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

Summary— 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.     

A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs)

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.     

Binding of mammalian brain microtubule-associated proteins (MAPs) to insect ovarian microtubules

In this study we have applied microtubule-associated proteins (MAPs) from mammalian brain to both native and reassembled insect ovarian microtubules. Such microtubules, which are normally smooth walled, become decorated with projections similar to those observed when mammalian brain MAPs are added back to assembling or assembled mammalian brain microtubules. The mammalian MAPs were also detected as components of insect microtubules when analyzed by polyacrylamide gel electrophoresis. Our observations suggest that mammalian brain MAPs have common binding sites on microtubules from two widely different sources and indicate the degree of evolutionary conservation of such sites.     

Preparation and characterization of native, fluorescently labelled brain tubulin and microtubule-associated proteins (MAPs)

Tubulin and Microtubule-Associated Proteins (MAPs) isolated from chick brain by cycles of assembly and disassembly in vitro have been stoichiometrically labelled with the fluorogenic compound fluorescamine. Under the conditions employed, tubulin can be labelled with up to 2 moles of fluorescamine/mole of dimer while the MAPs accept up to 8 moles/mole of protein, assuming an average molecular weight of 300 000 D. After the labelling procedure, both the tubulin and MAPs retain their native conformations as judged by several criteria: (a) the labelled proteins remain competent to participate in further rounds of the assembly-disassembly procedure in vitro, and the kinetics of this assembly are identical to those seen with an unlabelled control sample; (b) incubation of the fluorescent microtubule proteins with the antimitotic agent vinblastine sulfate results in the formation of birefringent vinblastine-tubulin paracrystals, indicating the binding site for this ligand is unaltered; (c) the mobilities of the fluorescent tubulin and MAPs on SDS-polyacrylamide gels are unaltered when compared to the mobilities of the respective unlabelled control proteins. The results are discussed in relation to the use of these fluorescent cytoskeletal proteins as easily detectable biochemical and histochemical probes.     

Microtubule dynamics and tubulin interacting proteins

Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op 18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.     

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.     

Amphitropic proteins: regulation by reversible membrane interactions (review).

What do Src kinase, Ras-guanine nucleotide exchange factor, cytidylyltransferase, protein kinase C, phospholipase C, vinculin, and DnaA protein have in common? These proteins are amphitropic, that is, they bind weakly (reversibly) to membrane lipids, and this process regulates their function. Proteins functioning in transduction of signals generated in cell membranes are commonly regulated by amphitropism. In this review, the strategies utilized by amphitropic proteins to bind to membranes and to regulate their membrane affinity are described. The recently solved structures of binding pockets for specific lipids are described, as well as the amphipathic alpha-helix motif. Regulatory switches that control membrane affinity include modulation of the membrane lipid composition, and modification of the protein itself by ligand binding, phosphorylation, or acylation. How does membrane binding modulate the protein's function? Two mechanisms are discussed: (1) localization with the substrate, activator, or downstream target, and (2) activation of the protein by a conformational switch. This paper also addresses the issue of specificity in the cell membrane targetted for binding.     

Microtubule-associated proteins (MAPs) and the organization of actin filaments in vitro

When purified muscle actin was mixed with microtubule-associated proteins (MAPs) prepared from brain microtubules assembled in vitro, actin filaments were organized into discrete bundles, 26 nm in diameter. MAP-2 was the principal protein necessary for the formation of the bundles. Analysis of MAP-actin bundle formation by sedimentation and electrophoresis revealed the bundles to be composed of approximately 20% MAP-2 and 80% actin by weight. Transverse striations were observed to occur at 28-nm intervals along negatively stained MAP- actin bundles, and short projections, approximately 12 nm long and spaced at 28-nm intervals, were resolved by high-resolution metal shadowing. The formation of MAP-actin bundles was inhibited by millimolar concentrations of ATP, AMP-PCP (beta, gamma-methylene- adenosine triphosphate), and pyrophosphate but not by AMP, ADP, or GTP. The addition of ATP to a solution containing MAP-actin bundles resulted in the dissociation of the bundles into individual actin filaments; discrete particles, presumably MAP-2, were periodically attached along the splayed filaments. These results demonstrate that MAPs can bind to actin filaments and can induce the reversible formation of actin filament bundles in vitro.     

Binding of microtubule-associated proteins (MAPs) to rat brain mitochondria: a comparative study of the binding of MAP2, its microtubule-binding and projection domains, and tau proteins

Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to be able to bind to purified rat brain mitochondria. The apparent dissociation constants of the binding of thermostable 32P-labeled MAP2 and tau are 0.9 +/- 0.04 x 10(-7) and 3.8 +/- 0.7 x 10(-7) M, respectively. 32P-labeled MAP2 and tau bound to the mitochondria can be displaced by phosphorylated, nonradioactive MAP2. The binding parameters of MAP2 prepared without heat treatment and those of the thermostable MAP2 were of the same order of magnitude. Microtubule-binding and projection domains of MAP2 were obtained by chymotryptic digestion of rat brain microtubules (Vallee, Proc. Natl. Acad. Sci. USA, 77:3206-3210, 1980). Displacement studies with these two domains show that MAP2 bound to mitochondria can be displaced by the microtubule-binding domain, whereas the projection domain does not displace MAP2. The two domains of MAP2 bind to the mitochondria with similar affinity constants; however, the Bmax for the projection domain was 10 times and 35 times lower than the Bmax of the binding of the intact MAP2 and the microtubule-binding domain, respectively. Chymotryptic digestion of MAP2 bound to the mitochondria yielded peptide fragments with molecular masses similar to those obtained by the digestion of MAP2 bound to the microtubules. The fragments corresponding to the projection domain were released into the extramitochondrial supernatant, whereas the fragments originating from the microtubule-binding domain remained bound to the mitochondria. These results suggest that MAP2 binds to mitochondria preferentially via its microtubule-binding domain.     

Effects of brain microtubule-associated proteins on microtubule dynamics and the nucleating activity of centrosomes

In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.     

Developmental regulation of two microtubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina

Previous studies with the mammalian brain have shown that the expression of a number of neuronal microtubule-associated proteins (MAPs) is developmentally regulated. For example, the low-molecular-weight form of MAP2 (MAP2c) is abundant in neonatal rat brains and is less abundant in adults. Similarly, MAP5 levels decrease during postnatal development. Using monoclonal antibodies, we have followed the time of first appearance, cellular distribution, and molecular form of MAP2 and MAP5 during the morphogenesis of the quail retina. MAP2 first appears in ganglion cell bodies and in the axons of the optic fibre layer (OFL) at embryonic day 4 (E4). Anti-MAP2 staining remains restricted to these sites until E10, when staining appears in the inner plexiform layer (IPL). At E14, one day before hatching, anti-MAP2 staining is found in three broad laminae in the IPL, as well as in photosensitive cells. MAP5 is present in ganglion cell axons from the onset of neurite elongation at E3 and is limited to the OFL until E10. The intensity of anti-MAP5 staining in the OFL and optic nerve decreases after E7, which corresponds with a decrease in the number of actively growing ganglion cell axons. By E14, anti-MAP5 stains five layers in the IPL that correspond with layers of amacrine cell process arborizations. Western blots of E10 brain microtubule proteins show that MAP2 is represented by both a 260 x 10(3) Mr protein and a 60-65 x 10(3) Mr protein; the latter is much more abundant. Anti-MAP5 recognizes a 320 x 10(3) Mr brain microtubule protein in both the quail and the rat. We conclude that the cellular distribution, developmental regulation and molecular forms of MAP2 and MAP5 are similar in the rat and quail, suggesting that these molecules have conserved and hence fundamental roles in the growth and differentiation of neuronal processes.     

Microtubule organization by cross-linking and bundling proteins.

To understand microtubule function the factors regulating their spatial organization and their interaction with cellular organelles, including other microtubules, must be elucidated. Many proteins are implicated in these organizational events and the known consequences of their actions within the cell are increasing. For example, the function of microtubule bundles at the surfaces of polarized cells has recently received attention, as has the action in cortical rotation of a transient arrangement of microtubules found beneath the vegetal surface of fertilized frog eggs. The in vivo association of microtubules during early Xenopus oogenesis has added interest as microtubules bundled in cell-free extracts are protected against the action of a severing protein found in this animal. A 52 kDa F-actin bundling protein purified from Physarum polycephalum organizes microtubules and causes the cobundling of microtubules and microfilaments. These observations, in concert with others that are presented, emphasize the diversity within the family of microtubule cross-linking proteins. The challenge is to determine which proteins are relevant from a physiological perspective, to ascertain their molecular mechanisms of action and to describe how they affect cytoplasmic organization and cell function. To realize this objective, the proteins which cross-link and bundle microtubules must be investigated by techniques which reveal different but related aspects of their properties. Cloning and sequencing of genes for cross-linking proteins, their subcellular localization especially as microtubule-related changes in cell morphology are occurring and the application of genetic studies are necessary. Study of the neural MAP provides the best example of just how powerful current experimental approaches are and at the same time shows their limits. The neural MAP have long been noted for their enhancement of tubulin assembly and microtubule stability. Their spatial distribution has been studied during the morphogenesis of neural cells. Sequencing of cloned genes has revealed the functional domains of neural MAP including carboxy-terminal microtubule-binding sites. Similarities to microtubule binding proteins from other cell types stimulate interest in the neural MAP and further suggest their importance in microtubule organization. For example, MAP4 enjoys a wide cellular distribution and has microtubule-binding sequences very similar to those in the neural MAP. Moreover, the nontubulin proteins of marginal bands are immunologically related to neural MAP, indicating shared structural/functional domains. Even with these findings the mechanism by which neural MAP cross-link microtubules remains uncertain. Indeed, some researchers express doubt that microtubule cross-linking is actually a function of neural MAP in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tether and trap: regulation of membrane-raft dynamics by actin-binding proteins

The existence of plasma-membrane-raft microdomains has been widely debated during the past few years. However, it is clear that during lymphocyte stimulation a lipid-based reorganization occurs at the plasma membrane, with markers of the membrane rafts being selectively recruited to key active regions of the cell. Recent reports have demonstrated that membrane-raft dynamics are controlled by proteins that are linked to the actin cytoskeleton and have suggested a new model for the plasma membrane based on protein-lipid interactions. This new and dynamic view of the plasma membrane may improve our understanding of the complex process leading to cell polarization during lymphocyte migration and activation.     

Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18

The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly.     

Microtubule assembly dynamics at the nanoscale

BACKGROUND: The labile nature of microtubules is critical for establishing cellular morphology and motility, yet the molecular basis of assembly remains unclear. Here we use optical tweezers to track microtubule polymerization against microfabricated barriers, permitting unprecedented spatial resolution. RESULTS: We find that microtubules exhibit extensive nanometer-scale variability in growth rate and often undergo shortening excursions, in some cases exceeding five tubulin layers, during periods of overall net growth. This result indicates that the guanosine triphosphate (GTP) cap does not exist as a single layer as previously proposed. We also find that length increments (over 100 ms time intervals, n = 16,762) are small, 0.81 +/- 6.60 nm (mean +/- standard deviation), and very rarely exceed 16 nm (about two dimer lengths), indicating that assembly occurs almost exclusively via single-subunit addition rather than via oligomers as was recently suggested. Finally, the assembly rate depends only weakly on load, with the average growth rate decreasing only 2-fold as the force increases 7-fold from 0.4 pN to 2.8 pN. CONCLUSIONS: The data are consistent with a mechanochemical model in which a spatially extended GTP cap allows substantial shortening on the nanoscale, while still preventing complete catastrophe in most cases.     

Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c).

Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.     

Putative microtubule-associated proteins from the Arabidopsis genome

Summary. 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 <1×10^–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.Present address: School of Biological Sciences, University of Manchester, Manchester, United Kingdom.Correspondence and reprints: School of Biological Sciences A12, University of Sydney, NSW 2006, Australia.Received October 21, 2002; accepted December 30, 2002; published online September 23, 2003     

Phosphorylation of tubulin and microtubule-associated proteins by the purified insulin receptor kinase

The purified insulin receptor kinase catalyzed the phosphorylation of native tubulin and microtubule-associated proteins (MAPs; MAP2, tau) on tyrosine residues. Insulin (10(-7) M) stimulated the reaction by 4-10-fold by increasing Vmax with little change in Km. alpha-Tubulin was preferred as a substrate for the kinase compared to beta-tubulin. MAP2 was found to be the best substrate among the cytoskeletal proteins tested; in the presence of insulin, the Vmax for MAP2 was 6.3 nmol/min/mg, its Km was 5.1 microM, and 1.7 mol of phosphate were incorporated per mol of MAP2. Under the same conditions used for this phosphorylation of tubulin and MAPs, actin and tropomyosin were very poorly phosphorylated. These data, coupled with previous evidence for potential functional relationships between insulin action and microtubules, raise the possibility that microtubule proteins may be cellular targets for the insulin receptor kinase.