scholarly journals Robust and conserved stochastic self-assembly mechanism for dynamic ParB-parS partition complexes on bacterial chromosomes and plasmids

2018 ◽  
Author(s):  
Roxanne Diaz ◽  
Aurore Sanchez ◽  
Jérôme Rech ◽  
Delphine Labourdette ◽  
Jérôme Dorignac ◽  
...  
Keyword(s):  
Self Assembly ◽  
Protein Complexes ◽  
Dynamic Structure ◽  
Partition System ◽  
Plasmid Segregation ◽  
Assembly Mechanism ◽  
Bacterial Chromosomes ◽  
Active Transcription ◽  
Dna Partitioning

SummaryChromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that ‘Nucleation & caging’ is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the ‘Nucleation & Caging’ model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.

scholarly journals Coilin oligomerization and remodeling by Nopp140 reveals a hybrid assembly mechanism for Cajal bodies

2021 ◽  
Author(s):  
Edward Courchaine ◽  
Martin Machyna ◽  
Korinna Straube ◽  
Sarah Sauyet ◽  
Jade Enright ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Protein Complexes ◽  
Scaffolding Protein ◽  
Single Amino Acid ◽  
Cajal Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Assembly Mechanism ◽  
Functional Components

Cajal bodies (CBs) are ubiquitous nuclear membraneless organelles (MLOs) that promote efficient biogenesis of RNA-protein complexes. Depletion of the CB scaffolding protein coilin is lethal for vertebrate embryogenesis, making CBs a strong model for understanding the structure and function of MLOs. Although it is assumed that CBs form through biomolecular condensation, the biochemical and biophysical principles that govern CB dynamics have eluded study. Here, we identify features of the coilin protein that drive CB assembly and shape. Focusing on coilin's N-terminal domain (NTD), we discovered its unexpected capacity for oligomerization in vivo. Single amino acid mutational analysis of coilin revealed distinct molecular interactions required for oligomerization and binding to the Nopp140 ligand, which facilitates CB assembly. We demonstrate that the intrinsically disordered regions of Nopp140 have substantial condensation properties and suggest that Nopp140 binding thereby remodels stable coilin oligomers to form a particle that recruits other functional components.

scholarly journals Tunable Extracellular Self-Assembly of Multi-Protein Conjugates from Bacillus subtilis

2016 ◽  
Author(s):  
Charlie Gilbert ◽  
Mark Howarth ◽  
Colin R. Harwood ◽  
Tom Ellis
Keyword(s):  
Bacillus Subtilis ◽  
Self Assembly ◽  
Culture Media ◽  
Protein Complexes ◽  
Modular System ◽  
Protein Conjugation ◽  
Novel Approach ◽  
Multifunctional Enzymes

The ability to stably and specifically conjugate recombinant proteins to one another is a powerful in vitro technique for engineering multifunctional enzymes, protein therapeutics and novel biological materials. However, for many applications spontaneous in vivo protein conjugation would be preferable to in vitro methods. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically-encoded to covalently conjugate in culture media following programmable secretion by Bacillus subtilis. Using this novel approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling and we show that cellular consortia can be engineered to self-assemble functional multi-protein complexes with tunable composition. This genetically-encoded modular system provides a new, flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

The morphogenesis of the chick primary corneal stroma. I. New observations on collagen organization in vivo help explain stromal deposition and growth

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 135-145
Author(s):  
J.B. Bard ◽  
M.K. Bansal
Keyword(s):  
Self Assembly ◽  
Corneal Stroma ◽  
Collagen Fibrils ◽  
Maximum Diameter ◽  
Collagen Organization ◽  
Assembly Mechanism ◽  
Freeze Dried ◽  
Collagen Molecules ◽  
20 Nm

The primary stroma of the avian cornea contains collagen fibrils in orthogonal array. While investigating the processes underlying its morphogenesis, we have found that stromal organization is not as expected in three important respects. First, the fibrils are not uniform: those near the epithelium (newly laid down) have a maximum diameter of about 20 nm (mean: 17.7 nm), while those near the endothelium (laid down for approx. 40 h) have diameters up to 40 nm (mean: 22.8 nm). Fibrils thus grow rapidly to 20 nm and then continue to enlarge slowly, presumably by diffusion of collagen molecules from the epithelium. Second, the collagen, although orthogonally organized, does not contain layers of parallel fibrils. Instead, SEM observation shows that only a few fibrils lie in a parallel array before this short-range order is broken by orthogonal fibrils in the same plane. Furthermore, fibrils in corneas that had been freeze dried but not critical-point dried for SEM were widely spaced and the intervening gaps were filled by an extensive matrix that was probably composed of the proteoglycans known to be in the stroma. Third, we have shown experimentally that the stromal undulations seen in sections are not present in vivo but are shrinkage artifacts: the less corneas were shrunk for SEM preparation, the less pronounced were the stromal undulations. We also noted that, even after the distortions required for the stroma to undulate, the constituent fibrils remained orthogonally organized. These results give insight into the mechanisms underlying stromal morphogenesis and growth. The observations on the growth of collagen fibrils and on collagen organization show that stromal deposition is a more stochastic process than previously thought and, hence, provides support for the view that a complex self-assembly mechanism underlies both fibrillogenesis and the generation of orthogonal organization. The experiments on, and the analysis of, stromal folding show that fibrils slide over one another as undulations form, with the extensive matrix of hydrated proteoglycans being the likely lubricant. This fluidity of the stromal components probably explains how growth can occur without the structure being distorted.

scholarly journals Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

2015 ◽  
Vol 112 (51) ◽  
pp. E7055-E7064 ◽  
Author(s):  
Longhua Hu ◽  
Anthony G. Vecchiarelli ◽  
Kiyoshi Mizuuchi ◽  
Keir C. Neuman ◽  
Jian Liu
Keyword(s):  
Copy Number ◽  
Detailed Mechanism ◽  
Ratchet Mechanism ◽  
Partition System ◽  
Plasmid Segregation ◽  
Emergent Phenomenon ◽  
Mechanochemical Coupling ◽  
Low Copy Number

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Cooperative Self-Assembly of Pyridine-2,6-Diimine-Linked Macrocycles into Mechanically Robust Nanotubes

2019 ◽  
Author(s):  
Michael J. Strauss ◽  
Darya Asheghali ◽  
Austin Evans ◽  
Rebecca Li ◽  
Anton Chavez ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
High Aspect Ratio ◽  
Self Assembly ◽  
Gel Permeation Chromatography ◽  
Synthetic Polymers ◽  
Young’S Moduli ◽  
Structural Rigidity ◽  
Assembly Mechanism ◽  
Low Dimensionality ◽  
Harsh Conditions

<p>Nanotubes assembled from macrocyclic precursors offer a unique combination of low dimensionality, structural rigidity, and distinct interior and exterior microenvironments. Usually the weak stacking energies of macrocycles limit the length or strength of the resultant nanotubes. Imine-linked macrocycles were recently found to assemble into high-aspect ratio (>10<sup>3</sup>), lyotropic nanotubes in the presence of excess acid. Yet these harsh conditions are incompatible with many functional groups and processing methods, and lower acid loadings instead catalyze macrocycle degradation. Here we report pyridine-2,6-diimine-linked macrocycles that assemble into high-aspect ratio nanotubes in the presence of less than 1 equiv of CF<sub>3</sub>CO<sub>2</sub>H per macrocycle. Analysis by gel permeation chromatography and fluorescence spectroscopy revealed a cooperative self-assembly mechanism. Nanofibers obtained by touch-spinning the pyridinium-based nanotubes exhibit Young’s moduli of 1.48 GPa, which exceeds that of many synthetic polymers and biological filaments. These findings will enable the design of structurally diverse nanotubes from synthetically accessible macrocycles. </p>

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