Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts

Microtubules are dynamically unstable polymers that interconvert stochastically between growing and shrinking states by the addition and loss of subunits from their ends. However, there is little experimental data on the relationship between microtubule end structure and the regulation of dynamic instability. To investigate this relationship, we have modulated dynamic instability in Xenopus egg extracts by adding a catastrophe-promoting factor, Op18/stathmin. Using electron cryomicroscopy, we find that microtubules in cytoplasmic extracts grow by the extension of a two- dimensional sheet of protofilaments, which later closes into a tube. Increasing the catastrophe frequency by the addition of Op18/stathmin decreases both the length and frequency of the occurrence of sheets and increases the number of frayed ends. Interestingly, we also find that more dynamic populations contain more blunt ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. Our results demonstrate for the first time that microtubule assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of catastrophes is directly correlated with the structural state of microtubule ends.     

Self‐assembly of regenerated silk fibroin from random coil nanostructures to antiparallel β‐sheet nanostructures

In this work, we studied the effects of incubation concentration and time on the self‐assembly behaviors of regenerated silk fibroin (RSF). Our results showed the assembly ways of RSF were concentration‐dependent and there were four self‐assembly ways of RSF: (i) At relatively low concentration (≤0.015%), RSF molecules assembled into protofilaments (random coil), and then the thickness decreased and the secondary conformation changed to antiparallel β‐sheet; (ii) at the concentration of 0.015%, RSF molecules assembled into protofilaments (random coil), and then assembled into protofibrils (antiparallel β‐sheet). The protofibrils experienced the appearance and disappearance of phase periodic intervals in turn; (iii) at the concentration of 0.03%, RSF molecules assembled into bead‐like oligomers (random coil), and then assembled into protofibrils (antiparallel β‐sheet), and finally the height and phase periodic intervals of RSF protofibrils disappeared in turn; and (iv) at the relatively high concentration (≥0.15%), RSF molecules assembled into protofilaments (random coil), then aggregated into blurry cuboid‐like micelles (random coil), and finally self‐arranged to form smooth and clear cuboid‐like micelles (antiparallel β‐sheet). These results provide useful insights into the process by which the RSF molecules self‐assemble into protofilaments, protofibrils and micelles. Furthermore, our work will be beneficial to basic understanding of the nanoscale structure formations in different silk‐based biomaterials. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1181–1192, 2014.     

Bacterial ancestry of actin and tubulin

The structural and functional resemblance between the bacterial cell-division protein FtsZ and eukaryotic tubulin was the first indication that the eukaryotic cytoskeleton may have a prokaryotic origin. The bacterial ancestry is made even more obvious by the findings that the bacterial cell-shape-determining proteins Mreb and Mbl form large spirals inside non-spherical cells, and that MreB polymerises in vitro into protofilaments very similar to actin. Recent advances in research on two proteins involved in prokaryotic cytokinesis and cell shape determination that have similar properties to the key components of the eukaryotic cytoskeleton are discussed.     

Transient Membrane-Linked FtsZ Assemblies Precede Z-Ring Formation in Escherichia coli

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A structural model of an amyloid protofilament of transthyretin

A docking-and-alignment protocol was devised in order to build amyloid protofilaments of Transthyretin (TTR), starting from partially disrupted TTR monomeric subunits and based on experimentally available information. The docking approach is driven by a combination of shape complementarity and energetic criteria, and uses constraints derived from experimental data obtained for the fibrillar state. The dimeric structures obtained were then subjected to an alignment scheme followed by clustering analysis, producing a collection of protofilaments with distinct geometric properties. The selected protofilament model presented here does agree with known experimental data and general amyloid properties; it is formed by two extended continuous beta-sheets with the beta-strands perpendicular to the main axis of the protofilament and a helical twist with a period of approximately 48 beta-strands. This TTR proto-filament model may be an important step in the understanding of the molecular mechanisms of TTR aggregation, as well as, a valuable instrument in drug design strategies against amyloid diseases.     

Tektin interactions and a model for molecular functions

Tektins from echinoderm flagella were analyzed for microheterogeneity, self-associations and association with tubulin, resulting in a general model of tektin filament structure and function applicable to most eukaryotic cilia and flagella. Using a new antibody to tektin consensus peptide RPNVELCRD, well-characterized chain-specific antibodies and quantitative gel densitometry, tektins A, B and C were found to be present in equimolar amounts in Sarkosyl-urea-stable filaments. In addition, two isoforms of tektin A are present in half-molar ratios to tektins B and C. Cross-linking of AB filaments indicates in situ nearest neighbor associations of tektin A1B and A2B heterodimers, -trimers, -tetramers and higher oligomers. Soluble purified tektin C is cross-linked as homodimers, trimers and tetramers, but not higher oligomers. Tektin filaments associate with both loosely bound and tightly bound tubulin, and with the latter in a 1:1 molar ratio, implying a specific, periodic association of tightly bound tubulin along the tektin axis. Similarly, in tektin-containing Sarkosyl-stable protofilament ribbons, two polypeptides ( approximately 67/73 kDa, homologues of rib72, efhc1 and efhc2) are present in equimolar ratios to each other and to individual tektins, co-fractionating with loosely bound tubulin. These results suggest a super-coiled arrangement of tektin filaments, the organization of which has important implications for the evolution, assembly and functions of cilia and flagella.     

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.     

Recent advances in the discovery and development of antibacterial agents targeting the cell-division protein FtsZ

With the emergence of multidrug-resistant bacterial strains, there is a dire need for new drug targets for antibacterial drug discovery and development. Filamentous temperature sensitive protein Z (FtsZ), is a GTP-dependent prokaryotic cell division protein, sharing less than 10% sequence identity with the eukaryotic cell division protein, tubulin. FtsZ forms a dynamic Z-ring in the middle of the cell, leading to septation and subsequent cell division. Inhibition of the Z-ring blocks cell division, thus making FtsZ a highly attractive target. Various groups have been working on natural products and synthetic small molecules as inhibitors of FtsZ. This review summarizes the recent advances in the development of FtsZ inhibitors, focusing on those in the last 5 years, but also includes significant findings in previous years.