from 75 to 450 nm and function to transport dietary fat and fat-soluble vitamins to blood. The size heterogeneity of the secreted particles depends on the rate of fat absorption, type and amount of fat absorbed. The fatty acid composition of triglycerides present in chylomicrons reflects the composition of dietary fat, whereas the fatty acid composition of chylomicron phospholipids does not. The differences in the fatty acid compositions are also observed when lipids are labeled with glycerol. Thus, the differences are not due to differential incorporation of dietary fatty acids into different lipids but are mainly due to different pools of lipids used for chylomicron assembly. It has been suggested that preformed phospholipids and nascent triglycerides are preferentially used for intestinal lipoprotein assembly. Biosynthesis of chylomicrons requires apoB48. ApoB48 is translated from apoB mRNA that is post-transcriptionally edited in the intestinal cells to incorporate a stop codon. Nascent apoB48 may be cotranslationally lipidated and this process is critically dependent on the presence of microsomal triglyceride transfer protein. Two different models have been proposed for the assembly of chylomicrons. In the independent model, intestinal cells are hypothesized to synthesize VLDL and chylomicron by two independent pathways. The chylomicron assembly pathway is hypothesized to be sensitive to a surfactant, Pluronic L81, but that of VLDL assembly is not. In the sequential assembly model, synthesis of all lipoproteins is hypothesized to begin with the assembly of apoB-containing primordial lipoprotein particles. The primordial particles are suggested to fuse with triglyceride-rich lipid droplets that are synthesized independently of apoB. This process results in the core expansion of primordial particles and the synthesis of nascent lipoproteins. Differences in the size of secreted lipoproteins may be due to differences in the size of triglyceride-rich lipid droplets. Pluronic L81 is hypothesized to inhibit the formation of large triglyceride-rich droplets that serve as precursors for chylomicron assembly. In this review, we have discussed some signposts that might be unique to different steps in the assembly of chylomicrons. First, it is proposed that the association of preformed phospholipids with nascent apoB in the endoplasmic reticulum may serve as a signpost for the very early steps in the assembly of chylomicrons. Second, association of large amounts of newly synthesized triglycerides compared to preformed triglycerides may serve as a signpost for the assembly of larger lipoproteins. Third, the incorporation of retinyl esters may serve as markers for the final stages of chylomicron assembly. These signposts may be helpful in the identification and characterization of various intermediates in the assembly of chylomicrons. The knowledge about the molecular assembly of chylomicrons may lead to better therapeutic agents for controlling various hyperlipidemias, obesity, and atherosclerosis.

Apoproteins↗
PubMed2019

Residues on Adeno-associated Virus Capsid Lumen Dictate Interactions and Compatibility with the Assembly-Activating Protein.

The adeno-associated virus (AAV) serves as a broadly used vector system for in vivo gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly.IMPORTANCE Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

Amino Acid Sequence↗