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SummaryProteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

Summary 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.

Microtubules are a major component of the cytoskeleton and vital to numerous cellular processes. The central dogma of microtubules is that all their functions are driven by dynamic instability; understanding its key phenomena (i.e. catastrophe, rescue, pause, differential behaviors at the plus and minus ends) distilled from a myriad of experiments under a consistent and unified scheme, however, has been unattainable. Here, we present a novel statistical-physics-based model uniquely constructed from conformational states deduced from existing tubulin structures, with transitions between them controlled by steric constraints and mechanical energy of the microtubule lattice. This mechano-chemical model allows, for the first time, all the key phenomena of dynamic instability to be coherently reproduced by the corresponding kinetic simulations. Long-puzzling phenomena, such as aging, small GTP-cap size, fast catastrophe upon dilution and temperature-induced ribbon-to-tube transition of GMPCPP-tubulins, robustly emerge and thus can be understood with confidence.

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-severing protein Katanin is now shown to possess microtubule depolymerising activity. Purified recombinant Katanin exerts both activities in vitro. In migrating cells from Drosophila, Katanin localizes at the leading edge where it negatively regulates cell motility. 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.

The direct interaction between the ERM protein Moesin and microtubules is required for spindle organization in metaphase and cell shape transformation after anaphase onset., 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.

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 μm, 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.