RESUMEN
Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of outer nuclear membrane KASH proteins, interacting in the nuclear envelope lumen with inner nuclear membrane SUN proteins and connecting the nucleus and cytoskeleton. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light-dark transitions and abscisic acid (ABA), which requires F-actin reorganization. SINE2 influences actin depolymerization and SINE1 actin repolymerization. The actin-related protein 2/3 (ARP2/3) complex, an actin nucleator, and the plant actin-bundling and -stabilizing factor SCAB1 are involved in stomatal aperture control. Here, we have tested the genetic interaction of SINE1 and SINE2 with SCAB1 and the ARP2/3 complex. We show that SINE1 and the ARP2/3 complex function in the same pathway during ABA-induced stomatal closure, while SINE2 and the ARP2/3 complex play opposing roles. The actin repolymerization defect observed in sine1-1 is partially rescued in scab1-2 sine1-1, while SINE2 is epistatic to SCAB1. In addition, SINE1 and ARP2/3 act synergistically in lateral root development. The absence of SINE2 renders trichome development independent of the ARP2/3 complex. Together, these data reveal complex and differential interactions of the two KASH proteins with the actin-remodeling apparatus and add evidence to the proposed differential role of SINE1 and SINE2 in actin dynamics.
Asunto(s)
Actinas , Proteínas de Arabidopsis , Actinas/metabolismo , Proteínas de Plantas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Nuclear/metabolismoRESUMEN
Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Nicotiana/metabolismo , Membrana Nuclear/metabolismo , Señales de Localización Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Agrobacterium tumefaciens/metabolismo , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMEN
Stomatal movement, which regulates gas exchange in plants, is controlled by a variety of environmental factors, including biotic and abiotic stresses. The stress hormone abscisic acid (ABA) initiates a signaling cascade, which leads to increased H2O2 and Ca2+ levels and F-actin reorganization, but the mechanism of, and connection between, these events is unclear. SINE1, an outer nuclear envelope component of a plant Linker of Nucleoskeleton and Cytoskeleton complex, associates with F-actin and is, along with its putative paralog SINE2, expressed in guard cells. Here, we have determined that Arabidopsis (Arabidopsis thaliana) SINE1 and SINE2 play an important role in stomatal opening and closing. Loss of SINE1 or SINE2 results in ABA hyposensitivity and impaired stomatal dynamics but does not affect stomatal closure induced by the bacterial elicitor flg22. The ABA-induced stomatal closure phenotype is, in part, attributed to impairments in Ca2+ and F-actin regulation. Together, the data suggest that SINE1 and SINE2 act downstream of ABA but upstream of Ca2+ and F-actin. While there is a large degree of functional overlap between the two proteins, there are also critical differences. Our study makes an unanticipated connection between stomatal regulation and nuclear envelope-associated proteins, and adds two new players to the increasingly complex system of guard cell regulation.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Estomas de Plantas/metabolismo , Transducción de Señal/genética , Ácido Abscísico/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/toxicidad , Calcio/metabolismo , Cloruro de Calcio/farmacología , Sequías , Peróxido de Hidrógeno/toxicidad , Microscopía Confocal , Mutación , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/genética , Estomas de Plantas/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Tiazolidinas/toxicidad , Regulación hacia ArribaRESUMEN
Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.
Asunto(s)
Medicago/fisiología , Complejos Multiproteicos/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/fisiología , Actinas/metabolismo , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Medicago/citología , Complejos Multiproteicos/genética , Matriz Nuclear/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Mapas de Interacción de Proteínas , Nódulos de las Raíces de las Plantas/metabolismo , Simbiosis , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
The nuclear envelope is much more than a simple barrier between nucleoplasm and cytoplasm. Nuclear envelope bridging complexes are protein complexes spanning both the inner and outer nuclear envelope membranes, thus directly connecting the cytoplasm with the nucleoplasm. In metazoans, they are involved in connecting the cytoskeleton with the nucleoskeleton, and act as anchoring platforms at the nuclear envelope for the positioning and moving of both nuclei and chromosomes. Recently, nucleocytoplasmic bridging complexes have also been identified in more evolutionarily diverse organisms, including land plants. Here, I discuss similarities and differences among and between eukaryotic supergroups, specifically of the proteins forming the cytoplasmic surface of these complexes. I am proposing a structure and function for a hypothetical ancestral nucleocytoplasmic bridging complex in the last eukaryotic common ancestor, with the goal to stimulate research in more diverse emerging model organisms.
Asunto(s)
Citoesqueleto/metabolismo , Eucariontes/metabolismo , Evolución Molecular , Complejos Multiproteicos/metabolismo , Matriz Nuclear/metabolismo , Animales , Centrosoma/metabolismo , HumanosRESUMEN
The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Alelos , Sustitución de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , Técnicas de Silenciamiento del Gen , Genotipo , Mutación , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Óvulo Vegetal/crecimiento & desarrollo , Óvulo Vegetal/metabolismo , Fenotipo , Plantones/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas Nucleares/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fertilidad/genética , Fertilidad/fisiología , Genes de Plantas , Mutación , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Plantas Modificadas Genéticamente , Polen/genética , Polen/fisiologíaRESUMEN
The nuclear envelope (NE) is a double membrane system enclosing the genome of eukaryotes. Besides nuclear pore proteins, which form channels at the NE, nuclear membranes are populated by a collection of NE proteins that perform various cellular functions. However, in contrast to well-conserved nuclear pore proteins, known NE proteins share little homology between opisthokonts and plants. Recent studies on NE protein complexes formed by Sad1/UNC-84 (SUN) and Klarsicht/ANC-1/Syne-1 Homology (KASH) proteins have advanced our understanding of plant NE proteins and revealed their function in anchoring other proteins at the NE, nuclear shape determination, nuclear positioning, anti-pathogen defence, root development, and meiotic chromosome organization. In this review, we discuss the current understanding of plant SUN, KASH, and other related NE proteins, and compare their function with the opisthokont counterparts.
Asunto(s)
Proteínas de la Membrana/genética , Membrana Nuclear/metabolismo , Proteínas de Plantas/genética , Plantas/genética , Proteínas de la Membrana/metabolismo , Membrana Nuclear/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismoRESUMEN
LINC (linker of nucleoskeleton and cytoskeleton) complexes play an essential role in nuclear migration by connecting the nucleus to the cytoskeleton and/or motor proteins. Plant LINC complexes have recently been identified in Arabidopsis thaliana, with the inner nuclear membrane SUN and outer nuclear membrane WIP proteins comprising the first identified complex. A recent study identified a nuclear movement defect in Arabidopsis pollen vegetative nuclei linked to the outer nuclear envelope WIP and WIT proteins. However, the role that SUN proteins may play in pollen nuclear migration has yet to be addressed. To explore this question, a SUN2 lumenal domain that was targeted to the ER specifically in pollen was over-expressed. It is shown that the ER-targeted SUN2 lumenal domain was able to displace WIP and WIT proteins from the pollen vegetative nuclear envelope. Expression of this dominant-negative transgene led to impaired VN mobility, impaired pollen tube guidance, and defective pollen tube reception. The observed pollen defects are similar to phenotypes observed in a wip1-1 wip2-1 wip3-1 wit1-1 wit2-1 mutant. It is also shown that these defects were dependent on the KASH-binding function of the SUN2 lumenal domain. These data support a model where LINC complexes formed by SUN, WIP, and WIT at the VNE are responsible for VN migration and suggest an important function of SUN, WIP, and WIT in pollen tube guidance and reception.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Tubo Polínico/metabolismo , Núcleo Celular/metabolismo , Fertilidad , Péptidos y Proteínas de Señalización IntracelularRESUMEN
SUMO is a small ubiquitin-related protein modifier that is involved in a number of biological processes, including transcription, DNA repair, genome stability, and chromatin organization. Its potential role in mRNA biogenesis is less well investigated. The biogenesis of mRNA is closely coupled to transcription as well as mRNA nuclear export and several of the involved proteins have dual roles and appear in several complexes. Recently, SUMO-proteome analyses have discovered a number of these proteins as putative targets of SUMO regulation. In the model plant Arabidopsis thaliana, several mutants as well as environmental conditions have been identified that show a close correlation between over- and under-sumoylation of nuclear proteins and mRNA export retention. Three new plant SUMO-proteome studies add to the list of potentially sumoylated RNA-related proteins. Here, the emerging connection between SUMO and mRNA export is compared across kingdoms and its potential mechanistic role is discussed. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Asunto(s)
Arabidopsis , ARN Mensajero , Sumoilación/genética , Ubiquitinas , Transporte Activo de Núcleo Celular/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma , Procesamiento Postranscripcional del ARN , Transporte de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
In eukaryotes, the spindle assembly checkpoint (SAC) ensures the fidelity of chromosome segregation through monitoring the bipolar attachment of microtubules to kinetochores. Recently, the SAC components Mitotic Arrest Deficient 1 and 2 (MAD1 and MAD2) were found to associate with the nuclear pore complex (NPC) during interphase and to require certain nucleoporins, such as Tpr in animal cells, to properly localize to kinetochores. In plants, the SAC components MAD2, BUR1, BUB3 and Mps1 have been identified, but their connection to the nuclear pore has not been explored. Here, we show that AtMAD1 and AtMAD2 are associated with the nuclear envelope during interphase, requiring the Arabidopsis homolog of Tpr, NUA. Both NUA and AtMAD2 loss-of-function mutants have a shorter primary root and a smaller root meristem, and this defect can be partially rescued by sucrose. Mild AtMAD2 over-expressors exhibit a longer primary root, and an extended root meristem. In BY-2 cells, AtMAD2 is associated with kinetochores during prophase and prometaphase, but not metaphase, anaphase and telophase. Protein-interaction assays demonstrate binding of AtMAD2 to AtMAD1 and AtMAD1 to NUA. Together, these data suggest that NUA scaffolds AtMAD1 and AtMAD2 at the nuclear pore to form a functional complex and that both NUA and AtMAD2 suppress premature exit from cell division at the Arabidopsis root meristem.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Inmunoprecipitación , Proteínas Mad2 , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Unión Proteica , Nicotiana/genética , Nicotiana/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The linker of nucleoskeleton and cytoskeleton (LINC) complex is a protein complex spanning the inner and outer membranes of the nuclear envelope. Outer nuclear membrane KASH proteins interact in the nuclear envelope lumen with inner nuclear membrane SUN proteins. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light-dark transitions and ABA. Previous studies have shown F-actin organization, cytoplasmic calcium (Ca2+) oscillations, and vacuolar morphology changes are involved in ABA-induced stomatal closure. Here, we show that SINE1 and SINE2 are both required for actin pattern changes during ABA-induced stomatal closure, but influence different, temporally distinguishable steps. External Ca2+ partially overrides the mutant defects. ABA-induced cytoplasmic Ca2+ oscillations are diminished in sine2-1 but not sine1-1, and this defect can be rescued by both exogenous Ca2+ and F-actin depolymerization. We show first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1. Vacuolar fragmentation is impaired in both mutants and is partially rescued by F-actin depolymerization. Together, these data indicate distinct roles for SINE1 and SINE2 upstream of this network of players involved in ABA-based stomatal closure, suggesting a role for the nuclear surface in guard cell ABA signaling.
RESUMEN
West Nile Virus (WNV) infections are increasingly detected in birds and horses in central Europe, with the first mosquito-borne autochthonous human infection detected in Germany in 2019. Human infections are typically asymptomatic, with occasional severe neurological disease. Because of a low number of cases in central Europe, awareness regarding potential cases is low and WNV diagnostic testing is not routine. We tested cerebrospinal fluid (CSF) samples from unsolved encephalitis and meningitis cases from Berlin from 2019 to 2020, and describe a WNV-encephalitis case in a 33-year-old kidney transplant recipient. The infectious course was resolved by serology, RT-PCR and sequencing of stored samples. Phylogenetic sequence analysis revealed a close relationship of the patient's WNV strain to German sequences from 2019 and 2020. A lack of travel history and patient self-isolation during the SARS-CoV-2 pandemic suggest the infection was acquired in the patient's home or garden. Serological tests of four people sharing the living space were negative. Retrospective RT-PCR and WNV-IgM testing of 671 CSF samples from unsolved encephalitis and meningitis cases from Berlin detected no additional infections. The recent increase of WNV cases illustrates the importance of considering WNV in cases of meningoencephalitis, especially in immunocompromised patients, as described here. Proper education and communication and a revised diagnostic strategy will help to raise awareness and to detect future WNV infections.
Asunto(s)
Trasplante de Riñón , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Adulto , Humanos , Fiebre del Nilo Occidental/diagnósticoRESUMEN
Protein sumoylation plays an important role in plant development, flowering-time regulation, and abiotic stress response. However, the molecular role of sumoylation in these pathways is largely unknown. It was shown previously that in mutants of the inner nuclear basket nucleoporin NUA a large increase in the abundance of high-molecular weight SUMO conjugated proteins correlated with nuclear retention of bulk mRNA. Here, the connection between sumoylation and mRNA export in plants was further investigated. Both SUMO-conjugate accumulation and mRNA retention were also found in a second nucleoporin mutant that does not affect NUA, and SUMO conjugates accumulated predominantly in the nucleus. Similarly, after heat and ethanol treatment, two abiotic stress treatments known to lead to the accumulation of sumoylated proteins, nuclear mRNA was retained. To establish a causal relationship between sumoylation and mRNA export, mutations in two enzymes in the SUMO pathway were tested. Mutating either SUMO E3 ligase or SUMO isopeptidase lead to nuclear mRNA retention, indicating that both an increase and a decrease in the pool of sumoylated nuclear proteins blocks mRNA export. Together, these data show that sumoylation acts upstream of mRNA export in plants, likely through the transient sumoylation status of one or more factors involved in mRNA trafficking.
Asunto(s)
Arabidopsis/genética , Núcleo Celular/metabolismo , Ambiente , Homeostasis/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Etanol/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Respuesta al Choque Térmico/efectos de los fármacos , Homeostasis/efectos de los fármacos , Peso Molecular , Mutación/genética , Proteínas de Complejo Poro Nuclear/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismo , Estrés Fisiológico/efectos de los fármacos , Sumoilación/efectos de los fármacosRESUMEN
RanGAP is the GTPase-activating protein of the small GTPase Ran and is involved in nucleocytoplasmic transport in yeast and animals via the Ran cycle and in mitotic cell division. Arabidopsis thaliana has two copies of RanGAP, RanGAP1 and RanGAP2. To investigate the function of plant RanGAP, T-DNA insertional mutants were analysed. Arabidopsis plants with a null mutant of either RanGAP1 or RanGAP2 had no observable phenotype. Analysis of segregating progeny showed that double mutants in RanGAP1 and RanGAP2 are female gametophyte defective. Ovule clearing with differential interference contrast optics showed that mutant female gametophytes were arrested at interphase, predominantly after the first mitotic division following meiosis. In contrast, mutant pollen developed and functioned normally. These results show that the two RanGAPs are redundant and indispensable for female gametophyte development in Arabidopsis but dispensable for pollen development. Nuclear division arrest during a mitotic stage suggests a role for plant RanGAP in mitotic cell cycle progression during female gametophyte development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas Activadoras de GTPasa/metabolismo , Meiosis , Mitosis , Óvulo Vegetal/citología , Óvulo Vegetal/crecimiento & desarrollo , Alelos , Proteínas de Arabidopsis/genética , Segregación Cromosómica/genética , Cruzamientos Genéticos , Proteínas Activadoras de GTPasa/genética , Gametogénesis en la Planta/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Genoma de Planta/genética , Genotipo , Mutación/genética , Óvulo Vegetal/genética , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the "molecular memory" that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , División Celular , Proteínas Activadoras de GTPasa/metabolismo , Arabidopsis/metabolismo , Interferencia de ARNRESUMEN
The Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1-4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5-8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9-14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15-18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Arabidopsis/ultraestructura , Diferenciación Celular , Datos de Secuencia Molecular , Poro Nuclear/ultraestructura , Raíces de Plantas/metabolismo , Raíces de Plantas/ultraestructura , Conformación ProteicaRESUMEN
The nuclear envelope and the nuclear pore are important structures that both separate and selectively connect the nucleoplasm and the cytoplasm. The requirements for specific targeting of proteins to the plant nuclear envelope and nuclear pore are poorly understood. How are transmembrane-domain proteins sorted to the nuclear envelope and nuclear pore membranes? What protein-protein interactions are involved in associating other proteins to the nuclear pore? Are there plant-specific aspects to these processes? We are using the case of the nuclear pore-associated Ran-cycle component RanGAP (Ran GTPase-activating protein) to address these fundamental questions. Plant RanGAP is targeted to the nuclear pore by a plant-specific mechanism involving two families of nuclear pore-associated proteins [WIP (WPP-domain-interacting protein) and WIT (WPP-domain-interacting tail-anchored protein)] not found outside the land plant lineage. One protein family (WIP or WIT) is sufficient for RanGAP targeting in differentiated root cells, whereas both families are necessary in meristematic cells. A C-terminal predicted transmembrane domain is sufficient for targeting WIP proteins to the nuclear envelope. Nuclear-envelope targeting of WIT proteins requires a coiled-coil domain and is facilitated by HSC70 (heat-shock cognate 70 stress protein) chaperones and a class of plant-specific proteins resembling the RanGAP-targeting domain (WPP proteins). Taken together, this sheds the first light on the requirements and interdependences of nuclear envelope and nuclear pore targeting in land plants.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Activadoras de GTPasa/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Unión Proteica , Alineación de SecuenciaRESUMEN
The nuclear pore complex is the gateway of macromolecular trafficking between the nucleus and the cytoplasm. Although its composition is well characterized in yeast and mammalian systems, little is known about the plant nuclear pore. Several recent reports describe complex whole-organism phenotypes based on mutations in plant nucleoporins. The pathways affected include plant-microbe interactions, auxin response, cold-stress tolerance and flowering-time regulation. The effects are probably based, at least in part, on changes in protein import and/or RNA export (including regulatory small RNAs). Here, we review these new findings while comparing and contrasting them with what is known about nucleoporin functions from non-plant organisms, including nucleoporin activities not linked to nucleocytoplasmic transport.