RESUMEN
Bone marrow transplantation (BMT) of healthy donor cells has been postulated as a strategy for treating osteogenesis imperfecta (OI) and other bone fragility disorders. The effect of engraftment by tail vein injection and/or marrow ablation by 6 Gy whole body irradiation were tested in Col1a2+/G610C (OI) mice as a model of mild-moderate OI. Dual-emission X-ray absorptiometry, microCT, and 4-point bending were used to measure bone volume (BV), bone mineral density (BMD), and biomechanical strength. BV, BMD, and mechanical strength were reduced in OI mice compared to wild type (WT) controls. BMT with and without irradiation yielded no difference in BV and BMD outcomes for both OI and WT mice, at 3 weeks. Transplantation of OI cells into OI mice to test for paracrine effects of BMT also showed no difference with non-transplanted OI mice. In a parallel cell tracking study, donor marrow was taken from transgenic mice constitutively expressing tdTomato and transplanted into WT mice. Lineage tracking demonstrated that irradiation considerably enhanced engraftment of tdTomato+ cells. However, tdTomato+ cells predominantly expressed TRAP and not AP, indicating engrafted donor cells were chiefly from the hematopoietic lineages. These data show that whole marrow transplantation fails to rescue the bone phenotype of Col1a2+/G610C (OI) mice and that osteopoietic engraftment is not significantly enhanced by irradiation. These findings are highly relevant to modern approaches focused on the gene repair of patient cells ex vivo and their subsequent reintroduction into the osteopoietic compartment via the circulation.
Asunto(s)
Trasplante de Médula Ósea , Huesos/metabolismo , Osteogénesis Imperfecta/terapia , Osteogénesis/fisiología , Animales , Densidad Ósea/fisiología , Trasplante de Médula Ósea/métodos , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteogénesis Imperfecta/genéticaRESUMEN
Tumor protein D52 (TPD52) is amplified and/or overexpressed in cancers of diverse cellular origins. Altered cellular metabolism (including lipogenesis) is a hallmark of cancer development, and protein-protein associations between TPD52 and known regulators of lipid storage, and differential TPD52 expression in obese versus non-obese adipose tissue, suggest that TPD52 might regulate cellular lipid metabolism. We found increased lipid droplet numbers in BALB/c 3T3 cell lines stably expressing TPD52, compared with control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 cells showed increased fatty acid storage in triglyceride (from both de novo synthesis and uptake) and formed greater numbers of lipid droplets upon oleic acid supplementation than control cells. TPD52 colocalised with Golgi, but not endoplasmic reticulum (ER), markers and also showed partial colocalisation with lipid droplets coated with ADRP (also known as PLIN2), with a proportion of TPD52 being detected in the lipid droplet fraction. Direct interactions between ADRP and TPD52, but not TPD52L1, were demonstrated using the yeast two-hybrid system, with ADRP-TPD52 interactions confirmed using GST pulldown assays. Our findings uncover a new isoform-specific role for TPD52 in promoting intracellular lipid storage, which might be relevant to TPD52 overexpression in cancer.
Asunto(s)
Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Neoplasias/biosíntesis , Triglicéridos/metabolismo , Animales , Células 3T3 BALB , Línea Celular Tumoral , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Ácidos Grasos/genética , Femenino , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Neoplasias/genética , Perilipina-2 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Triglicéridos/genéticaRESUMEN
Throughout the wheat-growing regions of Australia, chilling temperatures below 2 °C occur periodically on consecutive nights during the period of floral development in spring wheat (Triticum aestivumâ L.). In this study, wheat plants showed significant reductions in fertility when exposed to prolonged chilling temperatures in controlled environment experiments. Among the cultivars tested, the Australian cultivars Kite and Hartog had among the lowest levels of seed set due to chilling and their responses were investigated further. The developmental stage at exposure, the chilling temperature and length of exposure all influenced the level of sterility. The early period of booting, and specifically the +4 cm auricle distance class, was the most sensitive and corresponded to meiosis within the anthers. The response of microtubules to chilling during meiosis in Hartog was monitored, but there was little difference between chilled and control plants. Other abnormalities, such as plasmolysis and cytomixis increased in frequency, were associated with death of developing pollen cells, and could contribute to loss of fertility. The potential for an above-zero chilling sensitivity in Australian spring wheat varieties could have implications for exploring the tolerance of wheat flower development to chilling and freezing conditions in the field.
Asunto(s)
Congelación , Meiosis , Microtúbulos/metabolismo , Polen/citología , Polen/crecimiento & desarrollo , Triticum/citología , Triticum/crecimiento & desarrollo , Australia , ADN de Plantas/metabolismo , Geografía , Profase Meiótica I , Polinización , Triticum/fisiologíaRESUMEN
PURPOSE: Legg-Calve-Perthes disease is a paediatric condition encompassing idiopathic osteonecrosis of the femoral head (ONFH). Preventing collapse and the need for subsequent joint replacement remains the major goal of clinical management. This exploratory study utilises a porcine model of surgically induced ONFH. METHODS: rhBMP-2 with and without zoledronic acid (ZA) was delivered by intra-osseous injection in the phase-transitioning sucrose acetate isobutyrate (SAIB) in an attempt to prevent femoral head collapse. Epiphyseal quotient (EQ) at eight weeks post-surgery was the primary outcome measure. Heterotopic ossification in the joint capsule and bisphosphonate retention in the femoral head were key secondary outcomes. RESULTS: Femoral heads with ONFH and no treatment all collapsed (3/3, EQ < 0.4, P < 0.05 compared to no ONFH). Local delivery of rhBMP-2/SAIB into the femoral head prevented collapse by EQ measurement one of four samples; however, this specimen still showed evidence of significant collapse. In contrast, the combination of local rhBMP-2 and local ZA prevented collapse in two of four samples. Confocal fluorescence microscopy showed locally dosed bisphosphonate entered and was retained in the femoral head. This group also showed strong Calcein signal, indicating new bone formation. Treatment with rhBMP-2 was associated with a limited amount of heterotrophic ossification in the joint capsules in some specimens. CONCLUSIONS: Operators reported SAIB to be an efficient way to deliver rhBMP-2 to the femoral head. These data suggest that rhBMP-2 is ineffective for preventing femoral head collapse without the addition of bisphosphonate. Further research will be required to validate the clinical efficacy of a combined local rhBMP-2/bisphosphonate approach.
Asunto(s)
Conservadores de la Densidad Ósea/administración & dosificación , Proteína Morfogenética Ósea 2/administración & dosificación , Difosfonatos/administración & dosificación , Necrosis de la Cabeza Femoral/prevención & control , Imidazoles/administración & dosificación , Enfermedad de Legg-Calve-Perthes/tratamiento farmacológico , Factor de Crecimiento Transformador beta/administración & dosificación , Animales , Portadores de Fármacos , Necrosis de la Cabeza Femoral/etiología , Inyecciones , Enfermedad de Legg-Calve-Perthes/complicaciones , Proyectos Piloto , Proteínas Recombinantes/administración & dosificación , Sacarosa/análogos & derivados , Porcinos , Ácido ZoledrónicoRESUMEN
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, mostly caused by mutations in MECP2. The disorder mainly affects girls and it is associated with severe cognitive and physical disabilities. Modeling RTT in neural and glial cell cultures and brain organoids derived from patient- or mutation-specific human induced pluripotent stem cells (iPSCs) has advanced our understanding of the pathogenesis of RTT, such as disease-causing mechanisms, disease progression, and cellular and molecular pathology enabling the identification of actionable therapeutic targets. Brain organoid models that recapitulate much of the tissue architecture and the complexity of cell types in the developing brain, offer further unprecedented opportunity for elucidating human neural development, without resorting to conventional animal models and the limited resource of human neural tissues. This review focuses on the new knowledge of RTT that has been gleaned from the iPSC-based models as well as limitations of the models and strategies to refine organoid technology in the quest for clinically relevant disease models for RTT and the broader spectrum of neurodevelopmental disorders.
RESUMEN
Low-temperature stress during microspore development alters cellular organization in rice anthers. The major cellular damage includes unusual starch accumulation in the plastids of the endothecium in postmeiotic anthers, abnormal vacuolation and hypertrophy of the tapetum, premature callose (1,3-beta-glucan) breakdown and lack of normal pollen wall formation. These cellular lesions arise from damage to critical biochemical processes that include sugar metabolism in the anthers and its use by the microspores. Failure of utilization of the callose breakdown product and other microspore wall components like sporopollenin can also be considered as critical. In recent years, considerable progress has been made in the understanding of major biochemical processes including the expression of critical genes that are sensitive to low temperature in rice and cause male sterility. This paper combines a discussion of cellular organization and associated biochemical processes that are sensitive to low temperatures and provides an overview of the potential mechanisms of low-temperature-induced male sterility in rice.
Asunto(s)
Frío , Fertilidad/fisiología , Oryza/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Metabolismo de los Hidratos de Carbono , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Oryza/citología , Oryza/genética , Oryza/metabolismo , Polen/metabolismo , Polen/ultraestructuraRESUMEN
BACKGROUND: HSV is an important cause of brain infection. Virus entry is often through breeches in the skin. γδT cells play an immunoprotective role in mice after corneal, genital or footpad (subcutaneous) HSV infection. METHODS: Here we report that the presence of γδT cells in murine skin is associated with increased severity of herpetic disease, reduced protective cytokine responses and increased viral spread from the skin to the sensory ganglia in the zosteriform model. γδT cell-deficient (TCR δ -/-) mice displayed significantly decreased herpetic lesion severity after flank HSV infection compared to WT C57BL/6 controls at both primary and secondary skin infection sites. RESULTS: Viral titer at the primary skin site was similar to WT mice in γδT cell-deficient mice, but was significantly decreased in the ganglia and secondary skin site. γδT cell-deficient mice showed increased Th1 responses by both T cells and non-T cells at the primary site, and decreased T-cell Th17 responses and immune infiltration at the secondary site. CONCLUSION: Cytokine responses of epidermal and dermal γδT cells to HSV also differed in WT mice (Th1 in epidermis, and Th17 in the dermis), suggesting a functional dichotomy between these two subsets. Our data suggest that in contrast to other mouse models of HSV infection, skinresident γδT cells promote the pathogenesis of HSV in skin.
Asunto(s)
Herpes Simple/inmunología , Piel/inmunología , Piel/virología , Linfocitos T/inmunología , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunidad , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Índice de Severidad de la Enfermedad , Piel/citología , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Bone allografts are inferior to autografts for the repair of critical-sized defects. Prior studies have suggested that bone morphogenetic protein-2 (BMP-2) can be combined with allografts to produce superior healing. We created a bioactive coating on bone allografts using polycondensed deoxyribose isobutyrate ester (PDIB) polymer to deliver BMP-2 ± the bisphosphonate zoledronic acid (ZA) and tested its ability to enhance the functional utility of allografts in preclinical Wistar rat models. One ex vivo and two in vivo proof-of-concept studies were performed. First, PDIB was shown to be able to coat bone grafts (BGs). Second, PDIB was used to coat structural allogenic corticocancellous BG with BMP-2 ± ZA ± hydroxyapatite (HA) microparticles and compared with PDIB-coated grafts in a rat muscle pouch model. Next, a rat critical defect model was performed with treatment groups including (i) empty defect, (ii) BG, (iii) collagen sponge + BMP-2, (iv) BG + PDIB/BMP-2, and (v) BG + PDIB/BMP-2/ZA. Key outcome measures included detection of fluorescent bone labels, microcomputed tomography (CT) quantification of bone, and radiographic healing. In the muscle pouch study, BMP-2 did not increase net bone volume measured by microCT, however, fluorescent labeling showed large amounts of new bone. Addition of ZA increased BV by sevenfold (p < 0.01). In the critical defect model, allografts were insufficient to promote reliable union, however, union was achieved in collagen/BMP-2 and all BG/BMP-2 groups. Statement of clinical significance: These data support the concept that PDIB is a viable delivery method for BMP-2 and ZA delivery to enhance the bone forming potential of allografts. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2278-2286, 2019.
Asunto(s)
Conservadores de la Densidad Ósea/administración & dosificación , Proteína Morfogenética Ósea 2/administración & dosificación , Trasplante Óseo , Ácido Zoledrónico/administración & dosificación , Aloinjertos , Animales , Desoxirribosa/química , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Isobutiratos/química , Masculino , Polímeros/química , Ratas WistarRESUMEN
Tumor protein D52 (TPD52) is amplified and overexpressed in breast and prostate cancers which are frequently characterised by dysregulated lipid storage and metabolism. TPD52 expression increases lipid storage in mouse 3T3 fibroblasts, and co-distributes with the Golgi marker GM130 and lipid droplets (LDs). We examined the effects of Brefeldin A (BFA), a fungal metabolite known to disrupt the Golgi structure, in TPD52-expressing 3T3 cells, and in human AU565 and HMC-1-8 breast cancer cells that endogenously express TPD52. Five-hour BFA treatment reduced median LD numbers, but increased LD sizes. TPD52 knockdown decreased both LD sizes and numbers, and blunted BFA's effects on LD numbers. Following BFA treatment for 1-3 hours, TPD52 co-localised with the trans-Golgi network protein syntaxin 6, but after 5 hours BFA treatment, TPD52 showed increased co-localisation with LDs, which was disrupted by microtubule depolymerising agent nocodazole. BFA treatment also increased perilipin (PLIN) family protein PLIN3 but reduced PLIN2 detection at LDs in TPD52-expressing 3T3 cells, with PLIN3 recruitment to LDs preceding that of TPD52. An N-terminally deleted HA-TPD52 mutant (residues 40-184) almost exclusively targeted to LDs in both vehicle and BFA treated cells. In summary, delayed recruitment of TPD52 to LDs suggests that TPD52 participates in a temporal hierarchy of LD-associated proteins that responds to altered LD packaging requirements induced by BFA treatment.
Asunto(s)
Brefeldino A/farmacología , Proteínas Asociadas a Gotas Lipídicas/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Aparato de Golgi/metabolismo , Ratones , Mutación , Proteínas de Neoplasias/genética , Perilipina-3/metabolismo , Transporte de ProteínasRESUMEN
Mitochondria are crucial for cells, supplying up to 90% of the energy requirements for neurons. Their correct localisation is crucial and ensured by a transport system. Mitochondrial trafficking in neurons is particularly critical, because mitochondria must leave the soma and travel along the axon and dendritic network to facilitate neuronal function. Abnormal mitochondrial trafficking has been reported in several neurological disorders, therefore the ability to quantify and analyse mitochondrial trafficking is vital to improving our understanding of their pathogenesis. Commercial software currently lacks an automated approach for performing such quantitation. Here we demonstrate the development of the Mitochondrial Trafficking and Distribution (MiTrakD) analysis toolset, which consists of simple and free-to-use instructions for mitochondrial trafficking analysis using time-lapse microscopy. MiTrakD utilises existing Fiji (ImageJ) tools for semi-automated, fast and efficient analysis of mitochondrial trafficking and distribution, including velocity, abundance, localisation and distance travelled in neurons. We document MiTrakD's efficiency and accuracy by analysing mitochondrial trafficking using two-dimensional fluorescence images of cortical neurons of wild type mice after 6 days (DIV6), 10 days (DIV10) and 14 days (DIV14) of in vitro incubation. Using MiTrakD we have demonstrated that neurons at all developmental stages exhibited the same percentage of mobile mitochondria, all of which travel in equidistance. Interestingly, the mitochondria in neurons at DIV10 were in greater abundance and were faster than those at DIV6 and DIV14. We can also conclude that MiTrakD is more efficient than manual analysis and is an accurate and reliable tool for performing mitochondrial trafficking analysis in neuronal cells.
Asunto(s)
Mitocondrias/química , Neuronas/química , Animales , Rastreo Celular , Células Cultivadas , Corteza Cerebelosa/química , Instrucción por Computador , RatonesRESUMEN
Cathepsin K inhibitors (CKIs) are an emerging class of drugs that are potent antagonists of osteoclastic activity. We speculated that they may be beneficial in bone tissue engineering, where a stress shielded environment can lead to rapid resorption of new bone. Most CKIs require frequent dosing, so to achieve a sustained release we manufactured polymer nanoparticles encapsulating the CKI L006235 (CKI/nP). CKI/nP and the collagen matrices that were used to deliver them were characterized by electron microscopy and fluorescent confocal microscopy, and data indicated that the particles were evenly distributed throughout the collagen. Elution studies indicated a linear release of the inhibitor from the CKI/nP, with approximately 2% of the drug being released per day. In an in vivo study, mice were implanted with collagen scaffolds containing rhBMP-2 that were loaded with the CKI/nP. Measurement of bone volume (BV) by microCT showed no significant increase with CKI/nP incorporation, and other parameters similarly showed no statistical differences. Cell culture studies confirmed the activity of the drug, even at low concentrations. These data indicate that polymer nanoparticles are an effective method for sustained drug delivery of a CKI, however, this may not be readily translatable to substantively improved bone tissue engineering outcomes. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 136-144, 2017.
Asunto(s)
Benzamidas , Proteína Morfogenética Ósea 2 , Catepsina K/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos/métodos , Nanosferas/química , Osteoclastos/metabolismo , Poliglactina 910 , Tiazoles , Animales , Benzamidas/química , Benzamidas/farmacología , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/farmacología , Humanos , Ratones , Poliglactina 910/química , Poliglactina 910/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Bisphosphonates (BP) are antiresorptive drugs with a high affinity for bone. Despite the therapeutic success in treating osteoporosis and metabolic bone diseases, chronic BP usage has been associated with reduced repair of microdamage and atypical femoral fracture (AFF). The latter has a poor prognosis, and although anabolic interventions such as teriparatide (PTH(1-34) ) have been suggested as treatment options, there is a limited evidence base in support of their efficacy. Because PTH(1-34) acts to increase bone turnover, we hypothesized that it may be able to increase BP in turnover in the skeleton, which, in turn, may improve bone healing. To test this, we employed a mixture of fluorescent Alexa647-labelled pamidronate (Pam) and radiolabeled (14) C-ZA (zoledronic acid). These traceable BPs were dosed to Wistar rats in models of normal growth and closed fracture repair. Rats were cotreated with saline or 25 µg/kg/d PTH(1-34) , and the effects on BP liberation and bone healing were examined by X-ray, micro-CT, autoradiography, and fluorescent confocal microscopy. Consistent with increased BP remobilization with PTH(1-34) , there was a significant decrease in fluorescence in both the long bones and in the fracture callus in treated animals compared with controls. This was further confirmed by autoradiography for (14) C-ZA. In this model of acute BP treatment, callus bone volume (BV) was significantly increased in fractured limbs, and although we noted significant decreases in callus-bound BP with PTH(1-34) , these were not sufficient to alter this BV. However, increased intracellular BP was noted in resorbing osteoclasts, confirming that, in principle, PTH(1-34) increases bone turnover as well as BP turnover.
Asunto(s)
Difosfonatos , Fracturas del Fémur , Curación de Fractura/efectos de los fármacos , Imidazoles , Hormona Paratiroidea , Animales , Isótopos de Carbono/farmacocinética , Isótopos de Carbono/farmacología , Difosfonatos/farmacocinética , Difosfonatos/farmacología , Fracturas del Fémur/tratamiento farmacológico , Fracturas del Fémur/metabolismo , Imidazoles/farmacocinética , Imidazoles/farmacología , Marcaje Isotópico , Masculino , Pamidronato , Hormona Paratiroidea/farmacocinética , Hormona Paratiroidea/farmacología , Ratas , Ratas Wistar , Ácido ZoledrónicoRESUMEN
An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Colágeno/química , Difosfonatos/farmacología , Sistemas de Liberación de Medicamentos , Durapatita/química , Proteínas Recombinantes/farmacología , Fosfatasa Ácida/metabolismo , Animales , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Ratones , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Porosidad , Implantación de Prótesis , Ratas , Ratas Wistar , Fosfatasa Ácida Tartratorresistente , Andamios del Tejido , Microtomografía por Rayos XRESUMEN
A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)3: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.
Asunto(s)
Dextranos/metabolismo , Organogénesis/fisiología , Scrophulariaceae/metabolismo , Transporte Biológico , Fluoresceína , Fluoresceínas , Colorantes Fluorescentes , Ácido Glutámico , Meristema/citología , Meristema/crecimiento & desarrollo , Microinyecciones , Epidermis de la Planta/citología , Epidermis de la Planta/crecimiento & desarrollo , Brotes de la Planta/citología , Brotes de la Planta/crecimiento & desarrollo , Plasmodesmos/metabolismo , Scrophulariaceae/citología , Scrophulariaceae/crecimiento & desarrollo , Técnicas de Cultivo de TejidosRESUMEN
Pre-meiotic cellular organisation of rice anthers has a great significance in pollen formation. We have used a combination of confocal laser and transmission electron microscopy (TEM) to characterise and differentiate organelles in pre-meiotic rice anthers. Along with the characteristic organelles in the cytoplasm the epidermal cells of the pre-meiotic rice anther are coated on their outer surface by a conspicuous bi-lamellate cuticle. Chloroplasts of the endothecium contain immature grana, thylakoids and also starch granules. These plastids clearly contain photosynthetic pigments as shown by autofluorescence in confocal microscope studies. Both confocal and TEM studies reveal clusters of mitochondria in the middle layer. The tapetum contains electron opaque ribosomes, bundles of mitochondria and plastids. The nuclei of the tapetum occupy a large volume of the cytoplasm indicating the onset of mitotic prophase. Intense Rhodamine 123 staining reveals that a major portion of the structurally indistinguishable organelles that were seen throughout the densely ribosomic cytoplasm of sporogenous cells are mitochondria.
Asunto(s)
Flores/ultraestructura , Oryza/ultraestructura , Epidermis de la Planta/ultraestructura , Polen/ultraestructura , Ciclo Celular , Diferenciación Celular , Núcleo Celular/ultraestructura , Pared Celular/ultraestructura , Cloroplastos/química , Cloroplastos/ultraestructura , Citoplasma/ultraestructura , Flores/citología , Flores/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Oryza/citología , Oryza/metabolismo , Pigmentos Biológicos/análisis , Epidermis de la Planta/citología , Ribosomas/ultraestructuraRESUMEN
We have used fluorescent, confocal laser and transmission electron microscopy (TEM) to examine cellular organisations, including callose (1,3-beta-glucan) behaviour, in meiotic and early post-meiotic rice anthers. These features are critical for pollen formation and provide information to better understand pollen sterility caused by abiotic stress in rice and other monocotyledonous species. Among organelles during meiosis, abundant plastids, mitochondria and nuclei of the anther cells show distinctive features. Chloroplasts in the endothecium store starch and indicate a potential for photosynthetic activity. During meiosis, the middle layer cells are markedly compressed and at the tetrad stage are either vacuolated or filled with degenerating electron-opaque organelles. Viable mitochondria, stained with Rhodamine 123, are seen in the endothecium and tapetum, but the mitochondria in the middle layer are not stained during meiosis. The radial walls of the tapetum are disorganised and degenerating, indicating the formation of a syncytium; pro-orbicules are located at the locular walls at the tetrad stage. Immunohistochemical studies show that the sporogenous cells are entirely enveloped by a thick callosic layer at early meiosis. Cell plate callose was assembled in a plane between the dyad cells. In the tetrads, however, callose formed only at the centre, showing that the tetrad microspores are not enveloped but separated by callose walls. Thick, undulating electron-opaque walls around the tetrads indicate the beginning of exinous microspore wall differentiation.