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1.
Gene ; 727: 144241, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715301

RESUMO

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.


Assuntos
Katanina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Células HCT116 , Humanos , Katanina/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Transcrição Genética/genética
2.
Int J Mol Sci ; 21(1)2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31878228

RESUMO

The microtubule cytoskeleton is a dynamic system that plays vital roles in fundamental cellular processes and in responses to environmental stumili. Salt stress induced depolymerization and reorganization of microtubules are believed to function in the promotion of survival in Arabidopsis. Microtubule-severing enzyme ATKATANIN1 (AtKTN1) is recognized as a MAP that help to maintain organized microtubule structure. To date, whether AtKTN1 is involved in response to salt stress in Arabidopsis remains unknown. Here, our phenotypic analysis showed that the overexpression of AtKTN1 decreased tolerance to salt stress, whereas the knock-out of AtKTN1 increased salt tolerance in the early stage but decreased salt tolerance in the later stage. Microscopic analysis revealed that microtubule organization and dynamics are distorted in both overexpression and mutant cells which, in turn, resulted in an abnormal disassembly and reorganization under salt stress. Moreover, qRT analysis revealed that stress-responsive genes were down-regulated in overexpression and mutant cells compared to WT cells under salt stress. Taken together, our results indicated roles of AtKTN1 in modulating microtubule organization, salt-stress induced microtubule disruption and recovery, and its involvement in stress-related signaling pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Microtúbulos/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Katanina/genética , Katanina/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estresse Salino , Tolerância ao Sal , Cloreto de Sódio/farmacologia
3.
Int J Mol Sci ; 20(21)2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31684028

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Células Gigantes/metabolismo , Katanina/metabolismo , Raízes de Plantas/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Parede Celular/parasitologia , Regulação da Expressão Gênica de Plantas , Células Gigantes/parasitologia , Interações Hospedeiro-Parasita , Katanina/genética , Microtúbulos/metabolismo , Mutação , Pectinas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Polissacarídeos/metabolismo , Tylenchoidea/fisiologia
4.
Plant Physiol ; 181(3): 1191-1206, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31537749

RESUMO

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.


Assuntos
Parede Celular/metabolismo , Glucanos/metabolismo , Katanina/metabolismo , Microtúbulos/metabolismo , Xilanos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Homeostase , Katanina/genética , Meristema/enzimologia , Meristema/genética , Meristema/crescimento & desenvolvimento
5.
Mol Med Rep ; 20(4): 3573-3582, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485656

RESUMO

Microtubule­severing proteins (MTSPs) are a group of microtubule­associated proteins essential for multiple microtubule­related processes, including mitosis and meiosis. Katanin p60 ATPase­containing subunit A­like 1 (p60 katanin­like 1) is an MTSP that maintains the density of spindle microtubules at the poles in mitotic cells; however, to date, there have been no studies about its role in female meiosis. Using in vitro­matured (IVM) oocytes as a model, it was first revealed that p60 katanin­like 1 was predominant in the ovaries and oocytes, indicating its essential roles in oocyte meiosis. It was also revealed that p60 katanin­like 1 was concentrated at the spindle poles and co­localized and interacted with γ­tubulin, indicating that it may be involved in pole organization. Next, specific siRNA was used to deplete p60 katanin­like 1; the spindle organization was severely disrupted and characterized by an abnormal width:length ratio, multipolarity and extra aster microtubules out of the main spindles. Finally, it was determined that p60 katanin­like 1 knockdown retarded oocyte meiosis, reduced fertilization, and caused abnormal mitochondrial distribution. Collectively, these results indicated that p60 katanin­like 1 is essential for oocyte meiosis by ensuring the integrity of the spindle poles.


Assuntos
Katanina/metabolismo , Meiose , Microtúbulos/metabolismo , Oócitos/citologia , Polos do Fuso/metabolismo , Animais , Células Cultivadas , Feminino , Katanina/análise , Camundongos , Camundongos Endogâmicos ICR , Células NIH 3T3 , Oócitos/metabolismo , Oócitos/ultraestrutura , Polos do Fuso/ultraestrutura , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo
6.
Nat Cell Biol ; 21(9): 1086-1092, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481789

RESUMO

Tau is an intrinsically disordered protein, which diffuses on microtubules1. In neurodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules are correlated with axonal degeneration2. Tau can protect microtubules from microtubule-degrading enzymes such as katanin3. However, how tau carries out this regulatory function is still unclear. Here, using in vitro reconstitution, we show that tau molecules on microtubules cooperatively form cohesive islands that are kinetically distinct from tau molecules that individually diffuse on microtubules. Dependent on the tau concentration in solution, the islands reversibly grow or shrink by addition or release of tau molecules at their boundaries. Shielding microtubules from kinesin-1 motors and katanin, the islands exhibit regulatory qualities distinct from a comparably dense layer of diffusible tau. Superprocessive kinesin-8 motors penetrate the islands and cause their disassembly. Our results reveal a microtubule-dependent phase of tau that constitutes an adaptable protective layer on the microtubule surface. We anticipate that other intrinsically disordered axonal proteins display a similar cooperative behaviour and potentially compete with tau in regulating access to the microtubule surface.


Assuntos
Cinesina/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Katanina/metabolismo , Cinética , Doenças Neurodegenerativas/metabolismo
7.
Structure ; 27(9): 1375-1383.e3, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31353241

RESUMO

Katanin is a microtubule-severing enzyme that is crucial for many cellular processes. Katanin consists of two subunits, p60 and p80, that form a stable complex. The interaction between subunits is mediated by the p60 N-terminal microtubule-interacting and -trafficking domain (p60-MIT) and the p80 C-terminal domain (p80-CTD). Here, we performed a biophysical characterization of the mouse p60-MIT:p80-CTD heterodimer and show that this complex can assemble into heterotetramers. We identified two mutations that enhance heterotetramer formation and determined the X-ray crystal structure of this mutant complex. The structure revealed a domain-swapped heterotetramer consisting of two p60-MIT:p80-CTD heterodimers. Structure-based sequence alignments suggest that heterotetramerization of katanin might be a common feature of various species. Furthermore, we show that enhanced heterotetramerization of katanin impairs its microtubule end-binding properties and increases the enzyme's microtubule lattice binding and severing activities. Therefore, our findings suggest the existence of different katanin oligomers that possess distinct functional properties.


Assuntos
Katanina/química , Mutação , Animais , Sítios de Ligação , Cristalografia por Raios X , Katanina/genética , Katanina/metabolismo , Camundongos , Microtúbulos/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Alinhamento de Sequência
8.
PLoS Biol ; 17(7): e3000381, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31314751

RESUMO

The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption.


Assuntos
Axonema/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cílios/fisiologia , Transdução de Sinais/fisiologia , Animais , Axonema/metabolismo , Cálcio/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular , Cílios/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Katanina/genética , Katanina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência
9.
Eur J Med Chem ; 178: 177-194, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185410

RESUMO

Microtubule is one of the important targets for cancer treatment. A novel class of diaryl substituted imidazo[4,5-c]pyridin-2-ones and imidazo[4,5-c]pyridines were designed based on combination principles by merging the structures of ß-lactams and purine-type compounds known as tubulin polymerization inhibitor and katanin activity up-regulator, respectively. Their antitumor activities were evaluated in vitro and the mechanism was elucidated, leading to the identification of 1,6-diaryl-1H-imidazo[4,5-c]pyridin-2(3H)-one 20b as the first bifunctional agent that can target both tubulin and katanin simultaneously. The in vivo assays verified that compound 20b significantly inhibited xenograft tumor growth with good pharmacokinetic characteristics, demonstrating a promising potential for further development into anti-tumor drug candidates with a unique mechanism of dual-targeting microtubule.


Assuntos
Antineoplásicos/uso terapêutico , Imidazóis/uso terapêutico , Katanina/antagonistas & inibidores , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Carcinoma Endometrioide/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacocinética , Katanina/metabolismo , Ligantes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Estrutura Molecular , Neoplasias Ovarianas/tratamento farmacológico , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Mol Biol Cell ; 30(13): 1587-1597, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31017848

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Katanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Adenosina Trifosfatases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Katanina/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica
11.
Dev Cell ; 49(5): 731-747.e7, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31006649

RESUMO

Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. We show that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shot, absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, our study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Katanina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Poliploidia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocinese , Proteínas de Drosophila/genética , Feminino , Katanina/genética , Masculino , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Fuso Acromático
12.
PLoS One ; 14(2): e0212518, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30789974

RESUMO

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.


Assuntos
Katanina/metabolismo , Espastina/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , Metilação de DNA , Humanos , Katanina/química , Katanina/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espastina/química , Espastina/genética , Sumoilação , Transcrição Genética , Proteínas Elk-1 do Domínio ets/química , Proteínas Elk-1 do Domínio ets/genética
13.
Cell Rep ; 26(5): 1357-1367.e5, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699360

RESUMO

Katanin was the first microtubule (MT)-severing enzyme discovered, but how katanin executes MT severing remains poorly understood. Here, we report X-ray crystal structures of the apo and ATPγS-bound states of the catalytic AAA domain of human katanin p60 at 3.0 and 2.9 Å resolution, respectively. Comparison of the two structures reveals conformational changes induced by ATP binding and how such changes ensure hexamer stability. Moreover, we uncover structural details of pore loops (PLs) and show that Arg283, a residue unique to katanin among MT-severing enzymes, protrudes from PL1 and lines the entry of the catalytic pore. Functional studies suggest that PL1 and Arg283 play essential roles in the recognition and remodeling of the glutamylated, C-terminal tubulin tail and regulation of axon growth. In addition, domain-swapping experiments in katanin and spastin suggest that the non-homologous N-terminal region, which contains the MT-interacting and trafficking domain and a linker, confers specificity to the severing process.


Assuntos
Glutamatos/metabolismo , Katanina/química , Katanina/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Axônios/metabolismo , Células HeLa , Humanos , Camundongos Endogâmicos ICR , Modelos Moleculares , Mutação/genética , Domínios Proteicos , Multimerização Proteica , Células Receptoras Sensoriais/metabolismo , Espastina/metabolismo
14.
J Cell Biol ; 218(1): 8-9, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30573524

RESUMO

Microtubule reorientation into a longitudinal network during the phototropic response in Arabidopsis thaliana depends on their severing by katanin at crossovers. Lindeboom et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201805047) show that at newly generated plus ends, the anti-catastrophe activity of CLASP is essential for further growth.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Katanina , Proteínas Associadas aos Microtúbulos , Microtúbulos
15.
J Cell Biol ; 218(1): 190-205, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30377221

RESUMO

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Katanina/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Modelos Estatísticos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Reporter , Katanina/metabolismo , Luz , Transdução de Sinal Luminoso , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos da radiação , Microtúbulos/ultraestrutura , Mutação , Células Vegetais/metabolismo , Células Vegetais/efeitos da radiação , Células Vegetais/ultraestrutura , Estabilidade Proteica , Processos Estocásticos
16.
Dev Cell ; 47(4): 402-403, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30458134

RESUMO

The microtubule-severing proteins spastin and katanin were long thought to destabilize microtubules. Recent work demonstrates that these enzymes inflict nano-damage on the microtubule lattice that is then rapidly repaired by new GTP-tubulin incorporation, for a net stabilization of the polymer, a process that has implications for neurodegenerative disease.


Assuntos
Adenosina Trifosfatases , Tubulina (Proteína) , Guanosina Trifosfato , Katanina , Microtúbulos
17.
Biochem Biophys Res Commun ; 507(1-4): 389-394, 2018 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-30448058

RESUMO

Microtubule severing is essential for reorganization of microtubules during neuronal migration and process elongation. Katanin is a microtubule-severing enzyme, of which the major catalytic subunits are katanin A1 (KATNA1) and katanin A-like 1 (KATNAL1). The domain organization of the two subunits are almost the same; however, little is known about their functional difference. Here, we compared the expression pattern, microtubule-severing activity, intracellular degradation and knockdown phenotype in cultured cells of the two subunits. While KATNA1 was expressed ubiquitously among tissues of young adult mice, KATNAL1 was highly expressed in the brain and the testis. Neurons expressed almost only KATNAL1. When introduced into Neuro2a cells, KATNAL1 showed higher microtubule-severing activity. Cycloheximide chase analysis revealed that KATNAL1 is more stable in cells. To elucidate which part of the molecules are responsible for these characteristics, we generated chimeric molecules by swapping the amino-terminal and carboxyl-terminal halves between the two subunits. Experiments using these chimeras revealed that the amino-terminal half region is the determinant for their characteristics. Furthermore, KATNAL1 knockdown in Neuro2a cells resulted in enhancement of process elongation, while KATNA1 knockdown showed no effect. These data suggest that more active and more stable katanin subunit, KATNAL1, plays more important role in process elongation.


Assuntos
Katanina/genética , Neurônios/metabolismo , Animais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Katanina/metabolismo , Masculino , Camundongos Endogâmicos ICR , Microtúbulos/metabolismo , Neuritos/metabolismo , Estabilidade Proteica , Proteólise
18.
J Cell Biol ; 217(12): 4057-4069, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30373906

RESUMO

Microtubule-severing enzymes generate internal breaks in microtubules. They are conserved in eukaryotes from ciliates to mammals, and their function is important in diverse cellular processes ranging from cilia biogenesis to cell division, phototropism, and neurogenesis. Their mutation leads to neurodegenerative and neurodevelopmental disorders in humans. All three known microtubule-severing enzymes, katanin, spastin, and fidgetin, are members of the meiotic subfamily of AAA ATPases that also includes VPS4, which disassembles ESCRTIII polymers. Despite their conservation and importance to cell physiology, the cellular and molecular mechanisms of action of microtubule-severing enzymes are not well understood. Here we review a subset of cellular processes that require microtubule-severing enzymes as well as recent advances in understanding their structure, biophysical mechanism, and regulation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Katanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/enzimologia , Espastina/metabolismo , Animais , Humanos
19.
PLoS Genet ; 14(10): e1007705, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30296269

RESUMO

Plants have evolved diverse cell types with distinct sizes, shapes, and functions. For example, most flowering plants contain specialized petal conical epidermal cells that are thought to attract pollinators and influence light capture and reflectance, but the molecular mechanisms controlling conical cell shaping remain unclear. Here, through a genetic screen in Arabidopsis thaliana, we demonstrated that loss-of-function mutations in ANGUSTIFOLIA (AN), which encodes for a homolog of mammalian CtBP/BARs, displayed conical cells phenotype with wider tip angles, correlating with increased accumulation of reactive oxygen species (ROS). We further showed that exogenously supplied ROS generated similar conical cell phenotypes as the an mutants. Moreover, reduced endogenous ROS levels resulted in deceased tip sharpening of conical cells. Furthermore, through enhancer screening, we demonstrated that mutations in katanin (KTN1) enhanced conical cell phenotypes of the an-t1 mutants. Genetic analyses showed that AN acted in parallel with KTN1 to control conical cell shaping. Both increased or decreased ROS levels and mutations in AN suppressed microtubule organization into well-ordered circumferential arrays. We demonstrated that the AN-ROS pathway jointly functioned with KTN1 to modulate microtubule ordering, correlating with the tip sharpening of conical cells. Collectively, our findings revealed a mechanistic insight into ROS homeostasis regulation of microtubule organization and conical cell shaping.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/citologia , Forma Celular/fisiologia , Proteínas Repressoras/genética , Arabidopsis/fisiologia , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Flores/genética , Flores/metabolismo , Katanina/genética , Microtúbulos/genética , Espécies Reativas de Oxigênio/metabolismo
20.
Cytoskeleton (Hoboken) ; 75(12): 531-544, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30176123

RESUMO

Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant katanin p60. Our results demonstrate that targeting an active version of katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.


Assuntos
Katanina , Microtúbulos , Animais , Linhagem Celular , Drosophila melanogaster , Humanos , Katanina/genética , Katanina/metabolismo , Cinetocoros/enzimologia , Metáfase/fisiologia , Microtúbulos/enzimologia , Microtúbulos/genética
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