Microtubule-interacting Proteins Research Articles

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Inflammatory Glycoprotein 130 Signaling Links Changes in Microtubules and Junctophilin-2 to Altered Mitochondrial Metabolism and Right Ventricular Contractility.

Right ventricular dysfunction (RVD) is the leading cause of death in pulmonary arterial hypertension (PAH), but no RV-specific therapy exists. We showed microtubule-mediated junctophilin-2 dysregulation (MT-JPH2 pathway) causes t-tubule disruption and RVD in rodent PAH, but the druggable regulators of this critical pathway are unknown. GP130 (glycoprotein 130) activation induces cardiomyocyte microtubule remodeling in vitro; however, the effects of GP130 signaling on the MT-JPH2 pathway and RVD resulting from PAH are undefined. Immunoblots quantified protein abundance, quantitative proteomics defined RV microtubule-interacting proteins (MT-interactome), metabolomics evaluated the RV metabolic signature, and transmission electron microscopy assessed RV cardiomyocyte mitochondrial morphology in control, monocrotaline, and monocrotaline-SC-144 (GP130 antagonist) rats. Echocardiography and pressure-volume loops defined the effects of SC-144 on RV-pulmonary artery coupling in monocrotaline rats (8-16 rats per group). In 73 patients with PAH, the relationship between interleukin-6, a GP130 ligand, and RVD was evaluated. SC-144 decreased GP130 activation, which normalized MT-JPH2 protein expression and t-tubule structure in the monocrotaline RV. Proteomics analysis revealed SC-144 restored RV MT-interactome regulation. Ingenuity pathway analysis of dysregulated MT-interacting proteins identified a link between microtubules and mitochondrial function. Specifically, SC-144 prevented dysregulation of electron transport chain, Krebs cycle, and the fatty acid oxidation pathway proteins. Metabolomics profiling suggested SC-144 reduced glycolytic dependence, glutaminolysis induction, and enhanced fatty acid metabolism. Transmission electron microscopy and immunoblots indicated increased mitochondrial fission in the monocrotaline RV, which SC-144 mitigated. GP130 antagonism reduced RV hypertrophy and fibrosis and augmented RV-pulmonary artery coupling without altering PAH severity. In patients with PAH, higher interleukin-6 levels were associated with more severe RVD (RV fractional area change 23±12% versus 30±10%, P=0.002). GP130 antagonism reduces MT-JPH2 dysregulation, corrects metabolic derangements in the RV, and improves RVD in monocrotaline rats.

Affinity purification of tubulin from plant materials.

In the plant cytoskeleton research, mammalian brain tubulin has been widely used to study plant microtubule-interacting proteins in vitro since purification of tubulins from plant sources is generally considered to be challenging and time-consuming. A convenient method for affinity purification of tubulins was devised, which utilized the TOG domains of yeast Stu2 tubulin-binding protein as an affinity ligand (Widlund et al., 2012). We showed that this so-called TOG tubulin affinity chromatography worked efficiently with plant materials, especially actively-dividing cultured cells (Hotta et al., 2016). Plant tubulins purified with the TOG method is highly assembly-competent and thus can be used in various in vitro experiments. Here, we summarize purification strategies of native or tagged plant tubulins as well as an in vitro pull-down assay to monitor their polymerization activity.

Ciliary kinesins beyond IFT: Cilium length, disassembly, cargo transport and signalling.

Cilia and flagella are microtubule-based antenna which are highly conserved among eukaryotes. In vertebrates, primary and motile cilia have evolved to exert several key functions during development and tissue homoeostasis. Ciliary dysfunction in humans causes a highly heterogeneous group of diseases called ciliopathies, a class of genetic multisystemic disorders primarily affecting kidney, skeleton, retina, lung and the central nervous system. Among key ciliary proteins, kinesin family members (KIF) are microtubule-interacting proteins involved in many diverse cellular functions, including transport of cargo (organelles, proteins and lipids) along microtubules and regulating the dynamics of cytoplasmic and spindle microtubules through their depolymerising activity. Many KIFs are also involved in diverse ciliary functions including assembly/disassembly, motility and signalling. We here review these ciliary kinesins in vertebrates and focus on their involvement in ciliopathy-related disorders.

Deciphering the Tubulin Code

Enzymes of the tubulin tyrosine ligase-like (TTLL) family posttranslationally modify and thereby mark microtubules by glutamylation, generating specific recognition sites for microtubule-interacting proteins. Garnham et al. report the first structure of a TTLL protein alone and in complex with microtubules, elucidating their mechanism of action.

Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

BackgroundIn the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity.ResultsFollowing a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration.ConclusionsOur work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.

The centriolar satellite protein SSX2IP promotes centrosome maturation

Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.

Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α–tubulin. Tyrosination regulates recruitment of microtubule interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP-grasp fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.

Molekulare Progressionsmechanismen der humanen Hepatokarzinogenese

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors worldwide with poor prognosis. Based on high-throughput screening technology, we and others have identified factors and pathways that are pivotal for tumor progression including transcription factors and microtubule-interacting proteins. In addition, aberrant activation of the IGF signalling pathway is frequently observed in HCCs which is predominantly based on high level expression of its ligand IGF-II. Because protumorigenic effects of IGF-II such as proliferation, anti-apoptosis, and migration are transmitted through its receptor IGF-1R, selective inhibition of this tyrosine kinase by small molecule compounds might reduce IGF-II-driven tumor growth. Indeed, administration of IGF-1R-selective inhibitors reduces IGF-II-induced effects and was associated with a significant reduction of tumor growth in a xenograft transplantation model. In conclusion, the IGF-II/IGF-1R signalling pathway is critically involved in the regulation of tumor growth and tumor cell dissemination, representing a promising therapeutic target structure in the treatment of HCC.

APC2 plays an essential role in axonal projections through the regulation of microtubule stability.

Growth cones at the tip of growing axons are key cellular structures that detect guidance cues and mediate axonal growth. An increasing number of studies have suggested that the dynamic regulation of microtubules in the growth cone plays an essential role in growth cone steering. The dynamic properties of microtubules are considered to be regulated by variegated cellular factors but, in particular, through microtubule-interacting proteins. Here, we examined the functional role of adenomatous polyposis coli-like molecule 2 (APC2) in the development of axonal projections by using the chick retinotectal topographic projection system. APC2 is preferentially expressed in the nervous system from early developmental stages through to adulthood. Immunohistochemical analysis revealed that APC2 is distributed along microtubules in growth cones as well as axon shafts of retinal axons. Overexpression of APC2 in cultured cells induced the stabilization of microtubules, whereas the knockdown of APC2 in chick retinas with specific short hairpin RNA reduced the stability of microtubules in retinal axons. APC2 knockdown retinal axons showed abnormal growth attributable to a reduced response to ephrin-A2 in vitro. Furthermore, they showed drastic alterations in retinotectal projections without making clear target zones in the tectum in vivo. These results suggest that APC2 plays a critical role in the development of the nervous system through the regulation of microtubule stability.

Coordinated Expression of Stathmin Family Members by Far Upstream Sequence Element-Binding Protein-1 Increases Motility in Non–Small Cell Lung Cancer

Dynamic instability of the microtubule network modulates processes such as cell division and motility, as well as cellular morphology. Overexpression of the microtubule-destabilizing phosphoprotein stathmin is frequent in human malignancies and represents a promising therapeutic target. Although stathmin inhibition gives rise to antineoplastic effects, additional and functionally redundant microtubule-interacting proteins may attenuate the efficiency of this therapeutic approach. We have systematically analyzed the expression and potential protumorigenic effects of stathmin family members in human non-small cell lung cancer (NSCLC). Both stathmin and stathmin-like 3 (SCLIP) were overexpressed in adenocarcinoma as well as squamous cell carcinoma (SCC) tissues and induced tumor cell proliferation, migration, and matrix invasion in respective cell lines. Accordingly, reduced stathmin and SCLIP levels affected cell morphology and were associated with a less malignant phenotype. Combined inhibition of both factors caused additive effects on tumor cell motility, indicating partial functional redundancy. Because stathmin and SCLIP expression significantly correlated in NSCLC tissues, we searched for common upstream regulators and identified the far upstream sequence element-binding protein-1 (FBP-1) as a pivotal inducer of several stathmin family members. Our results indicate that the coordinated overexpression of microtubule-destabilizing factors by FBP-1 is a critical step to facilitate microtubule dynamics and subsequently increases proliferation and motility of tumor cells.

Antimitotic agent alters MIP levels In breast cancer cells

Progression through mitosis requires a balance of active microtubule-interacting proteins (MIPs) that stabilize and destabilize microtubules in mitotic spindles. Molecular interactions between MIPs and tubulin or microtubules are important for cell cycle progression. Insights into these interactions can contribute to understanding the mechanisms underlying cell cycle interference by antimitotic agents that halt cell cycle progression in mitosis. We examined the direct interaction of the antimitotic agent, vinblastine with tubulin and stathmin using AUC, in order to understand how changes in stathmin levels during the cell cycle might affect the cellular drug response. Vinblastine acts during G2/M phase of the cell cycle and reduces microtubule dynamics. At high doses it destabilizes microtubules in mitotic spindles. We found in vitro that stathmin reduces the potency of vinblastine. Vinblastine was found to compete for tubulin-stathmin oligomers, at the same time as it induced tubulin spiral formation. To extend these data to a cellular context, we investigated changes in intracelluar MIP levels in response to paclitaxel, an antimitotic agent known to stabilize microtubules at high concentrations. Using qRT-PCR we found that paclitaxel treatment of human breast cancer MCF7 cells leads to a significant reduction in MAP4 and stathmin mRNA levels. Interestingly, the levels return to pre-paclitaxel treatment levels after a 4-day drug washout, suggesting that paclitaxel alters transcript levels. We found that the ratios of MAP4/stathmin increased after or during drug treatment. These data suggest that changes in MIPs levels alter the cellular response to drugs. These results also suggest that disruption of the cell cycle by antimitotic agents can alter the relative amounts of MIPs and thus affect the balance needed for normal progression through the cell cycle. These results must be taken into account when modeling the cellular response to antimitotic drugs.

Drosophila Ankyrin 2 Is Required for Synaptic Stability

Synaptic connections are stabilized through transsynaptic adhesion complexes that are anchored in the underlying cytoskeleton. The Drosophila neuromuscular junction (NMJs) serves as a model system to unravel genes required for the structural remodeling of synapses. In a mutagenesis screen for regulators of synaptic stability, we recovered mutations in Drosophila ankyrin 2 (ank2) affecting two giant Ank2 isoforms that are specifically expressed in the nervous system and associate with the presynaptic membrane cytoskeleton. ank2 mutant larvae show severe deficits in the stability of NMJs, resulting in a reduction in overall terminal size, withdrawal of synaptic boutons, and disassembly of presynaptic active zones. In addition, lack of Ank2 leads to disintegration of the synaptic microtubule cytoskeleton. Microtubules and microtubule-associated proteins fail to extend into distant boutons. Interestingly, Ank2 functions downstream of spectrin in the anchorage of synaptic microtubules, providing the cytoskeletal scaffold that is essential for synaptic stability.

Functional evidence for in vitro microtubule severing by the plant katanin homologue.

Temporal and spatial assembly of microtubules in plant cells depends mainly on the activity of microtubule-interacting proteins, which either stabilize, destabilize or translocate microtubules. Recent data have revealed that the thale cress (Arabidopsis thaliana) contains a protein related to the p60 catalytic subunit of animal katanin, a microtubule-severing protein. However, effects of the plant p60 on microtubule assembly are not known. We report the first functional evidence that the recombinant A. thaliana p60 katanin subunit, Atp60, binds to microtubules and severs them in an ATP-dependent manner in vitro. ATPase activity of Atp60 is stimulated by low tubulin/katanin ratios, and is inhibited at higher ratios. Considering its properties in vitro, several functions of Atp60 in vivo are discussed.

D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo.

We identify Drosophila TACC (D-TACC) as a novel protein that is concentrated at centrosomes and interacts with microtubules. We show that D-TACC is essential for normal spindle function in the early embryo; if D-TACC function is perturbed by mutation or antibody injection, the microtubules emanating from centrosomes in embryos are short and chromosomes often fail to segregate properly. The C-terminal region of D-TACC interacts, possibly indirectly, with microtubules, and can target a heterologous fusion protein to centrosomes and microtubules in embryos. This C-terminal region is related to the mammalian transforming, acidic, coiled-coil-containing (TACC) family of proteins. The function of the TACC proteins is unknown, but the genes encoding the known TACC proteins are all associated with genomic regions that are rearranged in certain cancers. We show that at least one of the mammalian TACC proteins appears to be associated with centrosomes and microtubules in human cells. We propose that this conserved C-terminal 'TACC domain' defines a new family of microtubule-interacting proteins.

An anchoring factor targets protein phosphatase 2A to brain microtubules

Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase composed of a heterodimeric core enzyme that associates with a variety of regulatory subunits. A fraction of brain PP2A associates with microtubules and may play a role in regulating phosphorylation of microtubule-associated proteins. We examined the isoform specificity and the mechanism involved in the association of PP2A with brain microtubules. Only the R2α (B/PR55α) and R2β (B/PR55β) regulatory subunits associated with endogenous neural microtubules. Neither the R2γ (B/PR55γ) nor members of the R5 (B′/PR56) family of regulatory subunits co-sedimented with microtubules, although abundant amounts of these proteins were detected in brain. The efficient association of PP2A with microtubules in vitro was dependent on an anchoring activity present in a brain protein fraction containing microtubule-associated and microtubule-interacting proteins. Anchoring factor-dependent association of PP2A with microtubules was specific for the heterotrimeric form of PP2A. The core dimer and the isolated subunits of PP2A had very little affinity for microtubules. Characterization of a fraction enriched in the anchoring factor showed that the activity was a heat labile protein that does not correspond to classical microtubule-associated proteins. The anchoring factor associated with microtubules independently of PP2A. These results indicate the association of PP2A with microtubules can be mediated by an anchoring factor that interacts in an isoform-specific manner with heterotrimeric forms of the phosphatase.

Kinesin Is a Candidate for Cross-bridging Microtubules and Intermediate Filaments: SELECTIVE BINDING OF KINESIN TO DETYROSINATED TUBULIN AND VIMENTIN

We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an approximately 2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of approximately 120 kDa and a light chain of approximately 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster.

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes.

Microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of beta-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a 67 kDa microtubule-binding protein in the pancreas from different species

Microtubule-interacting proteins have been studied from a pancreas supernatant. These proteins were first identified by affinity chromatography on taxol-stabilized microtubules. Among these interacting polypeptides, we show, for the first time, the presence of a protein which has a molecular mass of 67 kDa, as determined by polyacrylamide slab gel electrophoresis. The heat stability and the ability of this 67 kDa polypeptide to copolymerize with phosphocellulose-purified tubulin suggest that this protein may be a microtubule-associated protein.