Microtubule-associated Proteins Function Research Articles

Emerging role of microtubule-associated proteins on cancer metastasis.

The major cause of death in cancer patients is strongly associated with metastasis. While much remains to be understood, microtubule-associated proteins (MAPs) have shed light on metastatic progression’s molecular mechanisms. In this review article, we focus on the role of MAPs in cancer aggressiveness, particularly cancer metastasis activity. Increasing evidence has shown that a growing number of MAP member proteins might be fundamental regulators involved in altering microtubule dynamics, contributing to cancer migration, invasion, and epithelial-to-mesenchymal transition. MAP types have been established according to their microtubule-binding site and function in microtubule-dependent activities. We highlight that altered MAP expression was commonly found in many cancer types and related to cancer progression based on available evidence. Furthermore, we discuss and integrate the relevance of MAPs and related molecular signaling pathways in cancer metastasis. Our review provides a comprehensive understanding of MAP function on microtubules. It elucidates how MAPs regulate cancer progression, preferentially in metastasis, providing substantial scientific information on MAPs as potential therapeutic targets and prognostic markers for cancer management.

The evolution of microtubule associated proteins \u2013 a reference proteomic perspective

Microtubule associated proteins (MAPs), defined as proteins that bind microtubules but are not molecular motors or severing enzymes, play a key role in regulating microtubule stability in neurons. Existing studies of the evolutionary relationships between these proteins are limited to genomic data from a small number of species. We therefore used a large collection of publicly available reference-quality eukaryotic proteomes to carry out a phylogenetic analysis of microtubule associated proteins in both vertebrates and invertebrates. Complete or near-complete reference quality proteomes were obtained from Uniprot. Microtubule associated proteins were identified using InterProtScan, aligned using MUSCLE and then phylogenetic trees constructed using the WAG algorithm. We identified 889 proteins with tubulin binding domains, of which 663 were in eukaryotes, including 168 vertebrates and 64 invertebrates. The vertebrate proteins separated into three families, resembling human MAP 2, MAP4 and MAPT, respectively, while invertebrate MAPs clustered separately. We found significant variation in number of microtubule associated proteins and number of microtubule binding domains between taxa, with fish and mollusks having an unexpectedly high number of MAPs and binding domains, respectively. Our findings represent a novel analysis of the evolution of microtubule associated proteins based on publicly available proteomics data sets. We were able to confirm the phylogeny of MAPs identified based on more limited genomic analyses, and in addition, derived several novel insights on the structure and function of MAPs.

A Crosslinking Mass Spectrometry Protocol for the Structural Analysis of Microtubule-Associated Proteins.

Microtubule-associated proteins (MAPs) engage microtubules (MTs) to regulate both the MT state and wide variety of cytoskeletal functions. A comprehensive understanding of MAPs function requires the structural characterization of physical contacts MAPs make with other proteins, particularly when engaged with the microtubule (MT) lattice. Most of the interaction between MAPs and MTs evade classical structural determination techniques, as the interactions can be both heterogenous and sub-stoichiometric. Crosslinking mass spectrometry (XL-MS) can aid in MAP-MT structure analysis by providing a wealth of residue-based distance restraints. This protocol provides an XL-MS workflow for accurate and unbiased sampling of an equilibrated MAP-MT interaction, involving modifications to the preparation and validation of a MAP-MT construct suitable for crosslinking with fast-sampling heterobifunctional crosslinkers. The distance restrains obtained by this protocol can be used to generate accurate models assembled with an integrative structural modeling approach.

Mapping the kinetochore MAP functions required for stabilizing microtubule attachments to chromosomes during metaphase.

In mitosis, faithful chromosome segregation is orchestrated by the dynamic interactions between the spindle microtubules (MTs) emanating from the opposite poles and the kinetochores of the chromosomes. However, the precise mechanism that coordinates the coupling of the kinetochore components to dynamic MTs has been a long-standing question. Microtubule-associated proteins (MAPs) regulate MT nucleation and dynamics, MT-mediated transport and MT cross-linking in cells. During mitosis, MAPs play an essential role not only in determining spindle length, position, and orientation but also in facilitating robust kinetochore-microtubule (kMT) attachments by linking the kinetochores to spindle MTs efficiently. The stability of MTs imparted by the MAPs is critical to ensure accurate chromosome segregation. This review primarily focuses on the specific function of nonmotor kinetochore MAPs, their recruitment to kinetochores and their MT-binding properties. We also attempt to synthesize and strengthen our understanding of how these MAPs work in coordination with the kinetochore-bound Ndc80 complex (the key component at the MT-binding interface in metaphase and anaphase) to establish stable kMT attachments and control accurate chromosome segregation during mitosis.

ReMAPping the microtubule landscape: How phosphorylation dictates the activities of microtubule-associated proteins.

Classical microtubule-associated proteins (MAPs) were originally identified based on their co-purification with microtubules assembled from mammalian brain lysate. They have since been found to perform a range of functions involved in regulating the dynamics of the microtubule cytoskeleton. Most of these MAPs play integral roles in microtubule organization during neuronal development, microtubule remodeling during neuronal activity, and microtubule stabilization during neuronal maintenance. As a result, mutations in MAPs contribute to neurodevelopmental disorders, psychiatric conditions, and neurodegenerative diseases. MAPs are post-translationally regulated by phosphorylation depending on developmental time point and cellular context. Phosphorylation can affect the microtubule affinity, cellular localization, or overall function of a particular MAP and can thus have profound implications for neuronal health. Here we review MAP1, MAP2, MAP4, MAP6, MAP7, MAP9, tau, and DCX, and how each is regulated by phosphorylation in neuronal physiology and disease. Developmental Dynamics 247:138-155, 2018. © 2017 Wiley Periodicals, Inc.

Asymmetric Friction of Nonmotor MAPs Can Lead to Their Directional Motion in Active Microtubule Networks

Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division. We find that frictional forces increase nonlinearly with MAP velocity across microtubules and depend on filament polarity, with NuMA's friction being lower when moving toward minus ends, EB1's lower toward plus ends, and PRC1's exhibiting no directional preference. Mathematical models predict, and experiments confirm, that MAPs with asymmetric friction can move directionally within actively moving microtubule pairs they crosslink. Our findings reveal how nonmotor MAPs can generate frictional resistance in dynamic cytoskeletal networks via micromechanical adaptations whose anisotropy may be optimized for MAP localization and function within cellular structures.

The growth speed of microtubules with XMAP215-coated beads coupled to their ends is increased by tensile force

The generation of pulling and pushing forces is one of the important functions of microtubules, which are dynamic and polarized structures. The ends of dynamic microtubules are able to form relatively stable links to cellular structures, so that when a microtubule grows it can exert a pushing force and when it shrinks it can exert a pulling force. Microtubule growth and shrinkage are tightly regulated by microtubule-associated proteins (MAPs) that bind to microtubule ends. Given their localization, MAPs may be exposed to compressive and tensile forces. The effect of such forces on MAP function, however, is poorly understood. Here we show that beads coated with the microtubule polymerizing protein XMAP215, the Xenopus homolog of Dis1 and chTOG, are able to link stably to the plus ends of microtubules, even when the ends are growing or shrinking; at growing ends, the beads increase the polymerization rate. Using optical tweezers, we found that tensile force further increased the microtubule polymerization rate. These results show that physical forces can regulate the activity of MAPs. Furthermore, our results show that XMAP215 can be used as a handle to sense and mechanically manipulate the dynamics of the microtubule tip.

Template-Free 13-Protofilament Microtubule-Map Assembly Visualised at 8Å Resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organising cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy (cryo-EM) and single particle algorithms, we solved the 8Å structure of doublecortin (DCX)-stabilized microtubules. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament (pf) MTs, our reconstruction provides unprecedented insight into the structure of MTs with their in vivo architecture and in the absence of a stabilizing drug. DCX specifically recognises the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly facilitates formation of conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

Template-free 13-protofilament microtubule–MAP assembly visualized at 8 Å resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

MAP2-mediated in vitro interactions of brain microtubules and their modulation by cAMP

Microtubule-associated proteins (MAPs) are involved in microtubule (MT) bundling and in crossbridges between MTs and other organelles. Previous studies have assigned the MT bundling function of MAPs to their MT-binding domain and its modulation by the projection domain. In the present work, we analyse the viscoelastic properties of MT suspensions in the presence or the absence of cAMP. The experimental data reveal the occurrence of interactions between MT polymers involving MAP2 and modulated by cAMP. Two distinct mechanisms of action of cAMP are identified, which involve on one hand the phosphorylation of MT proteins by the cAMP-dependent protein kinase A (PKA) bound to the end of the N-terminal projection of MAP2, and on the other hand the binding of cAMP to the RII subunit of the PKA affecting interactions between MTs in a phosphorylation-independent manner. These findings imply a role for the complex of PKA with the projection domain of MAP2 in MT-MT interactions and suggest that cAMP may influence directly the density and bundling of MT arrays in dendrites of neurons.

Mars promotes dTACC dephosphorylation on mitotic spindles to ensure spindle stability

Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. We show that mars, which encodes a Drosophila melanogaster member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with minispindles and Drosophila transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. We propose that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization.

Xnf7 Contributes to Spindle Integrity through Its Microtubule-Bundling Activity

Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly understood. One interesting class of MAPs is known to localize to the nucleus during interphase yet fulfill important spindle functions during mitosis. We have identified Xenopus nuclear factor 7 (Xnf7), a developmental regulator of dorsal-ventral patterning, as a microtubule-binding protein that also associates with the nuclear import receptor importin alpha/beta. Xnf7 localized to interphase nuclei and metaphase spindles both in Xenopus egg extracts and cultured cells. Xnf7-depleted spindles were hypersensitive to microtubule-depolymerizing agents. Functional characterization of Xnf7 revealed that it binds directly to microtubules, exhibits RING-finger-dependent E3-ubiquitin-ligase activity, and has C-terminal-dependent microtubule-bundling activity. The minimal microtubule-bundling domain of Xnf7 was sufficient to rescue the spindle-hypersensitivity phenotype. Thus, we have identified Xnf7 as a nuclear MAP whose microtubule-bundling activity, but not E3-ligase activity, contributes to microtubule organization and spindle integrity. Characterization of the multiple activities of Xnf7 may have implications for understanding human diseases caused by mutations in related proteins.

Regulation of microtubule-associated proteins, protein kinases and protein phosphatases during differentiation of SY5Y cells

Regulation of expression and function of microtubule-associated proteins (MAPs) is critical for neurons to maintain normal cytoskeletal architecture and functions. We have shown previously that in differentiated human neuroblastoma SY5Y cells, the expression of tau, a major neuronal MAP, is dramatically increased, and tau phosphorylation is differentially regulated. In the present study, we investigated the expression, the subcellular distribution and the microtubule-binding activities of several MAPs in SY5Y cells upon differentiation. We also studied the activities of protein kinases and phosphatases that are involved in regulation of tau phosphorylation during cell differentiation. We found that the expression of MAP1b in addition to tau was upregulated upon differentiation. Tau, MAP1a, MAP1b and MAP2 had distinct immunocytochemical staining patterns in differentiated SY5Y cells, suggesting differential biological functions. The microtubule-binding activity of tau increased after cell differentiation, whereas the activities of MAP1a and MAP2 decreased. Upon differentiation, the phosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 was increased, but that at Ser262/Ser356 was decreased. These changes in tau phosphorylation were accompanied by an upregulation of activities of several protein kinases (cdk5, MAPK, PKC and CK-1) as well as protein phosphatases PP-1 and PP-2A. These results suggest that the expression, post-translational modifications and biological activities of various MAPs are differentially regulated to meet the biological needs during cell differentiation.

Putative microtubule-associated proteins from the Arabidopsis genome.

Plant microtubule-associated proteins (MAPs) are important in modulating the function of the microtubule cytoskeleton. Various plant MAPs have already been described. However, because of the complexity of the plant microtubule cytoskeleton and its responses to developmental and environmental stimuli, there are undoubtedly many more MAPs to be discovered. We have used a literature search and the BLAST protein comparison program to identify which model MAPs from other taxa have close homologues in Arabidopsis thaliana. The search revealed Arabidopsis homologues of 14 model MAPs, with E values (numbers of proteins that will match the model protein merely by chance) of <1 x 10(-10) and homologous domains spanning 98-599 amino acid residues, representing 57.1-97.0% of the model MAP sequence, as well as 22.5-72.8% amino acid identities and 76.3-96.2% conservation of secondary structure in the homologous domain. All of the Arabidopsis homologues have either a full cDNA clone or an expressed sequence tag in the GenBank database and therefore are expressed. The proteins are likely to regulate a variety of functions, including tubulin folding, microtubule nucleation and polymerisation dynamics, microtubule-dependent cell cycle control, organisation of microtubule arrays, interaction of microtubules with plasma-membrane-associated protein complexes, and interactions with various other proteins. The exact functions of these putative MAPs in the plant cell remain to be elucidated empirically. The identification of these putative MAPs opens new avenues for the investigation of the complexities of the plant microtubule cytoskeleton.

E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics.

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

Analysis of microtubule-associated protein 1a gene in hereditary spastic paraplegia.

Development and maintenance of neuronal shape depend on regulated assembly and disassembly of microtubules. Microtubule-associated proteins (MAPs) bind to microtubules, regulate microtubule assembly and disassembly, and play critical roles in neuronal development and the balance between axonal plasticity and stability. In general, MAPs that are present postnatally (e.g., MAP1a) are considered to enhance microtubule (and axonal) stability.1 Disturbance of MAP structure or function could contribute to degenerative neurologic disorders such as hereditary spastic paraplegia (HSP). HSP is characterized by axonal degeneration that is maximal in the terminal portions of the longest descending and ascending axons in the CNS: crossed and uncrossed corticospinal tracts to the legs, fasciculus gracilis fibers, and to a lesser extent spinocerebellar fibers (see Fink et al.2 for review). Distal degeneration of CNS axons suggests that the primary disturbance of HSP may involve abnormal axonal transport, neurotrophic factors, their receptors, or impaired maintenance of axonal cytoskeleton. Loci for autosomal dominant HSP are present on chromosomes 2p, 14q, and proximal 15q (see Fink et al.2 for references). MAP1a has been localized to proximal chromosome 15q by somatic cell hybrid mapping, and was sublocalized using a chromosome 15 deletion hybrid panel.3 We …

Microtubule-associated proteins in developing oligodendrocytes: transient expression of a MAP2c isoform in oligodendrocyte precursors.

The morphological differentiation of oligodendrocytes is characterized by the formation of multiple, microtubule-rich processes which endow these cells with the ability to myelinate many axons simultaneously. Since microtubule-associated proteins (MAPs) strongly influence the structure and function of microtubules, we have investigated their expression in cultured differentiating oligodendrocytes in order to gain insights into MAP function during process formation and stabilization. MAP1B has been compared with two other structural MAPs: MAP4, which is an ubiquitously expressed protein, and MAP2, which hitherto was thought to be confined to neurons and reactive astrocytes. Immunofluorescence microscopy showed that the colocalization of MAP4 with microtubules in oligodendrocyte processes is not as extensive as found previously for MAP1B (Vouyiouklis and Brophy: J Neurosci Res 35:257-267, 1993). Nevertheless, like MAP1B, the expression of MAP4 increases during oligodendrocyte differentiation. In contrast, the expression of MAP2 is transiently elevated in preoligodendrocytes but declines precipitously at the onset of terminal differentiation. Cells of the oligodendrocyte lineage exclusively express a novel isoform of MAP2c which is primarily localized in the cell bodies of preoligodendrocytes. This suggests that MAP2c assists in the initiation of process extension rather than in the stabilization of microtubules in the cytoplasm-filled membranous extensions of mature cells. MAP-tau was not expressed at any developmental stage by oligodendrocytes. The distinct subcellular localizations and patterns of developmental expression of MAP1B, MAP4, and MAP2c suggest that these MAPs have different roles in the regulation of the microtubule network during the differentiation of myelin-forming oligodendrocytes.

Postnatal changes in serine/threonine protein phosphatases and their association with the microtubules

The activities and protein levels of three serine/threonine protein phosphatases were determined in homogenates and microtubule preparations from rat brain at various ages from postnatal day 1 (P1) through adulthood. The activities and levels of the calcium/calmodulin-dependent protein phosphatase, phosphatase 2B increased significantly from P1 to P21 in brain homogenates and remained elevated in the adult. The association of phosphatase 213 with microtubules was also found to be increased in the adult compared to the neonate (P3). In contrast, protein phosphatase 2A in brain homogenates decreased significantly from P1 to adult. However, the association of phosphatase 2A with the microtubules was found to increase with age. Finally, the activity and levels of phosphatase 1 in brain homogenates did not change with postnatal age, although the association of phosphatase 1 with microtubules was significantly decreased in the adult brain compared to P3. These studies clearly indicate that the activity, levels and association of these serine/threonine phosphatases with microtubules are independently regulated during postnatal development and suggest unique roles of phosphatase 1, phosphatase 2A and phosphatase 213 in modulating the phosphorylation state and function of microtubule-associated proteins at different postnatal ages.

Microtubule organization and dynamics dependent on microtubule-associated proteins

High-resolution microscopic analysis has precisely revealed the control of microtubule dynamics by individual microtubule-associated proteins (MAPs) in vitro. Furthermore, transfection of MAP cDNA into fibroblasts and subsequent analysis using microinjection of caged fluorescein-labeled tubulin and photoactivation have enabled the function of MAPs in microtubule dynamics to be studied in detail in vivo. Systematic, quantitative studies using transfection of various kinds of MAP cDNA deletion mutants have demonstrated the complex mechanism for microtubule bundling in vivo, and have shown the involvement in microtubule bundling of both microtubule binding and projection regions of MAPs. A similar approach, combined with detailed structural analysis, has indicated clearly that differences in the amino-terminal projection region of MAPs can determine differential organization of MT bundles, and thus influence the characteristic organization of microtubule domains in dendrites and axons.

Non-motor microtubule-associated proteins

This past year, the structure and function of microtubule-associated proteins (MAPs) have been investigated in studies probing their phosphorylation, patterns of expression, and the function of the microtubule-binding domain. Cellular studies have also contributed new insights into the roles of these proteins in process outgrowth.