Neuronal Microtubule Organization Research Articles

Super-resolution imaging and quantitative analysis of microtubule arrays in model neurons show that epothilone D increases the density but decreases the length and straightness of microtubules in axon-like processes

Microtubules are essential for the development of neurons and the regulation of their structural plasticity. Microtubules also provide the structural basis for the long-distance transport of cargo. Various factors influence the organization and dynamics of neuronal microtubules, and disturbance of microtubule regulation is thought to play a central role in neurodegenerative diseases. However, imaging and quantitative assessment of the microtubule organization in the densely packed neuronal processes is challenging. The development of super-resolution techniques combined with the use of nanobodies offers new possibilities to visualize microtubules in neurites in high resolution. In combination with recently developed computational analysis tools, this allows automated quantification of neuronal microtubule organization with high precision. Here we have implemented three-dimensional DNA-PAINT (Point Accumulation in Nanoscale Topography), a single-molecule localization microscopy (SMLM) technique, which allows us to acquire 3D arrays of the microtubule lattice in axons of model neurons (neuronally differentiated PC12 cells) and dendrites of primary neurons. For the quantitative analysis of the microtubule organization, we used the open-source software package SMLM image filament extractor (SIFNE). We found that treatment with nanomolar concentrations of the microtubule-targeting drug epothilone D (EpoD) increased microtubule density in axon-like processes of model neurons and shifted the microtubule length distribution to shorter ones, with a mean microtubule length of 2.39 µm (without EpoD) and 1.98 µm (with EpoD). We also observed a significant decrease in microtubule straightness after EpoD treatment. The changes in microtubule density were consistent with live-cell imaging measurements of ensemble microtubule dynamics using a previously established Fluorescence Decay After Photoactivation (FDAP) assay. For comparison, we determined the organization of the microtubule array in dendrites of primary hippocampal neurons. We observed that dendritic microtubules have a very similar length distribution and straightness compared to microtubules in axon-like processes of a neuronal cell line. Our data show that super-resolution imaging of microtubules followed by algorithm-based image analysis represents a powerful tool to quantitatively assess changes in microtubule organization in neuronal processes, useful to determine the effect of microtubule-modulating conditions. We also provide evidence that the approach is robust and can be applied to neuronal cell lines or primary neurons, both after incorporation of labeled tubulin and by anti-tubulin antibody staining.

TIAM-1 differentially regulates dendritic and axonal microtubule organization in patterning neuronal development through its multiple domains.

Axon and dendrite development require the cooperation of actin and microtubule cytoskeletons. Microtubules form a well-organized network to direct polarized trafficking and support neuronal processes formation with distinct actin structures. However, it is largely unknown how cytoskeleton regulators differentially regulate microtubule organization in axon and dendrite development. Here, we characterize the role of actin regulators in axon and dendrite development and show that the RacGEF TIAM-1 regulates dendritic patterns through its N-terminal domains and suppresses axon growth through its C-terminal domains. TIAM-1 maintains plus-end-out microtubule orientation in posterior dendrites and prevents the accumulation of microtubules in the axon. In somatodendritic regions, TIAM-1 interacts with UNC-119 and stabilizes the organization between actin filaments and microtubules. UNC-119 is required for TIAM-1 to control axon growth, and its expression levels determine axon length. Taken together, TIAM-1 regulates neuronal microtubule organization and patterns axon and dendrite development respectively through its different domains.

The HAUS Complex Is a Key Regulator of Non-centrosomal Microtubule Organization during Neuronal Development

SummaryNeuron morphology and function are highly dependent on proper organization of the cytoskeleton. In neurons, the centrosome is inactivated early in development, and acentrosomal microtubules are generated by mechanisms that are poorly understood. Here, we show that neuronal migration, development, and polarization depend on the multi-subunit protein HAUS/augmin complex, previously described to be required for mitotic spindle assembly in dividing cells. The HAUS complex is essential for neuronal microtubule organization by ensuring uniform microtubule polarity in axons and regulation of microtubule density in dendrites. Using live-cell imaging and high-resolution microscopy, we found that distinct HAUS clusters are distributed throughout neurons and colocalize with γ-TuRC, suggesting local microtubule nucleation events. We propose that the HAUS complex locally regulates microtubule nucleation events to control proper neuronal development.

Tubulin isotype specificity in neuronal migration: Tuba8 can't fill in for Tuba1a.

Several tubulin isotypes, including Tuba1a, are associated with brain malformations. In this issue, Belvindrah et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607074) show that Tuba1a and Tuba8 differentially regulate microtubule organization in neurons, and they provide insights into the mechanisms by which Tuba1a mutations disrupt adult mouse brain morphology.

Microtubule Organization Determines Axonal Transport Dynamics

Axonal microtubule (MT) arrays are the major cytoskeleton substrate for cargo transport. How MTorganization, i.e., polymer length, number, and minus-end spacing, is regulated and how it impinges on axonal transport are unclear. We describe a method for analyzing neuronal MT organization usinglight microscopy. This method circumvents the needfor electron microscopy reconstructions and is compatible with live imaging of cargo transport and MT dynamics. Examination of a C.elegans motor neuron revealed how age, MT-associated proteins, and signaling pathways control MT length, minus-end spacing, and coverage. In turn, MT organization determines axonal transport progression: cargoes pause at polymer termini, suggesting that switching MT tracks is rate limiting for efficient transport. Cargo run length is set by MT length, and higher MT coverage correlates with shorter pauses. These results uncover the principles and mechanisms of neuronal MT organization and its regulation of axonal cargo transport.

Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy.

The formation and maintenance of highly polarized neurons critically depends on the proper organization of the microtubule (MT) cytoskeleton. In axons, MTs are uniformly oriented with their plus-end pointing outward whereas in mature dendrites MTs have mixed orientations. MT organization and dynamics can be regulated by MT-associated proteins (MAPs). Plus-end tracking proteins are specialized MAPs that decorate plus-ends of growing MTs and regulate neuronal polarity, neurite extension, and dendritic spine morphology. Conventional fluorescence microscopy enables observation of specific cellular components through molecule-specific labeling but provides limited resolution (∼250 nm). Therefore, electron microscopy has until now provided most of our knowledge about the precise MT organization in neurons. In the past decade, super-resolution fluorescence microscopy techniques have emerged that circumvent the diffraction limit of light and enable high-resolution reconstruction of the MT network combined with selective protein labeling. However, preserving MT ultrastructure, MAP binding, high labeling density, and antibody specificity after fixation protocols is still quite challenging. In this chapter, we provide an optimized protocol for two-color direct stochastic optical reconstruction microscopy imaging of neuronal MTs together with their growing plus-ends to probe MT architecture and polarity.

Microtubule minus-end binding protein CAMSAP2 controls axon specification and dendrite development.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

Microtubules are organized independently of the centrosome in Drosophilaneurons

BackgroundThe best-studied arrangement of microtubules is that organized by the centrosome, a cloud of microtubule nucleating and anchoring proteins is clustered around centrioles. However, noncentrosomal microtubule arrays are common in many differentiated cells, including neurons. Although microtubules are not anchored at neuronal centrosomes, it remains unclear whether the centrosome plays a role in organizing neuronal microtubules. We use Drosophila as a model system to determine whether centrosomal microtubule nucleation is important in mature neurons.ResultsIn developing and mature neurons, centrioles were not surrounded by the core nucleation protein γ-tubulin. This suggests that the centrioles do not organize functional centrosomes in Drosophila neurons in vivo. Consistent with this idea, centriole position was not correlated with a specific region of the cell body in neurons, and growing microtubules did not cluster around the centriole, even after axon severing when the number of growing plus ends is dramatically increased. To determine whether the centrosome was required for microtubule organization in mature neurons, we used two approaches. First, we used DSas-4 centriole duplication mutants. In these mutants, centrioles were present in many larval sensory neurons, but they were not fully functional. Despite reduced centriole function, microtubule orientation was normal in axons and dendrites. Second, we used laser ablation to eliminate the centriole, and again found that microtubule polarity in axons and dendrites was normal, even 3 days after treatment.ConclusionWe conclude that the centrosome is not a major site of microtubule nucleation in Drosophila neurons, and is not required for maintenance of neuronal microtubule organization in these cells.

UNC-33 (CRMP) and ankyrin organize microtubules and localize kinesin to polarize axon-dendrite sorting.

The polarized distribution of neuronal proteins to axons and dendrites relies upon microtubule-binding proteins such as CRMP, directed motors such as kinesin UNC-104/Kif1A, and diffusion barriers such as ankyrin. The causative relationships between these molecules are unknown. We show here that Caenorhabditis elegans CRMP (UNC-33) acts early in neuronal development, together with ankyrin (UNC-44), to organize microtubule asymmetry and axon-dendrite sorting. In unc-33 and unc-44 mutants, axonal proteins are present in dendrites and vice versa, suggesting bidirectional failures of axon-dendrite identity. UNC-33 protein is localized to axons by unc-44, and enriched in a region that resembles the axon initial segment. unc-33 and unc-44 establish the asymmetric dynamics of axonal and dendritic microtubules; in their absence, microtubules are disorganized, the axonal kinesin UNC-104 invades dendrites, and inappropriate UNC-104 activity randomizes axonal protein sorting. We suggest that UNC-44 and UNC-33 direct polarized sorting through their global effects on neuronal microtubule organization.

Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein)

Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.

Human, but not bovine, oxidized cerebral spinal fluid lipoproteins disrupt neuronal microtubules.

Cerebral spinal fluid (CSF) lipoproteins have become a focus of research since the observation that inheritance of particular alleles of the apolipoprotein E gene affects the risk of Alzheimer's disease (AD). There is evidence of increased lipid peroxidation in CSF lipoproteins from patients with AD, but the biological significance of this observation is not known. A characteristic of the AD brain is a disturbance of the neuronal microtubule organization. We have shown previously that 4-hydroxy-2(E)-nonenal, a major product of lipid peroxidation, causes disruption of neuronal microtubules and therefore tested whether oxidized CSF lipoproteins had the same effect. We exposed Neuro 2A cells to human CSF lipoproteins and analyzed the microtubule organization by immunofluorescence. In vitro oxidized human CSF lipoproteins caused disruption of the microtubule network, while their native (nonoxidized) counterparts did not. Microtubule disruption was observed after short exposures (1 h) and lipoprotein concentrations were present in CSF (20 microg/mL), conditions that did not result in loss of cell viability. Importantly, adult bovine CSF lipoproteins, oxidized under identical conditions, had no effect on the microtubule organization of Neuro 2A cells. Comparison of human and bovine CSF lipoproteins revealed similar oxidation-induced modifications of apolipoproteins E and A-I as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Fatty acid analysis revealed substantially lower amounts of unsaturated fatty acids in bovine CSF lipoproteins, when compared to their human counterparts. Our data therefore indicate that oxidized human CSF lipoproteins are detrimental to neuronal microtubules. This effect is species-specific, since equally oxidized bovine CSF lipoproteins left the neuronal microtubule organization unchanged.

The lipid peroxidation product 4-hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin.

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.

Postmortem changes in the phosphorylation state of tau-protein in the rat brain

The phosphorylation state of tau-protein is crucial for the regulation of neuronal microtubule organization. Functional conclusions on tau-protein require an accurate assessment of phosphorylated sites. Therefore, the in vivo distribution and postmortem preservation of some phospho-epitopes on tau-protein were examined in the rat brain under different fixation and preparation conditions. Detection of tau-protein with a phosphorylation-independent antiserum revealed both axonal and somatodendritic localizations, which were not influenced by a postmortem interval of 30 min. The phospho-epitopes recognized by 12E8, AT8, and PHF-1 were mainly localized in the somatodendritic compartment. The binding sites of AT8 and PHF-1 were rapidly dephosphorylated postmortem, whereas the Tau-1 epitope was unmasked in the somatodendritic region. The axonally located phospho-epitope of AT270 and the nuclear epitope of AT100 were still detectable after a postmortem interval of 30 min. Postmortem dephosphorylation and inhibition of this process by PP1 and/or PP2A was further demonstrated on Western blot. In conclusion, rapid processing of tau-protein is essential for the correct assessment of investigations on phospho-isoforms.

Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.