In silico investigation of molecular interactions of Volatile Anesthetics: Effects on phospholipid membranes and subcellular targets

The ability of anesthetics to reversibly suppress consciousness must reside in the effects exerted onto specific molecular targets. Interactions between Volatile Anesthetics and the phospholipid membrane as well as intracellular tubulin, were investigated using Computational Molecular Modelling, which showed rapid ligand partitioning inside the membrane and significant effects on the mechanical characteristics thereof, while transient binding locations have been found on the tubulin dimer.

Microtubule Disassembling Through Stathmin Bending Bow: A Molecular Dynamic Study

By this work, molecular modeling has been used to interpret the dynamic instabilities of these macromolecules in their structures. By this investigation, multi-dimension structures of microtubules are fixed in both length and width. Via Monte Carlo simulation, the tubulins have been added from the first side of the tubule towards the opposite side by gradually growing a random position. This method is theoretically accomplished via generating a uniform random number between (0, 1) based on the Monte Carlo approach. Our calculations have been done by proper dimension around 5×10-6 meters of length that consists of 2000 tubulin dimers. The structure growth rates are based on soluble tubulin dimer concentration. Hereby all results were run between 6-12 times in our modeling of any conditions. There have been recorded value numbers, average length, free tubulin concentration, and the important data of thermodynamic parameters for each simulation.

Fast leaps between millisecond confinements govern Ase1 diffusion along microtubules

Diffusion is the most fundamental mode of protein translocation within cells. Confined diffusion of proteins along the electrostatic potential constituted by the surface of microtubules, although modeled meticulously in molecular dynamics simulations, has not been experimentally observed in real-time. Here, we used interferometric scattering microscopy to directly visualize the movement of the microtubule-associated protein Ase1 along the microtubule surface at nanometer and microsecond resolution. We resolved millisecond confinements of Ase1 and fast leaps between these positions of dwelling preferentially occurring along the microtubule protofilaments, revealing Ase1’s mode of diffusive translocation along the microtubule’s periodic surface. The derived interaction potential closely matches the tubulin-dimer periodicity and the distribution of the electrostatic potential on the microtubule lattice. We anticipate that mapping the interaction landscapes for different proteins on microtubules, finding plausible energetic barriers of different positioning and heights, will provide valuable insights into regulating the dynamics of essential cytoskeletal processes, such as intracellular cargo trafficking, cell division, and morphogenesis, all of which rely on diffusive translocation of proteins along microtubules.

Acetylated α-tubulin residue K394 regulates microtubule stability to shape the growth of axon terminals

Microtubules are essential to neuron shape and function. Therefore, the stability of the microtubule cytoskeleton must be carefully regulated. Acetylation of tubulin has the potential to directly tune microtubule stability, and proteomic studies have identified several acetylation sites in α-tubulin. This includes the highly conserved residue lysine 394 (K394), which is located at the αβ-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons by reducing microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394 mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

Study of dynamic reaction of tubulins in microtubules. A qm/mm simulation

In this model one-dimensional microtubule is fixed at one of the two and simulated while the opposite end is allowed for growing in random situation. By this study at each step one tubulin has been added to the length for growing microtubule length. Computationally this can be done through generating a uniform random number between (0, 1). Microtubules are demonstrated as straight macromolecules consist of the linear chains of tubulin subunits in the length. QM/MM simulation has been applied to study dynamic instability of the microtubule length. It has been calculated a correct dimension around 10-6 meter of microtubules length consist of around 1650 tubulin dimers. Microtubule growth rate is related to the soluble tubulin dimer concentration and for all results shown here, simulation of any single condition was run 5–10 times.

Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a–tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

The large loop-8 insert in Saccharomyces cerevisiae kinesin-5 Cin8 is necessary and sufficient to promote noncanonical microtubule interactions

ABSTRACTSaccharomyces cerevisiae kinesin-5 Cin8 displays unconventional biochemical behavior including bidirectional motility and ability to bind multiple motor domains per αβ tubulin dimer in the microtubule lattice. Previous research suggested that a large loop-8 insert near the microtubule binding interface of Cin8 was critical for its noncanonical microtubule binding behavior. Here we utilized mutagenesis, thermodynamic, and kinetic assays to further understand the mechanism for how this loop-8 insert promotes super-stoichiometric microtubule binding in Cin8. This loop-8 insert that interrupts the conserved β5a/b hairpin was swapped between Cin8, Eg5 (KIF11, a human kinesin-5) and Kip1 (another S. cerevisiae kinesin-5). Cin8 with the loop-8 insert from Eg5 (Cin8-EL8) binds one motor per tubulin dimer, whereas Eg5 with the loop-8 insert from Cin8 (Eg5-CL8) binds approximately 2-4 motors per tubulin dimer. Eg5-CL8 bound the canonical and noncanonical sites on the microtubule lattice with weakened oligomerization between motors, while Cin8-EL8 showed only canonical site binding. These results demonstrate that the large loop-8 insert in Cin8 is necessary and sufficient to promote noncanonical microtubule binding behavior.

Self-repair protects microtubules from their destruction by molecular motors:

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

Lattice defects induce microtubule self-renewal

ABSTRACTThe dynamic instability of microtubules is powered by the addition and removal of tubulin dimers at the ends of the microtubule. Apart from the end, the microtubule shaft is not considered to be dynamic. However recent evidence suggests that free dimers can be incorporated into the shaft of a microtubule damaged by mechanical stress. Here we explored whether dimer exchange was a core property of the microtubule lattice independently of any external constraint. We found that dimers can be removed from and incorporated into the lattice at sites along the microtubule shaft. Furthermore, we showed by experiment and by modeling that rapid dimer renewal requires structural defects in the lattice, which occur in fast growing microtubules. Hence long-lived microtubules have the capacity to self-renew despite their apparent stability and thereby can potentially regulate signaling pathways and structural rearrangements associated with tubulin-dimer exchange at sites along their entire length.