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1.
Proc Natl Acad Sci U S A ; 121(29): e2320470121, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38990951

RESUMO

Although the formation of new walls during plant cell division tends to follow maximal tensile stress direction, analyses of individual cells over time reveal a much more variable behavior. The origin of such variability as well as the exact role of interphasic microtubule behavior before cell division have remained mysterious so far. To approach this question, we took advantage of the Arabidopsis stem, where the tensile stress pattern is both highly anisotropic and stable. Although cortical microtubules (CMTs) generally align with maximal tensile stress, we detected a specific time window, ca. 3 h before cell division, where cells form a radial pattern of CMTs. This microtubule array organization preceded preprophase band (PPB) formation, a transient CMT array predicting the position of the future division plane. It was observed under different growth conditions and was not related to cell geometry or polar auxin transport. Interestingly, this cortical radial pattern correlated with the well-documented increase of cytoplasmic microtubule accumulation before cell division. This radial organization was prolonged in cells of the trm678 mutant, where CMTs are unable to form a PPB. Whereas division plane orientation in trm678 is noisier, we found that cell division symmetry was in contrast less variable between daughter cells. We propose that this "radial step" reflects a trade-off in robustness for two essential cell division attributes: symmetry and orientation. This involves a "reset" stage in G2, where an increased cytoplasmic microtubule accumulation transiently disrupts CMT alignment with tissue stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Divisão Celular , Microtúbulos , Arabidopsis/metabolismo , Arabidopsis/citologia , Microtúbulos/metabolismo , Divisão Celular/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Ácidos Indolacéticos/metabolismo
2.
Nat Plants ; 10(1): 100-117, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38172572

RESUMO

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Separação de Fases , Parede Celular/metabolismo , Microtúbulos/metabolismo , Xilema/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
3.
Sci Rep ; 11(1): 2873, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536509

RESUMO

Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. Additionally, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species. In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and flow cytometry measurements were completed within 10 min. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Citometria de Fluxo/métodos , Haemophilus influenzae/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Antibacterianos/uso terapêutico , Contagem de Colônia Microbiana , Estudos de Viabilidade , Infecções por Haemophilus/diagnóstico , Infecções por Haemophilus/tratamento farmacológico , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Sensibilidade e Especificidade , Streptococcus pneumoniae/efeitos dos fármacos
4.
Curr Biol ; 31(3): R143-R159, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33561417

RESUMO

Plants produce organs of various shapes and sizes. While much has been learned about genetic regulation of organogenesis, the integration of mechanics in the process is also gaining attention. Here, we consider the role of forces as instructive signals in organ morphogenesis. Turgor pressure is the primary cause of mechanical signals in developing organs. Because plant cells are glued to each other, mechanical signals act, in essence, at multiple scales, through cell wall contiguity and water flux. In turn, cells use such signals to resist mechanical stress, for instance, by reinforcing their cell walls. We show that the three elemental shapes behind plant organs - spheres, cylinders and lamina - can be actively maintained by such a mechanical feedback. Combinations of this 3-letter alphabet can generate more complex shapes. Furthermore, mechanical conflicts emerge at the boundary between domains exhibiting different growth rates or directions. These secondary mechanical signals contribute to three other organ shape features - folds, shape reproducibility and growth arrest. The further integration of mechanical signals with the molecular network offers many fruitful prospects for the scientific community, including the role of proprioception in organ shape robustness or the definition of cell and organ identities as a result of an interplay between biochemical and mechanical signals.


Assuntos
Desenvolvimento Vegetal , Plantas , Fenômenos Biomecânicos , Parede Celular , Células Vegetais , Reprodutibilidade dos Testes , Estresse Mecânico
5.
Plant Cell Rep ; 40(3): 575-582, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33439322

RESUMO

KEY MESSAGE: We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitive channel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Gravitropismo/fisiologia , Proteínas de Membrana/genética , Meristema/crescimento & desenvolvimento , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Estresse Fisiológico
6.
Dev Cell ; 56(1): 67-80.e3, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33434527

RESUMO

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Ácidos Indolacéticos/metabolismo , Katanina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Morfogênese/genética , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Sinais (Psicologia) , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Katanina/genética , Proteínas de Membrana Transportadoras/genética , Microfibrilas/metabolismo , Microscopia Confocal , Microtúbulos/enzimologia , Microtúbulos/metabolismo , Morfogênese/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Plântula/genética , Plântula/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microtomografia por Raio-X
7.
Curr Biol ; 30(8): 1491-1503.e2, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32169210

RESUMO

Growth variability generates mechanical conflicts in tissues. In plants, cortical microtubules usually align with maximal tensile stress direction, thereby mechanically reinforcing cell walls, and channeling growth rate and direction. How this is achieved remains largely unknown and likely involves microtubule regulators. The NIMA-related microtubule-associated kinase NEK6 phosphorylates tubulin, leading to the depolymerization of a subset of microtubules. We found that cortical microtubules exhibit a hyper-response to mechanical stress in the nek6 mutant. This response contributes to local cell protrusions in slow-growing regions of the nek6 mutant hypocotyl. When growth amplitude is higher, the hyper-alignment of microtubules leads to variable, stop-and-go, phenotypes, resulting in wavy hypocotyl shapes. After gravistimulation or touch, the nek6 mutant also exhibits a hyperbent hypocotyl phenotype, consistent with an enhanced perception of its own deformation. Strikingly, we find that NEK6 exhibits a novel form of polarity, being recruited at the ends of a subset of microtubules at cell edges. This pattern can be modified after local ablation, matching the new maximal tensile stress directions. We propose that NEK6 depolymerizes cortical microtubules that best align with maximal tensile stress to generate a noisier network of microtubules. This prevents an overreaction of microtubules to growth fluctuations and, instead, promotes the buffering of growth variations.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hipocótilo/crescimento & desenvolvimento , Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Hipocótilo/genética , Quinases Relacionadas a NIMA/metabolismo , Propriocepção , Estresse Fisiológico/genética
8.
Plant Cell Physiol ; 59(8): 1581-1591, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30011034

RESUMO

Mechanical sensing is one of the most fundamental processes for sessile plants to survive and grow. The response is known to involve calcium elevation in the cell. Arabidopsis seedlings grown horizontally on agar plates covered with a dialysis membrane show a 2-fold reduction in root growth compared with those grown vertically, a response to mechanical stress generated due to gravitropism of the root. To understand the molecular mechanism of how plant roots sense and respond to mechanical stimuli, we screened chemical libraries for compounds that affect the horizontal root growth in this experimental system and found that, while having no effect on root gravitropism, omeprazole known as a proton pump inhibitor significantly enhanced the mechanical stress-induced root growth reduction especially in lower pH media. In contrast, omeprazole reversed neither the alleviation of the mechanical stress-induced growth reduction caused by calcium depletion nor the insensitivity to the mechanical stress in the ethylene signaling mutant ein2. Together with the finding that omeprazole increased expression of touch-induced genes and ETHYLENE RESPONSE FACTOR1, our results suggest that the target of omeprazole mediates ethylene signaling in the root growth response to mechanical stress.


Assuntos
Omeprazol/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Estresse Mecânico , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gravitropismo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
9.
Development ; 145(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29440300

RESUMO

Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants.


Assuntos
Marchantia , Quinases Relacionadas a NIMA/fisiologia , Rizoma/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Sequência Conservada , Embriófitas , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Marchantia/genética , Marchantia/crescimento & desenvolvimento , Quinase 1 Relacionada a NIMA/genética , Quinases Relacionadas a NIMA/genética , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , Rizoma/genética
10.
Sci Rep ; 7(1): 7826, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798328

RESUMO

Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in ß-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant ß-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of ß-tubulin and the resulting destabilization of cortical microtubules.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Tubulina (Proteína)/química , Anisotropia , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Mutação , Fosforilação , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
11.
J Plant Res ; 128(6): 875-91, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26354760

RESUMO

Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both protein kinases and phosphatases. Never in mitosis A (NIMA)-related kinases (NEKs) are a family of serine/threonine kinases that regulate microtubule-related mitotic events in fungi and animal cells (e.g. centrosome separation and spindle formation). Although plants contain multiple members of the NEK family, their functions remain elusive. Recent studies revealed that NEK6 of Arabidopsis thaliana regulates cell expansion and morphogenesis through ß-tubulin phosphorylation and microtubule destabilization. In addition, plant NEK members participate in organ development and stress responses. The present phylogenetic analysis indicates that plant NEK genes are diverged from a single NEK6-like gene, which may share a common ancestor with other kinases involved in the control of microtubule organization. On the contrary, another mitotic kinase, polo-like kinase, might have been lost during the evolution of land plants. We propose that plant NEK members have acquired novel functions to regulate cell growth, microtubule organization, and stress responses.


Assuntos
Evolução Molecular , Microtúbulos/metabolismo , Plantas/classificação , Plantas/enzimologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Fosforilação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Proteínas Quinases/genética
12.
Sci Rep ; 5: 11364, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26068445

RESUMO

Abscisic acid (ABA) regulates seed maturation, germination and various stress responses in plants. The roles of ABA in cellular growth and morphogenesis, however, remain to be explored. Here, we report that ABA induces the ectopic outgrowth of epidermal cells in Arabidopsis thaliana. Seedlings of A. thaliana germinated and grown in the presence of ABA developed ectopic protrusions in the epidermal cells of hypocotyls, petioles and cotyledons. One protrusion was formed in the middle of each epidermal cell. In the hypocotyl epidermis, two types of cell files are arranged alternately into non-stoma cell files and stoma cell files, ectopic protrusions being restricted to the non-stoma cell files. This suggests the presence of a difference in the degree of sensitivity to ABA or in the capacity of cells to form protrusions between the two cell files. The ectopic outgrowth was suppressed in ABA insensitive mutants, whereas it was enhanced in ABA hypersensitive mutants. Interestingly, ABA-induced ectopic outgrowth was also suppressed in mutants in which microtubule organization was compromised. Furthermore, cortical microtubules were disorganized and depolymerized by the ABA treatment. These results suggest that ABA signaling induces ectopic outgrowth in epidermal cells through microtubule reorganization.


Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Germinação/fisiologia , Microtúbulos/metabolismo , Epiderme Vegetal/metabolismo , Ácido Abscísico/farmacologia , Germinação/efeitos dos fármacos
13.
Plant Signal Behav ; 7(12): 1552-5, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23072999

RESUMO

NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the ß-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Benzamidas/farmacologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hipocótilo/efeitos dos fármacos , Hipocótilo/enzimologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Quinases Relacionadas a NIMA , Paclitaxel/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteínas Quinases/genética
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