scholarly journals Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

2020 ◽  
Author(s):  
Raviv Dharan ◽  
Asaf Shemesh ◽  
Abigail Millgram ◽  
Yael Levi-Kalisman ◽  
Israel Ringel ◽  
...  
Keyword(s):  
Progressive Increase ◽  
Hexagonal Symmetry ◽  
Time Resolved ◽  
Hierarchical Assembly ◽  
X Ray Scattering ◽  
Cellular Factors ◽  
Hierarchical Architectures ◽  
Assembly Pathways ◽  
Insight Into

<p>Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with cellular factors. Spermine, an endogenous polyamine, promotes and stabilizes tubulin assemblies. Yet, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced tubulin to assemble <i>in vitro</i> into hierarchical architectures, based on a tubulin conical-spiral (TCS) subunit. Using solution X-ray scattering and cryo-TEM, we showed that with progressive increase of spermine concentration, tubulin-dimers assembled into a tubulin helical-pitch (or a short TCS), TCSs, TCS that stacked into tubes through base-to-top packing, antiparallel bundles of TCS tubes in a quasi-hexagonal symmetry, and eventually twisted hexagonal bundles of inverted tubulin tubules. Time-resolved experiments revealed that tubulin assemblies formed at low spermine concentrations were precursors of the assemblies formed at higher spermine concentrations. The results provide insight into the variety of morphologies that tubulin can form, and contribute to our understanding of the fundamental interactions that control the composition and construction of protein-based biomaterials.</p>

scholarly journals Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

2020 ◽  
Author(s):  
Raviv Dharan ◽  
Asaf Shemesh ◽  
Abigail Millgram ◽  
Yael Levi-Kalisman ◽  
Israel Ringel ◽  
...  
Keyword(s):  
Progressive Increase ◽  
Hexagonal Symmetry ◽  
Time Resolved ◽  
Hierarchical Assembly ◽  
X Ray Scattering ◽  
Cellular Factors ◽  
Hierarchical Architectures ◽  
Assembly Pathways ◽  
Insight Into

<p>Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with cellular factors. Spermine, an endogenous polyamine, promotes and stabilizes tubulin assemblies. Yet, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced tubulin to assemble <i>in vitro</i> into hierarchical architectures, based on a tubulin conical-spiral (TCS) subunit. Using solution X-ray scattering and cryo-TEM, we showed that with progressive increase of spermine concentration, tubulin-dimers assembled into a tubulin helical-pitch (or a short TCS), TCSs, TCS that stacked into tubes through base-to-top packing, antiparallel bundles of TCS tubes in a quasi-hexagonal symmetry, and eventually twisted hexagonal bundles of inverted tubulin tubules. Time-resolved experiments revealed that tubulin assemblies formed at low spermine concentrations were precursors of the assemblies formed at higher spermine concentrations. The results provide insight into the variety of morphologies that tubulin can form, and contribute to our understanding of the fundamental interactions that control the composition and construction of protein-based biomaterials.</p>

scholarly journals Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

2019 ◽  
Author(s):  
Raviv Dharan ◽  
Asaf Shemesh ◽  
Abigail Millgram ◽  
Yael Levi-Kalisman ◽  
Israel Ringel ◽  
...  
Keyword(s):  
Self Assembly ◽  
Hexagonal Lattice ◽  
Building Blocks ◽  
Time Resolved ◽  
X Ray ◽  
Hierarchical Assembly ◽  
Research Fields ◽  
X Ray Scattering ◽  
Assembly Pathways ◽  
Ray Scattering

<p>Tubulin dimers are flexible entities serving as building blocks for construction of cellular polymers essential for the cytoskeleton. The conformational state of the dimer dictates the exact formation of assembly and can be regulated by cellular factors including spermine. Using solution X-ray scattering and cryo-TEM measurements we studied the behavior of tubulin assembly in the presence of millimolar spermine concentrations. The results discovered novel structural architectures of tubulin polymers and revealing fascinating hierarchical self-associations based on unique tubulin conical-spiral (TCS) subunits.</p> <p> </p> <p>We followed the assembly pathways of tubulin dimers with different spermine concentrations, from milliseconds to days, and discovered multiple phase transitions with increasing spermine concentration. At 1 mM spermine, tubulin assembled into tubulin helical-pitch (THP) structures, resembling tubulin-rings. Above 1.5 mM spermine, tubulin assembled into TCS architectures. TCS is a unique tubulin assembly, serving as a new building block subunit. TCS assembled into different architectures . The predominant structure was TCS-tube (TCST) that further assembled in a remarkable antiparallel orientation which formed bundles with 2D-cubic and unique quasi-2D hexagonal lattices. Each TCST in the quasi-2D hexagonal lattice was surrounded by four antiparallel TCSTs and two parallel TCSTs. All the above assemblies have never been observed before. At higher spermine concentrations, tubulin assembled into twisted inverted tubulin tubules (ITTs).</p> <p>Here we also show for the first time, the hierarchical assembly pathways from tubulin dimer to each of the above structures, using time-resolved experiments with millisecond temporal resolution. We discovered that the structures that formed at low spermine concentrations were transient precursors of the structures formed at higher spermine concentrations. </p> <p> </p> <p>The results are based on high quality cryo-TEM images, cutting edge synchrotron solution X-ray scattering measurements and state-of-the-art data analysis, using our home developed groundbreaking analysis software, D+. </p> <p>The findings can be relevant to a broad research fields including studies which explore different arrangements of the cytoskeletal network, or studies exploring the attraction forces between proteins that dictate their mode of assembly and molecular designed self-assembly of natural and/or synthetic analogous.</p>

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

Real-time insight into nanostructure evolution during the rapid formation of ultra-thin gold layers on polymers

2021 ◽  
Author(s):  
Matthias Schwartzkopf ◽  
Sven-Jannik Wöhnert ◽  
Vivian Waclawek ◽  
Niko Carstens ◽  
André Rothkirch ◽  
...  
Keyword(s):  
Polymer Matrix ◽  
Hybrid Material ◽  
Time Resolved ◽  
X Ray ◽  
Polymer Hybrid ◽  
X Ray Scattering ◽  
Rapid Formation ◽  
Metal Polymer ◽  
Insight Into

At the nascence of a metal–polymer hybrid material primarily vertical Au dimers and free adatoms diffuse on and into the polymer matrix revealed in situ by sub-millisecond time-resolved surface-sensitive X-ray scattering (GISAXS).

scholarly journals Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

2006 ◽  
Vol 188 (12) ◽  
pp. 4198-4206 ◽  
Author(s):  
Peter Redder ◽  
Roger A. Garrett
Keyword(s):  
Sulfolobus Solfataricus ◽  
Mobile Element ◽  
Progressive Increase ◽  
Mobile Elements ◽  
Is Elements ◽  
Copy Numbers ◽  
Different Types ◽  
Single Deletion ◽  
Insight Into

ABSTRACT The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed “in vitro library” strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such as S. solfataricus with a continually changing genome.

Insight into Fast Nucleation and Growth of Zeolitic Imidazolate Framework-71 by In Situ Time-Resolved Light and X-ray Scattering Experiments

2016 ◽  
Vol 16 (4) ◽  
pp. 2002-2010 ◽  
Author(s):  
Sanjib Saha ◽  
Sergej Springer ◽  
Maria E. Schweinefuß ◽  
Diego Pontoni ◽  
Michael Wiebcke ◽  
...  
Keyword(s):  
Nucleation And Growth ◽  
Zeolitic Imidazolate Framework ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Insight Into ◽  
Ray Scattering

Microtubule nucleation sequence studied by time-resolved x-ray scattering and electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 766-767
Author(s):  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
Joan Bordas
Keyword(s):  
Electron Microscopy ◽  
Dynamic Equilibrium ◽  
Time Resolved ◽  
X Ray ◽  
Additional Information ◽  
X Ray Scattering ◽  
Low Concentrations ◽  
Microtubule Growth ◽  
Ray Patterns ◽  
Ray Scattering

When a solution of microtubule protein is changed from non-polymerising to polymerising conditions (e.g. by temperature jump or mixing with GTP) there is a series of structural transitions preceding microtubule growth. These have been detected by time-resolved X-ray scattering using synchrotron radiation, and they may be classified into pre-nucleation and nucleation events. X-ray patterns are good indicators for the average behavior of the particles in solution, but they are difficult to interpret unless additional information on their structure is available. We therefore studied the assembly process by electron microscopy under conditions approaching those of the X-ray experiment. There are two difficulties in the EM approach: One is that the particles important for assembly are usually small and not very regular and therefore tend to be overlooked. Secondly EM specimens require low concentrations which favor disassembly of the particles one wants to observe since there is a dynamic equilibrium between polymers and subunits.

In situ Time-Resolved Small-Angle X-ray Scattering Reveals the Formation Kinetics of Polyelectrolyte Complex Micelles

2019 ◽  
Author(s):  
Hao Wu ◽  
Jeffrey Ting ◽  
Siqi Meng ◽  
Matthew Tirrell
Keyword(s):  
Small Angle ◽  
Self Assembly ◽  
Electrostatic Interactions ◽  
Polyelectrolyte Complex ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Kinetics Of ◽  
Ray Scattering

We have directly observed the <i>in situ</i> self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. This work has elucidated one general kinetic pathway for the process of PEC micelle formation, which provides useful physical insights for increasing our fundamental understanding of complexation and self-assembly dynamics driven by electrostatic interactions that occur on ultrafast timescales.

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