Microtubule Severing Research Articles

NeuroD1 overexpression in spinal neurons accelerates axonal regeneration after sciatic nerve injury

Neurogenic differentiation 1 (NeuroD1) is mainlyexpressed in developing neurons where it plays critical roles in neuronal maturation and neurite elongation. The potential role and mechanism of NeuroD1 in adult axonal regeneration is not clear. The present study used synapsin (SYN) Cre and AAV9-Flex vectors to conditionally overexpress NeuroD1 in adult spinal neurons and found that NeuroD1 overexpression significantly accelerated axonal regeneration and functional recovery after sciatic nerve injury. Further in vitro and in vivo experiments suggested that the mechanism of NeuroD1 promotion on axonal regeneration was related to its regulation of the expression of neurotrophin BDNF and its receptor TrkB as well as a microtubule severing protein spastin.

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.

Expression of recombinant mouse spastin in E. coli

Mutations of the gene SPAST that encodes a microtubule severing enzyme, spastin, are the most frequent cause of Hereditary spastic paraplegia (HSP) disease. HSP is heterogeneous group of inherited neurodegenerative disorders characterized predominantly by progressive lower limb spasticity and weakness. Spastin belongs ATPase associated with various cellular activities (AAA) protein family and catalyzes microtubule severing. Spastin in mouse and human are highly identical in protein sequence and several spastin mutation models in mice have been generated in order to evaluate the significance of spastin loss-of-function in mammals. Expression and purification of spastin and the mutant variants determined in patients will facilitate the structure-function relationship study of spastin. Here I systemically optimized the expression condition of a truncated version of mouse spastin in E. coli. The recombinant protein and a mutant were further purified for ATPase activity assay.

P53 regulates katanin-p60 promoter in HCT 116 cells

Tumor suppressor protein p53, which functions in the cell cycle, apoptosis and neuronal differentiation via transcriptional regulations of target genes or interactions with several proteins, has been associated with neurite outgrowth through microtubule re-organization. We previously demonstrated in neurons that upon p53 induction, the level of microtubule severing protein Katanin-p60 increases, indicating that p53 might be a transcriptional regulator of the KATNA1 gene encoding Katanin-p60. In this context, we firstly elucidated the activity of KATNA1 regulatory regions and endogenous KATNA1 mRNA levels in the presence or absence of p53 using HCT 116 WT and HCT 116 p53 (−/−) cells. Next, we demonstrated the binding of p53 to the KATNA1 promoter and then investigated the role of p53 on KATNA1 gene expression by ascertaining KATNA1 mRNA and Katanin-p60 protein levels upon p53 overexpression and activation in both cells. Moreover, we showed changes in microtubule network upon increased Katanin-p60 level due to p53 overexpression. Also, the changes in KATNA1 mRNA and Katanin-p60 protein levels upon p53 knockdown were investigated. Our results indicate that p53 is an activator of KATNA1 gene expression and we show that both p53 and Katanin-p60 expression have strict regulations and are maintained at balanced levels as they are vital proteins to orchestrate either survival and apoptosis or differentiation.

The postmitotic midbody: Regulating polarity, stemness, and proliferation.

Abscission, the final stage of cell division, requires well-orchestrated changes in endocytic trafficking, microtubule severing, actin clearance, and the physical sealing of the daughter cell membranes. These processes are highly regulated, and any missteps in localized membrane and cytoskeleton dynamics often lead to a delay or a failure in cell division. The midbody, a microtubule-rich structure that forms during cytokinesis, is a key regulator of abscission and appears to function as a signaling platform coordinating cytoskeleton and endosomal dynamics during the terminal stages of cell division. It was long thought that immediately following abscission and the conclusion of cell division, the midbody is either released or rapidly degraded by one of the daughter cells. Recently, the midbody has gained prominence for exerting postmitotic functions. In this review, we detail the role of the midbody in orchestrating abscission, as well as discuss the relatively new field of postabscission midbody biology, particularly focusing on how it may act to regulate cell polarity and its potential to regulate cell tumorigenicity or stemness.

The Microtubule Severing Protein Katanin Regulates Proliferation of Neuronal Progenitors in Embryonic and Adult Neurogenesis

Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

Cell Wall Modifications in Giant Cells Induced by the Plant Parasitic Nematode Meloidogyne incognita in Wild-Type (Col-0) and the fra2 Arabidopsis thaliana Katanin Mutant.

Meloidogyne incognita is a root knot nematode (RKN) species which is among the most notoriously unmanageable crop pests with a wide host range. It inhabits plants and induces unique feeding site structures within host roots, known as giant cells (GCs). The cell walls of the GCs undergo the process of both thickening and loosening to allow expansion and finally support nutrient uptake by the nematode. In this study, a comparative in situ analysis of cell wall polysaccharides in the GCs of wild-type Col-0 and the microtubule-defective fra2 katanin mutant, both infected with M. incognita has been carried out. The fra2 mutant had an increased infection rate. Moreover, fra2 roots exhibited a differential pectin and hemicellulose distribution when compared to Col-0 probably mirroring the fra2 root developmental defects. Features of fra2 GC walls include the presence of high-esterified pectic homogalacturonan and pectic arabinan, possibly to compensate for the reduced levels of callose, which was omnipresent in GCs of Col-0. Katanin severing of microtubules seems important in plant defense against M. incognita, with the nematode, however, to be nonchalant about this “katanin deficiency” and eventually induce the necessary GC cell wall modifications to establish a feeding site.

Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis.

Klebsiella pneumoniae has become a growing concern within hospitals due to multidrug resistant strains and increasing mortality rates. Recently, we showed that at the subcellular level, K. pneumoniae compromises the integrity of the epithelia by disassembling the microtubule networks of cells through the actions of katanin microtubule severing proteins. In this study, we report on the observation that mitotic cells are targeted by K. pneumoniae and that during infections, the katanin proteins are excluded from the microtubule organizing centers of dividing cells, resulting in the alteration of the microtubule cytoskeleton. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1859-1864, 2020. © 2019 American Association for Anatomy.

Kif2a Scales Meiotic Spindle Size in Hymenochirus boettgeri.

Size is a fundamental feature of biological systems that affects physiology at all levels. For example, the dynamic, microtubule-based spindle that mediates chromosome segregation scales to a wide range of cell sizes across different organisms and cell types. Xenopus frog species possess a variety of egg and meiotic spindle sizes, and differences in activities or levels of microtubule-associated proteins in the egg cytoplasm between Xenopus laevis and Xenopus tropicalis have been shown to account for spindle scaling [1]. Increased activity of the microtubule severing protein katanin scales the X.tropicalis spindle smaller compared to X.laevis [2], as do elevated levels of TPX2, a protein that enriches the cross-linking kinesin-5 motor Eg5 at spindle poles [3]. To examine the conservation of spindle scaling mechanisms more broadly across frog species, we have utilized the tiny, distantly related Pipid frog Hymenochirus boettgeri. We find that egg extracts from H.boettgeri form meiotic spindles similar in size to X.tropicalis but that TPX2 and katanin-mediated scaling is not conserved. Instead, the microtubule depolymerizing motor protein kif2a functions to modulate spindle size. H.boettgeri kif2a possesses an activating phosphorylation site that is absent from X.laevis. Comparison of katanin and kif2a phosphorylation sites across a variety of species revealed strong evolutionary conservation, with X.laevis and X.tropicalis possessing distinct and unique alterations. Our study highlights the diversity and complexity of spindle assembly and scaling mechanisms, indicating that there is more than one way to assemble a spindle of a particular size.

Edwardsiella induces microtubule-severing in host epithelial cells

Edwardsiella bacteria cause economic losses to a variety of commercially important fish globally. Human infections are rare and result in a gastroenteritis-like illness. Because these bacteria are evolutionarily related to other Enterobacteriaceae and the host cytoskeleton is a common target of enterics, we hypothesized that Edwardsiella may cause similar phenotypes. Here we use HeLa and Caco-2 infection models to show that microtubules are severed during the late infections. This microtubule alteration phenotype was not dependant on the type III or type VI secretion system (T3SS and T6SS) of the bacteria as ΔT3SS and ΔT6SS mutants of E. piscicida EIB202 and E. tarda ATCC15947 that lacks both also caused microtubule disassembly. Immunolocalization experiments showed the host katanin catalytic subunits A1 and A like 1 proteins at regions of microtubule severing, suggesting their involvement in the microtubule disassembly events. To identify bacterial components involved in this phenotype, we screened a 2,758 transposon library of E. piscicida EIB202 and found that 4 single mutations in the atpFHAGDC operon disrupted microtubule disassembly in HeLa cells. We then constructed three atp deletion mutants; they all could not disassemble host microtubules. This work provides the first clear evidence of host cytoskeletal alterations during Edwardsiella infections.

Spastin uses its network

Structural Biology AAA ATPases are adenosine triphos phatases associated with diverse cellular activities that have a conserved module of amino acid residues. Spastin is an AAA ATPase protein that severs microtubules. Mutations in spastin cause hereditary spastic paraplegias. Microtubule severing seems to occur when spastin protomers form a hexamer around a polyglutamated tubulin tail and pull the tubulin subunit out of the microtubule lattice. Sandate et al. report a cryo–electron microscopy structure of the spastin hexamer bound to ATP and a polyglutamate peptide. Comparison to an apo-monomer structure shows that ATP binding drives the conformational changes required for oligomerization. Each protomer has three conserved loops. Two loops form a double helical staircase that surrounds the substrate, and the third links the substrate-interacting loops to the nucleotide binding pocket. Thus, ATP and peptide binding establish a network of interactions that stabilize the hexamer and facilitate efficient severing. Nat. Struct. Mol. Biol. 26 , 671 (2019).

Reorganization of paclitaxel-stabilized microtubule arrays at mitotic entry: roles of depolymerizing kinesins and severing proteins

ABSTRACTPaclitaxel is a widely used anti-cancer treatment that disrupts cell cycle progression by blocking cells in mitosis. The block at mitosis, with spindles assembled from short microtubules, is surprising given paclitaxel’s microtubule stabilizing activity and the need to depolymerize long interphase microtubules prior to spindle formation. Cells must antagonize paclitaxel’s microtubule stabilizing activity during a brief window of time at the transition from interphase to mitosis, allowing microtubule reorganization into a mitotic spindle, although the mechanism underlying microtubule depolymerization in the presence of paclitaxel has not been examined. Here we test the hypothesis that microtubule severing and/or depolymerizing proteins active at mitotic entry are necessary to clear the interphase array in paclitaxel-treated cells and allow subsequent formation of mitotic spindles formed of short microtubules. A549 and LLC-PK1 cells treated with 30nM paclitaxel approximately 4 h prior to mitotic entry successfully progress through the G2/M transition by clearing the interphase microtubule array from the cell interior outward to the cell periphery, a spatial pattern of reorganization that differs from that of cells possessing dynamic microtubules. Depletion of kinesin-8s, KIF18A and/or KIF18B obstructed interphase microtubule clearing at mitotic entry in paclitaxel-treated cells, with KIF18B making the larger contribution. Of the severing proteins, depletion of spastin, but not katanin, reduced microtubule loss as cells entered mitosis in the presence of paclitaxel. These results support a model in which KIF18A, KIF18B, and spastin promote interphase microtubule array disassembly at mitotic entry and can overcome paclitaxel-induced microtubule stability specifically at the G2/M transition.

An allosteric network in spastin couples multiple activities required for microtubule severing.

The AAA+ ATPase spastin remodels microtubule arrays through severing, and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.

Microtubule bundling by MAP65-1 protects against severing by inhibiting the binding of katanin.

The microtubule-severing enzyme katanin (KTN1) regulates the organization and turnover of microtubule arrays by the localized breakdown of microtubule polymers. In land plants, KTN1 activity is essential for the formation of linearly organized cortical microtubule arrays that determine the axis of cell expansion. Cell biological studies have shown that even though KTN1 binds to the sidewalls of single and bundled microtubules, severing activity is restricted to microtubule cross-over and nucleation sites, indicating that cells contain protective mechanisms to prevent indiscriminate microtubule severing. Here, we show that the microtubule-bundling protein MAP65-1 inhibits KTN1-mediated microtubule severing in vitro. Severing is inhibited at bundled microtubule segments and the severing rate of nonbundled microtubules is reduced by MAP65-1 in a concentration-dependent manner. Using various MAP65-1 mutant proteins, we demonstrate that efficient cross-linking of microtubules is crucial for this protective effect and that microtubule binding alone is not sufficient. Reduced severing due to microtubule bundling by MAP65-1 correlated to decreased binding of KTN1 to these microtubules. Taken together, our work reveals that cross-linking of microtubules by MAP65-1 confers resistance to severing by inhibiting the binding of KTN1 and identifies the structural features of MAP65-1 that are important for this activity.

068 Depletion of the Microtubule Severing Enzyme Fidgetin-like 2 Promotes Nerve Regeneration and Improves Erectile Function in a Rodent Model of Radical Prostatectomy

068 Depletion of the Microtubule Severing Enzyme Fidgetin-like 2 Promotes Nerve Regeneration and Improves Erectile Function in a Rodent Model of Radical Prostatectomy

Multicolour three dimensional structured illumination microscopy of immunolabeled plant microtubules and associated proteins

BackgroundIn the present work, we provide an account of structured illumination microscopy (SIM) imaging of fixed and immunolabeled plant probes. We take advantage of SIM, to superresolve intracellular structures at a considerable z-range and circumvent its low temporal resolution capacity during the study of living samples. Further, we validate the protocol for the imaging of fixed transgenic material expressing fluorescent protein-based markers of different subcellular structures.ResultsFocus is given on 3D imaging of bulky subcellular structures, such as mitotic and cytokinetic microtubule arrays as well as on the performance of SIM using multichannel imaging and the quantitative correlations that can be deduced. As a proof of concept, we provide a superresolution output on the organization of cortical microtubules in wild-type and mutant Arabidopsis cells, including aberrant preprophase microtubule bands and phragmoplasts in a cytoskeletal mutant devoid of the p60 subunit of the microtubule severing protein KATANIN and refined details of cytoskeletal aberrations in the mitogen activated protein kinase (MAPK) mutant mpk4. We further demonstrate, in a qualitative and quantitative manner, colocalizations between MPK6 and unknown dually phosphorylated and activated MAPK species and we follow the localization of the microtubule associated protein 65-3 (MAP65-3) in telophase and cytokinetic microtubular arrays.Conclusions3D SIM is a powerful, versatile and adaptable microscopy method for elucidating spatial relationships between subcellular compartments. Improved methods of sample preparation aiming to the compensation of refractive index mismatches, allow the use of 3D SIM in the documentation of complex plant cell structures, such as microtubule arrays and the elucidation of their interactions with microtubule associated proteins.

Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations.

Microtubule severing, which is highly critical for the survival of both mitotic and post-mitotic cells, has to be precisely adjusted by regulating the expression levels of severing proteins, katanin and spastin. Even though severing mechanism is relatively well-studied, there are limited studies for the transcriptional regulation of microtubule severing proteins. In this study, we identified the main regulatory region of KATNA1 gene encoding katanin-p60 as 5’ UTR, which has a key role for its expression, and showed Elk1 binding to KATNA1. Furthermore, we identified that Elk1 decreased katanin-p60 and spastin protein expressions, while mRNA levels were increased upon Elk1 overexpression. In addition, SUMOylation is a known post-translational modification regulating Elk1 activity. A previous study suggested that K230, K249, K254 amino acids in the R domain are the main SUMOylation sites; however, we identified that these amino acids are neither essential nor substantial for Elk1 SUMOylation. Also, we determined that KATNA1 methylation results in the reduction of Elk1 binding whereas SPG4 methylation does not. Together, our findings emphasizing the impacts of both transcriptional and post-transcriptional regulations of katanin-p60 and spastin suggest that Elk1 has a key role for differential expression patterns of microtubule severing proteins, thereby regulating cellular functions through alterations of microtubule organization.

Katanin Spiral and Ring Structures Shed Light on Power Stroke for Microtubule Severing

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

Exploring the Unfoldase Mechanism of Microtubule Severing by Coarse-Grained Simulations

Exploring the Unfoldase Mechanism of Microtubule Severing by Coarse-Grained Simulations

CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

SummaryThe coordinated reformation of the nuclear envelope (NE) after mitosis re-establishes the structural integrity and the functionality of the nuclear compartment. The endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodeling pathway that is highly conserved in eukaryotes, has been recently involved in NE resealing by mediating the annular fusion of the nuclear membrane (NM). We show here that CC2D1B, a regulator of ESCRT polymerization, is required to re-establish the nuclear compartmentalization by coordinating endoplasmic reticulum (ER) membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE. Accordingly, CC2D1B determines the spatiotemporal distribution of the CHMP7-ESCRT-III axis during NE reformation. Crucially, in CC2D1B-depleted cells, ESCRT activity is uncoupled from Spastin-mediated severing of spindle microtubules, resulting in persisting microtubules that compromise nuclear morphology. Therefore, we reveal CC2D1B as an essential regulatory factor that licenses the formation of ESCRT-III polymers to ensure the orderly reformation of the NE.