Plant Microtubule-associated Proteins Research Articles

The role of intrinsic disorder in binding of plant microtubule-associated proteins to the cytoskeleton.

Microtubules (MTs) represent one of the main components of the eukaryotic cytoskeleton and support numerous critical cellular functions. MTs are in principle tube-like structures that can grow and shrink in a highly dynamic manner; a process largely controlled by microtubule-associated proteins (MAPs). Plant MAPs are a phylogenetically diverse group of proteins that nonetheless share many common biophysical characteristics and often contain large stretches of intrinsic protein disorder. These intrinsically disordered regions are determinants of many MAP-MT interactions, in which structural flexibility enables low-affinity protein-protein interactions that enable a fine-tuned regulation of MT cytoskeleton dynamics. Notably, intrinsic disorder is one of the major obstacles in functional and structural studies of MAPs and represents the principal present-day challenge to decipher how MAPs interact with MTs. Here, we review plant MAPs from an intrinsic protein disorder perspective, by providing a complete and up-to-date summary of all currently known members, and address the current and future challenges in functional and structural characterization of MAPs.

Phosphorylation of Plant Microtubule-Associated Proteins During Cell Division.

Progression of mitosis and cytokinesis depends on the reorganization of cytoskeleton, with microtubules driving the segregation of chromosomes and their partitioning to two daughter cells. In dividing plant cells, microtubules undergo global reorganization throughout mitosis and cytokinesis, and with the aid of various microtubule-associated proteins (MAPs), they form unique systems such as the preprophase band (PPB), the acentrosomal mitotic spindle, and the phragmoplast. Such proteins include nucleators of de novo microtubule formation, plus end binding proteins involved in the regulation of microtubule dynamics, crosslinking proteins underlying microtubule bundle formation and members of the kinesin superfamily with microtubule-dependent motor activities. The coordinated function of such proteins not only drives the continuous remodeling of microtubules during mitosis and cytokinesis but also assists the positioning of the PPB, the mitotic spindle, and the phragmoplast, affecting tissue patterning by controlling cell division plane (CDP) orientation. The affinity and the function of such proteins is variably regulated by reversible phosphorylation of serine and threonine residues within the microtubule binding domain through a number of protein kinases and phosphatases which are differentially involved throughout cell division. The purpose of the present review is to provide an overview of the function of protein kinases and protein phosphatases involved in cell division regulation and to identify cytoskeletal substrates relevant to the progression of mitosis and cytokinesis and the regulation of CDP orientation.

Multifunctional Microtubule-Associated Proteins in Plants.

Microtubules (MTs) are involved in key processes in plant cells, including cell division, growth and development. MT-interacting proteins modulate MT dynamics and organization, mediating functional and structural interaction of MTs with other cell structures. In addition to conventional microtubule-associated proteins (MAPs) in plants, there are many other MT-binding proteins whose primary function is not related to the regulation of MTs. This review focuses on enzymes, chaperones, or proteins primarily involved in other processes that also bind to MTs. The MT-binding activity of these multifunctional MAPs is often performed only under specific environmental or physiological conditions, or they bind to MTs only as components of a larger MT-binding protein complex. The involvement of multifunctional MAPs in these interactions may underlie physiological and morphogenetic events, e.g., under specific environmental or developmental conditions. Uncovering MT-binding activity of these proteins, although challenging, may contribute to understanding of the novel functions of the MT cytoskeleton in plant biological processes.

Identification of Plant Homologues of Dual Specificity Yak1-Related Kinases

Currently, Dual Specificity YAK1-Related Kinases (MNB/DYRK) were found in slime molds, protista, fungi, and animals, but the existence of plant homologues is still unclear. In the present study, we have identified 14 potential plant homologues with the previously unknown functions, based on the strong sequence similarity. The results of bioinformatics analysis revealed their correspondence to DYRK1A, DYRK1B, DYRK3, and DYRK4. For two plant homologues of animal DYRK1A from Physcomitrella patens and Arabidopsis thaliana spatial structures of catalytic domains were predicted, as well as their complexes with ADP and selective inhibitor d15. Comparative analysis of 3D-structures of the human DYRK1A and plant homologues, their complexes with the specific inhibitors, and results of molecular dynamics confirm their structural and functional similarity with high probability. Preliminary data indicate the presence of potential MNB/DYRK specific phosphorylation sites in such proteins associated with plant cytoskeleton as plant microtubule-associated proteins WVD2 and WDL1, and FH5 and SCAR2 involved in the organization and polarity of the actin cytoskeleton and some kinesin-like microtubule motor proteins.

Lessons from in vitro reconstitution analyses of plant microtubule-associated proteins.

Plant microtubules, composed of tubulin GTPase, are irreplaceable cellular components that regulate the directions of cell expansion and cell division, chromosome segregation and cell plate formation. To accomplish these functions, plant cells organize microtubule structures by regulating microtubule dynamics. Each microtubule localizes to the proper position with repeated growth and shortening. Although it is possible to reconstitute microtubule dynamics with pure tubulin solution in vitro, many microtubule-associated proteins (MAPs) govern microtubule dynamics in cells. In plants, major MAPs are identified as microtubule stabilizers (CLASP and MAP65 etc.), microtubule destabilizers (kinesin-13, katanin, MAP18 and MDP25), and microtubule dynamics promoters (EB1, MAP215, MOR1, MAP200, SPR2). Mutant analyses with forward and reverse genetics have shown the importance of microtubules and individual MAPs in plants. However, it is difficult to understand how each MAP regulates microtubule dynamics, such as growth and shortening, through mutant analyses. In vitro reconstitution analyses with individual purified MAPs and tubulin are powerful tools to reveal how each MAP regulates microtubule dynamics at the molecular level. In this review, I summarize the results of in vitro reconstitution analyses and introduce current models of how each MAP regulates microtubule dynamic instability.

Dynamic interaction of NtMAP65-1a with microtubules in vivo

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

Identification of a novel family of 70 kDa microtubule‐associated proteins in Arabidopsis cells

Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected 'unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules.

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.

Proteomic analysis of plant microtubule-associated proteins.

Proteomic analysis of plant microtubule-associated proteins.

The plant cytoskeleton: recent advances in the study of the plant microtubule-associated proteins MAP-65, MAP-190 and the Xenopus MAP215-like protein, MOR1.

The microtubule cytoskeleton is a dynamic filamentous structure involved in many key processes in plant cell morphogenesis including nuclear and cell division, deposition of cell wall, cell expansion, organelle movement and secretion. The principal microtubule protein is tubulin, which associates to form the wall of the tubule. In addition, various associated proteins bind microtubules either to anchor, cross-link or regulate the microtubule network within cells. Biochemical, molecular biological and genetic approaches are being successfully used to identify these microtubule-associated proteins (MAPs) in plants, and we describe recent progress on three of these proteins.

Microtubule-associated proteins in plants — why we need a map

Plants have four main microtubule assemblies. Three are involved in arranging when and where the cell wall is laid down and have no direct homologues in animals. Microtubule-associated proteins are important components of these assemblies, and we are now starting to uncover what these proteins are and how they might work.

A new class of microtubule-associated proteins in plants.

In plants there are three microtubule arrays involved in cellular morphogenesis that have no equivalent in animal cells. In animals, microtubules are decorated by another class of proteins - the structural MAPS - which serve to stabilize microtubules and assist in their organization. The best-studied members of this class in plants are the MAP-65 proteins that can be purified together with plant microtubules after several cycles of polymerization and depolymerization. Here we identify three similar MAP-65 complementary DNAs representing a small gene family named NtMAP65-1, which encode a new set of proteins, collectively called NtMAP65-1. We show that NtMAP65-1 protein localizes to areas of overlapping microtubules, indicating that it may function in the behaviour of antiparallel microtubules in the mitotic spindle and the cytokinetic phragmoplast.

The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules.

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation. MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile. In the electron microscope, MAP65 appears as filamentous cross-bridges, maintaining an intermicrotubule spacing of 25-30 nm. Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains in vitro the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that this MAP is important for the organization of the cortical array in vivo.

Higher plant microtubule-associated proteins: In vitro functional assays

Microtubule-associated proteins (MAPs) can account for the assembly and stabilization of microtubules at low tubulin concentration, for their ability to interact with other microtubules and/or cytoskeletal polymers or organelles and also for regulating microtubule anchoring and bundling properties. The data concerning higher plant MAPs remain limited so far to a few examples. Motor MAPs such as dynein or kinesin remain poorly documented in plants and are not to be discussed here. In this manuscript, the attention is focused on structural MAPs which co-assemble with tubulin during microtubule assembly. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in a cytosolic extract of maize cultured cells and could be used as a native matrix for the isolation of putative higher plant MAPs. Seven polypeptides with molecular masses ranging between 60–125 kDa were found in this MAP-enriched fraction. These putative plant MAPs were shown to co-assemble with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of non-efficient self-assembly and induced bundling of both plant and neural microtubules. One of these polypeptides (83 kDa) was found to be immunologically related to neural suggesting the presence of common epitopes between neural and plant MAPs. Such epitopes may be present at the microtubule-binding domains, as the higher plant MAPs co-assemble with brain tubulin. Plant microtubules exhibit an important in situ bundling activity, as in cortical or pre-prophase band arrays, or during the drastic reorganization of the cytoskeleton during mitosis induction. It is argued that different plant MAPs may regulate stability and function of these distinct microtubule populations. As higher plant cells lack defined centrosomes, specific MAPs could be involved in the control of the critical concentration of intracellular plant tubulin.

Purification of a mung bean protein binding to microtubules through two defined sites in the caboxyl-terminal domain of ß-tubulin

A protein purified from mung beans binds to monomeric tubulin and microtubues through two sites near the C-terminus of ß-tubulin. The 34-kDa protein cosediments with taxol-stabilized brain microtubules and is purified by ion-exchange chromatography and two steps of affinity chromatography using tubulin and S-tubulin (tubulin cleaved with subtilisin at Gln-433). The 34-kDa protein binds to tubulin but not to S-tubulin whether monomeric or polymeric. A rabbit antiserum was raised against a synthetic peptide of 11 amino acids corresponding to β(422–432), the highly conserved region which lies just outside the subtilisin-cleaved C-terminal peptide. Partially-purified antibodies were used to generate murine anti-idiotypic monoclonal antibodies. One monoclonal antibody, which does not bind to sheep brain or mung bean tubulin, reacts in ELISA's with microtubule-associated proteins from the brain and with the 34-kDa protein from mung beans. On immunoblots, the anti-idiotypic antibody recognizes several bands two of which have similar M r values to previously reported plant microtubule-associated proteins. The results indicate that the mung bean protein, like neuronal MAP-2 and tau-protein, binds to both the variable, (highly acidic region cleaved by subtilisin) and to the highly conserved β(422–432) sequence. We conclude that the 34-kDa protein is likely to be a plant MAP that binds to a site known to be important in the regulation of tubulin polymerization. Anti-idiotypic antibodies and tubulin/S-tubulin affinity chromatography may have wide applicability in the identification of plant MAPs.

Higher plant microtubule-associated proteins (MAPs): A survey

Microtubule-associated proteins (MAPs) are one of the factors which regulate the different properties of microtubules during cell cycle and differentiation. They have been characterized as proteins which promote tubulin assembly in a concentration-dependent manner and bind to the outer surface of the polymers in vitro. Most of our knowledge comes from studies of neural microtubule-associated proteins and recent results highlight their implication in neuronal morphogenesis. In contrast, until recently, few data are available about the proteins that associate with plant tubulins. This is due principally to the fact that plant microtubule-associated proteins cannot be purified by the standard procedures used for neural microtubule-associated proteins. First, we will describe methods which have been used to isolate these proteins in plant cells. We will then discuss the biochemical and immunological properties of the plant microtubule-associated proteins which have been isolated. From these results, putative functions can be proposed for these proteins n the particular plant cytoskeleton activities.