John Gardiner

A 90-kD Phospholipase D from Tobacco Binds to Microtubules and the Plasma Membrane

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipid-metabolizing enzyme and a Ca2+-dependent lipid binding domain and is identical to Arabidopsis PLDδ. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

CLASP interacts with sorting nexin 1 to link microtubules and auxin transport via PIN2 recycling in Arabidopsis thaliana.

Polarized movement of auxin generates concentration gradients within plant tissues to control cell division patterns and growth direction by modulating microtubule organization. In this study, we identify a reverse mechanism, wherein microtubules influence polar auxin transport. We show that the microtubule-associated protein CLASP interacts with the retromer component sorting nexin 1 (SNX1) to mediate an association between endosomes and microtubules. clasp-1 null mutants display aberrant SNX1 endosomes, as do wild-type plants treated with microtubule-depolymerizing drugs. Consistent with SNX1s role in trafficking of the auxin efflux carrier PIN-FORMED2 (PIN2), clasp-1 mutant plants have enhanced PIN2 degradation, and PIN2 movement to lytic vacuoles is rapidly induced by depolymerization of microtubules. clasp-1 mutants display aberrant auxin distribution and exhibit numerous auxin-related phenotypes. In addition to mechanistically linking auxin transport and microtubules, our data identify a ubiquitous endosome-microtubule association in plants.

Potential role of tubulin acetylation and microtubule-based protein trafficking in familial dysautonomia.

Familial dysautonomia (FD), a disease of the autonomic and sensory nervous systems, involves mutations in the protein IκB kinase complex‐associated protein, which is a component of the human Elongator acetylase complex. We suggest a hypothesis in which defects in tubulin acetylation and impairment of microtubule‐based protein trafficking may be an underlying cause of FD. In addition, an Arabidopsis homolog of the Elongator subunit ELP3 has been found to bind to the αβ‐tubulin heterodimer, suggesting that α‐tubulin may be a cytoplasmic target of Elongator acetylase activity. Studies of synergistic double mutants in yeast indicate a novel role for Elongator in cytoskeletal dynamics, although this is probably because of an effect on actin rather than microtubules. Finally, we suggest that tubulin deacetylase inhibitors may prove useful in the treatment of FD.

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.

Neurotrophic Support and Oxidative Stress: Converging Effects in the Normal and Diseased Nervous System

Oxidative stress and loss of neurotrophic support play major roles in the development of various diseases of the central and peripheral nervous systems. In disorders of the central nervous system such as Alzheimers, Parkinsons, and Huntingtons diseases, oxidative stress appears inextricably linked to the loss of neurotrophic support. A similar situation is seen in the peripheral nervous system in diseases of olfaction, hearing, and vision. Neurotrophic factors act to up-regulate antioxidant enzymes and promote the expression of antioxidant proteins. On the other hand, oxidative stress can cause down-regulation of neurotrophic factors. We propose that normal functioning of the nervous systems involves a positive feedback loop between antioxidant processes and neurotrophic support. Breakdown of this feedback loop in disease states leads to increased oxidative stress and reduced neurotrophic support.

Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachment

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.

The Microtubule Cytoskeleton Acts as a Key Downstream Effector of Neurotransmitter Signaling

Microtubules are well known to play a key role in the trafficking of neurotransmitters to the synapse. However, less attention has been paid to their role as downstream effectors of neurotransmitter signaling in the target neuron. Here, we show that neurotransmitter‐based signaling to the microtubule cytoskeleton regulates downstream microtubule function through several mechanisms. These include tubulin posttranslational modification, binding of microtubule‐associated proteins, release of microtubule‐interacting second messenger molecules, and regulation of tubulin expression levels. We review the evidence for neurotransmitter regulation of the microtubule cytoskeleton, focusing on the neurotransmitters serotonin, melatonin, dopamine, glutamate, glycine, and acetylcholine. Some evidence suggests that microtubules may even play a more direct role in propagating action potentials through conductance of electric current. In turn, there is evidence for the regulation of neurotransmission by the microtubule cytoskeleton. Synapse, 2010.

The evolution and diversification of plant microtubule-associated proteins

Plant evolution is marked by major advances in structural characteristics that facilitated the highly successful colonization of dry land. Underlying these advances is the evolution of genes encoding specialized proteins that form novel microtubular arrays of the cytoskeleton. This review investigates the evolution of plant families of microtubule-associated proteins (MAPs) through the recently sequenced genomes of Arabidopsis thaliana, Oryza sativa, Selaginella moellendorffii, Physcomitrella patens, Volvox carteri and Chlamydomonas reinhardtii. The families of MAPs examined are AIR9, CLASP, CRIPT, MAP18, MOR1, TON, EB1, AtMAP70, SPR2, SPR1, WVD2 and MAP65 families (abbreviations are defined in the footnote to Table 1). Conjectures are made regarding the evolution of MAPs in plants in relation to the evolution of multicellularity, oriented cell division and vasculature. Angiosperms in particular have high numbers of proteins that are involved in promotion of helical growth or its suppression, and novel plant microtubular structures may have acted as a catalyst for the development of novel plant MAPs. Comparisons of plant MAP gene families with those of animals show that animals may have more flexibility in the structure of their microtubule cytoskeletons than plants, but with both plants and animals possessing many MAP splice variants.

Disruption of Normal Cytoskeletal Dynamics May Play a Key Role in the Pathogenesis of Epilepsy

Epilepsy, a common disease affecting 1% to 2% of the population, is characterized by seizures, hyperexcitability at synapses, and aberrant extension of neurons following seizures. Much work has been done on the role of synaptic components in the pathogenesis of epilepsy, but relatively little attention has been given to the potential role of the cytoskeleton. The neuronal cytoskeleton consists of microtubules, actin filaments, intermediate filaments, and associated proteins. A number of mutations in both microtubule-associated proteins (MAPs) and actin-binding proteins, as well as altered expression levels of several cytoskeletal proteins, are known to be involved in epilepsy. These changes will affect the dynamics of the neuronal cytoskeleton and therefore are likely to contribute to the pathogenesis of epilepsy through mechanisms such as increased neurotrophic support to neurons and increased sprouting of mossy fibers. These changes may also contribute to hyperexcitability of neurons through an as yet unidentified mechanism.

Arabidopsis thaliana, a plant model organism for the neuronal microtubule cytoskeleton?

The microtubule cytoskeleton is an important component of both neuronal cells and plant cells. While there are large differences in the function of microtubules between the two groups of organisms, for example plants coordinate the ordered deposition of cellulose through the microtubule cytoskeleton, there are also some notable similarities. It is suggested that Arabidopsis thaliana, with its superior availability of knockout lines, may be a suitable model organism for some aspects of the neuronal microtubule cytoskeleton. Some cellular processes that involve the neuronal microtubule cytoskeleton including neurotransmitter signalling and neurotrophic support may have homologous processes in plant cells. A number of microtubule-associated proteins (MAPs) are conserved, including katanin, EB1, CLASP, spastin, gephyrin, CRIPT, Atlastin/RHD3, and ELP3. As a demonstration of the usefulness of a plant model system for neuronal biology, an analysis of plant tubulin-binding proteins was used to show that Charcot-Marie-Tooth disease type 2D and spinal muscular atrophy may be due to microtubule dysfunction and suggest that indeed the plant microtubule cytoskeleton may be particularly similar to that of motor neurons as both are heavily reliant upon motor proteins.