The C-terminus of stathmin-like proteins governs the stability of their complexes with tubulin.

Microtubule dynamics is modulated by many cellular factors including stathmin family proteins. Vertebrate stathmins sequester two αß-tubulin heterodimers into a tight complex that cannot be incorporated in microtubules. Stathmins are regulated at the expression level during development and among tissues; they are also regulated by phosphorylation. Here, we study the dissociation kinetics of tubulin:stathmin assemblies in presence of different tubulin-binding proteins and identify a critical role of the C-terminus of the stathmin partner. Destabilizing this C-terminal region may represent an additional regulatory mechanism of the interaction with tubulin of stathmin proteins.

Modeling Study of the Dynamics of Kinesin-14 Molecular Motors.

Kinesin-14s constitute a subfamily of the large superfamily of adenosine triphosphate-dependent microtubule-based motor proteins. Kinesin-14s have the motor domain at the C-terminal end of the peptide, playing key roles during spindle assembly and maintenance. Some of them are nonprocessive motors, whereas others can move processively on microtubules. Here, we take budding yeast Cik1-Kar3 and human HSET as examples to study theoretically the dynamics of the processive kinesin-14 motor moving on the single microtubule under load, the dynamics of the motor coupled with an Ndc80 protein moving on the single microtubule, the dynamics of the motor moving in microtubule arrays, and so on. The dynamics of the nonprocessive Drosophila Ncd motor is also discussed. The studies explain well the available experimental data and, moreover, provide predicted results. We show that the processive kinesin-14 motors can move efficiently in microtubule arrays toward the minus ends, and after reaching the minus ends, they can stay there stably, thus performing the function of organizing the microtubules in the bipolar spindle into polar arrays at the spindle poles.

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Tetrazine-trans-cyclooctene Mediated Conjugation of Antibodies to Microtubules Facilitates Subpicomolar Protein Detection.

Engineering cargo-loading strategies is crucial to developing nanotechnological applications of microtubule-based biomolecular transport systems. Here, we report a highly efficient and robust bioconjugation scheme to load antibodies to microtubules. Our method takes advantage of the inverse-electron-demand Diels-Alder addition reaction between tetrazine and trans-cyclooctene: the fastest known bioorthogonal reaction, characterized by its excellent selectivity and biocompatibility. As proof of concept, we performed kinesin-1 gliding motility assays with antibody-conjugated microtubules and demonstrated the highly sensitive detection of fluorescent protein analyte down to 0.1 pM in microliter sample volumes. Importantly, the detection selectivity was retained in the presence of other fluorescent background proteins. We envision the applicability of our fast, simple, and robust conjugation method to a wide range of biosensing platforms based on biomolecular transport systems.

Microtubules stabilize cell polarity by localizing rear signals.

Microtubules are known to play an important role in cell polarity; however, the mechanism remains unclear. Using cells migrating persistently on micropatterned strips, we found that depolymerization of microtubules caused cells to change from persistent to oscillatory migration. Mathematical modeling in the context of a local-excitation-global-inhibition control mechanism indicated that this mechanism can account for microtubule-dependent oscillation, assuming that microtubules remove inhibitory signals from the front after a delayed generation. Experiments further supported model predictions that the period of oscillation positively correlates with cell length and that oscillation may be induced by inhibiting retrograde motors. We suggest that microtubules are required not for the generation but for the maintenance of cell polarity, by mediating the global distribution of inhibitory signals. Disassembly of microtubules induces cell oscillation by allowing inhibitory signals to accumulate at the front, which stops frontal protrusion and allows the polarity to reverse.

Mdp3 is a novel microtubule-binding protein that regulates microtubule assembly and stability.

Microtubule-binding proteins are a group of molecules that associate with microtubules, regulate the structural properties of microtubules, and thereby participate in diverse microtubule-mediated cellular activities. A recent mass spectrometry-based proteomic study has identified microtubule-associated protein 7 (MAP7) domain-containing 3 (Mdp3) as a potential microtubule-binding protein. However, its subcellular localization and functional importance are not characterized. In this study, by GST-pulldown assays, we found that Mdp3 interacted with tubulin both in cells and in vitro. Immunofluorescence microscopy and microtubule cosedimentation assays revealed that Mdp3 also associated with microtubules. Serial deletion experiments showed that the two coiled coil motifs of Mdp3 were critical for its interaction with tubulin and microtubules. Cold recovery and nocodazole washout assays further demonstrated an important role for Mdp3 in regulating cellular microtubule assembly. Our data also showed that Mdp3 significantly enhanced the stability of cellular microtubules. By tubulin turbidity assay, we found that Mdp3 could promote microtubule assembly and stability in the purified system. In addition, we found that Mdp3 expression varied during the cell cycle and in primary tissues. These findings thus establish Mdp3 as a novel microtubule-binding protein that regulates microtubule assembly and stability.

The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly.

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.

Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy.

The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.

Isolation of microtubules and microtubule proteins.

This unit describes various protocols for the isolation and purification of the main constituents of microtubules, chiefly alpha- and beta-tubulin, and the most significant microtubule associated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We include a classical isolation method for soluble tubulin heterodimer as the first basic purification protocol. In addition, we show how to analyze the tubulin and MAPs obtained after a phosphocellulose chromatography purification procedure. This unit also details a powerful and simple method to determine the native state of the purified tubulin based on one-dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last protocol describes the application of a new technique that allows visualizing the quality of polymerized microtubules based on atomic force microscopy (AFM).

Mass spectrometric analysis of microtubule co-sedimented proteins from rat brain.

Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures.

Molecular encounters at microtubule ends in the plant cell cortex.

The cortical arrays that accompany plant cell division and elongation are organized by a subtle interplay between intrinsic properties of microtubules, their self-organization capacity and a variety of cellular proteins that interact with them, modify their behaviour and drive organization of diverse, higher order arrays during the cell cycle, cell growth and differentiation. As a polar polymer, the microtubule has a minus and a plus end, which differ in structure and dynamic characteristics, and to which different sets of partners and activities associate. Recent advances in characterization of minus and plus end directed proteins provide insights into both plant microtubule properties and the way highly organized cortical arrays emerge from the orchestrated activity of individual microtubules.

Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa.

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.

Jupiter, a new Drosophila protein associated with microtubules.

In this study we describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-associated proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a soluble unfolded molecule with the high net positive charge, rich in Glycine. It possesses two degenerated repeats around the sequence PPGG, separated by a Serine-rich region. Jupiter associates with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the "protein-trap" technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concentrations in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established two permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo analysis of microtubule dynamics.

Truncation of the projection domain of MAP4 (microtubule-associated protein 4) leads to attenuation of microtubule dynamic instability.

MAP4, a ubiquitous heat-stable MAP, is composed of an asymmetric structure common to the heat-stable MAPs, consisting of an N-terminal projection (PJ) domain and a C-terminal microtubule (MT)-binding (MTB) domain. Although the MTB domain has been intensively studied, the role of the PJ domain, which protrudes from MT-wall and does not bind to MTs, remains unclear. We investigated the roles of the PJ domain on the dynamic instability of MTs by dark-field microscopy using various PJ domain deletion constructs of human MAP4 (PJ1, PJ2, Na-MTB and KDM-MTB). There was no obvious difference in the dynamic instability between the wtMAP4 and any fragments at 0.1 microM, the minimum concentration required to stabilize MTs. The individual MTs stochastically altered between polymerization and depolymerization phases with similar profiles of length change as had been observed in the presence of MAP2 or tau. We also examined the effects at the increased concentrations of 0.7 microM, and found that in some cases the dynamic instability was almost entirely attenuated. The length of both the polymerization and depolymerization phases decreased and "pause-phases" were occasionally observed, especially in the case of PJ1, PJ2 or Na-MTB. No obvious change was observed in the increased concentration of wtMAP4 and KDM-MTB. Additionally, the profiles of MT length change were quite different in 0.7 microM PJ2. Relatively rapid and long depolymerization phases were sometimes observed among quite slow length changes. Perhaps, this unusual profile could be due to the uneven distribution of PJ2 along the MT lattice. These results indicate that the PJ domain of MAP4 participates in the regulation of the dynamic instability.

Proteome analysis of the human mitotic spindle.

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies.

Neuronal-associated microtubule proteins class III beta-tubulin and MAP2c in neuroblastoma: role in resistance to microtubule-targeted drugs.

Advanced stage neuroblastoma has a poor clinical outcome and microtubule-destabilizing agents, such as the Vinca alkaloids, are an important component in the treatment of this childhood cancer. Vinca alkaloids bind to beta-tubulin on the alpha/beta-tubulin heterodimer and disrupt microtubule dynamics, leading to cell death. To date, studies examining the contribution of microtubules and associated proteins to the efficacy of microtubule-destabilizing agents in neuroblastoma have been limited. In this study, BE2-C neuroblastoma cells previously selected for resistance to either vincristine (BE/VCR10) or colchicine (BE/CHCb0.2) were found to display significant decreases in neuronal-specific class III beta-tubulin. Interestingly, vincristine-selected cells exhibited increased levels of polymerized tubulin that were not due to alpha-tubulin and class I, II, or III beta-tubulin mutations. Expression levels of the microtubule-depolymerizing protein stathmin were significantly increased in BE/VCR10 cells. In contrast, levels of MAP2a and MAP2b were relatively unaltered. A marked decrease in the neuronal protein, MAP2c, was identified in the vincristine-selected cells and, to a lesser extent, in the colchicine-selected cells. This is the first report describing specific microtubule alterations in neuroblastoma cells resistant to tubulin-targeted agents. The results indicate a need to identify the factors responsible for resistance to tubulin-targeted agents in neuroblastoma so that improved and novel treatment strategies can be developed for this drug refractory disease.

Differential localization of decidual stathmin during pregnancy in rats.

The present study was undertaken to determine the precise localization of stathmin, a protein associated with microtubule dynamics, during decidualization in rat uterus and to compare it with that of cyclin D3. Immunohistochemical analysis revealed that stathmin is exclusively localized in decidual cells, especially in the primary decidual zone surrounding the embryo, on days 7 and 9 of pregnancy. The intensity of staining was much higher on day 9 than day 7. On day 14, when the endometrial stromal cells had completely differentiated into decidual cells, the staining of decidual cells was faint. Cyclin D3 was expressed in decidual cells of the secondary but not the primary decidual zone on days 7 and 9. On day 14, cyclin D3 levels were low in decidua. Proliferating cell nuclear antigen (PCNA) was broadly detected in the uterus on days 7 and 9, and in the placenta and fetus on day 14. In an artificial decidualization model, cyclin D3 expression was stimulated as deciduoma was formed after an artificial stimulus. Stathmin mRNA levels also increased within 24 h and peaked at 48 h. The specific spatio-temporal uterine expression of stathmin and cyclin D3 suggest that they have a specific role in decidualization in rats.

Cloning and characterization of testis-specific tektin in Bombyx mori.

A testis-specific cDNA library of Bombyx mori was constructed by an mRNA subtraction technique. Several clones were randomly selected and determined for their nucleotide sequences. One of them, designated as BmTST, contained a 3'-part of an open reading frame homologous to tektin, the protein known to form filamentous polymers in the walls of ciliary and flagellar microtubules. Also isolated was a genomic fragment, which contains the 5'-part of the coding sequence of BmTST and its promoter region. As a whole, the complete open reading frame was found to encode 508 amino acid residues, whose sequence had 28, 28 and 30% identities with the Strongylocentrotus purpuratus tektins A1, B1 and C1, respectively. Expression analysis by reverse transcription polymerase chain reaction with the cDNA and Western blotting with a polyclonal antibody indicated that the BmTST gene was expressed specifically in the testis during sperm maturation. The protein was immunologically detected exclusively in the fraction expected to contain the 9 + 2 flagellar axonemes of sperms. We infer that the BmTst protein is possibly involved in the spermatogenesis of B. mori.

Decreasing oncoprotein 18/stathmin levels reduces microtubule catastrophes and increases microtubule polymer in vivo.

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein which destabilizes microtubules. To characterize the function of Op18 in living cells, we used microinjection of anti-Op18 antibodies or antisense oligonucleotides to block either Op18 activity or expression in interphase newt lung cells. Anti-tubulin staining of cells microinjected with anti-Op18 and fixed 1-2 hours after injection showed an increase in total microtubule polymer. In contrast, microinjection of either non-immune IgG or anti-Op18 preincubated with bacterially-expressed Op18 had little effect on microtubule polymer level. Cells treated with Op18 antisense oligonucleotides for 4 days had (greater than or equal to)50% reduced levels of Op18 with no change in the soluble tubulin level. Measurement of MT polymer level in untreated, antisense or nonsense oligonucleotide treated cells demonstrated that reduced Op18 levels resulted in a 2.5-fold increase in microtubule polymer. Next, the assembly dynamics of individual microtubules at the peripheral regions of living cells were examined using video-enhanced contrast DIC microscopy. Microinjection of antibodies against oncoprotein 18 resulted in a 2.2-fold reduction in catastrophe frequency and a slight reduction in plus end elongation velocity compared to uninjected cells or cells microinjected with non-immune IgG. Preincubation of anti-Op18 antibody with recombinant Op18 greatly diminished the effects of the antibody. Similarly, treatment of cells with antisense oligonucleotides reduced catastrophes 2.5- to 3-fold compared to nonsense oligonucleotide treated or untreated cells. The other parameters of dynamic instability were unchanged after reducing Op18 with antisense oligonucleotides. These studies are consistent with Op18 functioning to regulate microtubule catastrophes during interphase in vivo.