Dynamic Instability from Non-equilibrium Structural Transitions on the Energy Landscape of Microtubule.

Microtubules are the backbone of the cytoskeleton and vital to numerous cellular processes. The central dogma of microtubules is that all their functions are driven by dynamic instability, but its mechanism has remained unresolved for over 30 years because of conceptual difficulties inherent in the dominant GTP-cap framework. We present a physically rigorous structural mechanochemical model: dynamic instability is driven by non-equilibrium transitions between the bent (B), straight (S), and curved (C) forms of tubulin monomers and longitudinal interfaces in the two-dimensional lattice of microtubule. All the different phenomena (growth, shortening, catastrophe, rescue, and pausing) are controlled by the kinetic pathways for B↔S↔C transitions and corresponding energy landscapes. Different kinetics at minus end are due to different B↔S↔C pathways imposed by the polarity of microtubule lattice. This model enables us to reproduce all the observed phenomena of dynamic instability of purified tubulins in kinetic simulations.

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex.

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis.

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics.

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH(2)-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration.

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.

Mechanistic insights from a quantitative analysis of pollen tube guidance.

BACKGROUND: Plant biologists have long speculated about the mechanisms that guide pollen tubes to ovules. Although there is now evidence that ovules emit a diffusible attractant, little is known about how this attractant mediates interactions between the pollen tube and the ovules. RESULTS: We employ a semi-in vitro assay, in which ovules dissected from Arabidopsis thaliana are arranged around a cut style on artificial medium, to elucidate how ovules release the attractant and how pollen tubes respond to it. Analysis of microscopy images of the semi-in vitro system shows that pollen tubes are more attracted to ovules that are incubated on the medium for longer times before pollen tubes emerge from the cut style. The responses of tubes are consistent with their sensing a gradient of an attractant at 100-150 mum, farther than previously reported. Our microscopy images also show that pollen tubes slow their growth near the micropyles of functional ovules with a spatial range that depends on ovule incubation time. CONCLUSIONS: We propose a stochastic model that captures these dynamics. In the model, a pollen tube senses a difference in the fraction of receptors bound to an attractant and changes its direction of growth in response; the attractant is continuously released from ovules and spreads isotropically on the medium. The model suggests that the observed slowing greatly enhances the ability of pollen tubes to successfully target ovules. The relation of the results to guidance in vivo is discussed.

Lattice model for self-assembly with application to the formation of cytoskeletal-like structures.

We introduce a stochastic approach for self-assembly in systems far from equilibrium. The building blocks are represented by a lattice of discrete variables (Potts-like spins), and physically meaningful mechanisms are obtained by restricting transitions through spatially local rules based on experimental data. We use the method to study nucleation of filopodia-like bundles in a system consisting of purified actin, fascin, actin-related protein 2/3 , and beads coated with Wiskott-Aldrich syndrome protein. Consistent with previous speculation based on static experimental images, we find that bundles derive from Lambda-precursor-like patterns of spins on the lattice. The ratcheting of the actin network relative to the surface that represents beads plays an important role in determining the number and orientation of bundles due to the fact that branching is the primary means for generating barbed ends pointed in directions that allow rapid filament growth. By enabling the de novo formation of coexisting morphologies without the computational cost of explicit representation of proteins, the approach introduced complements earlier models of cytoskeletal behavior in vitro and in vivo.