RESUMO
RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.
Assuntos
Quinases Ciclina-Dependentes , Fosfoproteínas , Precursores de RNA , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Cromatina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Quinolonas/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/efeitos dos fármacos , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Treonina/metabolismoRESUMO
Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.
Assuntos
Antineoplásicos , Quinase 1 do Ponto de Checagem , Cisplatino , Neoplasias da Próstata , Ligante Indutor de Apoptose Relacionado a TNF , Humanos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Compostos Organoplatínicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacosRESUMO
We report flexible synthesis of new unsymmetrically 2,6-disubstituted benzoquinones (33 examples) and a systematic study of their reactivity in the Diels-Alder reaction. The Diels-Alder reactions of selected unsymmetrical benzoquinones with seemingly similar substituents were found to proceed with high regioselectivity and the formation of selected experimentally observed main products was rationalized by theoretical (DFT) calculations. The findings can be exploited in the convenient preparation of densely substituted and stereochemically defined decalins with unique angular substituents at ring fusion. We also demonstrate the usefulness of this methodology in complex molecule synthesis through the total synthesis of a novel forskolin analog possessing an ethyl group at the fusion of the rings B and C.
RESUMO
We report a new synthetic strategy for the flexible preparation of forskolin-like molecules. The approach is different from the previously published works and employs a convergent assembly of the tricyclic labdane-type core from pre-functionalized cyclic building blocks. Stereoselective Michael addition enabled the fragment coupling with excellent control over three newly created contiguous stereocenters, all-carbon quaternary centers included. Silyl enol ether-promoted ring-opening metathesis paired with ring closure were the other key steps enabling concise assembly of the tricyclic core. Late-stage functionalization sequences transformed the tricyclic intermediates into a set of different forskolin-like molecules. The modular nature of the synthetic scheme described herein has the potential to become a general platform for the preparation of analogs of forskolin and other complex tricyclic labdanes.
Assuntos
Diterpenos , Colforsina , Estereoisomerismo , ÉteresRESUMO
Casein kinases 1 (CK1) are key signaling molecules that have emerged recently as attractive therapeutic targets in particular for the treatment of hematological malignancies. Herein, we report the identification of a new class of potent and highly selective inhibitors of CK1α, δ and ϵ. Based on their optimal in vitro and in vivo profiles and their exclusive selectivity, MU1250, MU1500 and MU1742 were selected as quality chemical probes for those CK1 isoforms. At proper concentrations, MU1250 and MU1500 allow for specific targeting of CK1δ or dual inhibition of CK1δ/ϵ in cells. The compound MU1742 also efficiently inhibits CK1α and, to our knowledge, represents the first potent and highly selective inhibitor of this enzyme. In addition, we demonstrate that the central 1H-pyrrolo[2,3-b]pyridine-imidazole pharmacophore can be used as the basis of highly selective inhibitors of other therapeutically relevant protein kinases, e.g. p38α, as exemplified by the compound MU1299.
Assuntos
Caseína Quinase I , Transdução de Sinais , Caseína Quinase I/metabolismo , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/química , HumanosRESUMO
CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Células HCT116 , Humanos , Fosforilação , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect FriedreichÌs ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.
Assuntos
Cinetina/farmacologia , Purinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoproteção , Relação Dose-Resposta a Droga , Humanos , Cinetina/síntese química , Cinetina/química , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Purinas/síntese química , Purinas/química , Relação Estrutura-AtividadeRESUMO
Protein kinases represent a very pharmacologically attractive class of targets; however, some members of the family still remain rather unexplored. The biology and therapeutic potential of cdc-like kinases (CLKs) have been explored mainly over the last decade and the first CLK inhibitor, compound SM08502, entered clinical trials only recently. This review summarizes the biological roles and therapeutic potential of CLKs and their heretofore published small-molecule inhibitors, with a focus on the compounds' potential to be utilized as quality chemical biology probes.
Assuntos
Antineoplásicos/farmacologia , Carcinogênese/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genéticaRESUMO
Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.
Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mutação , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Reported is the identification of the furo[3,2-b]pyridine core as a novel scaffold for potent and highly selective inhibitors of cdc-like kinases (CLKs) and efficient modulators of the Hedgehog signaling pathway. Initially, a diverse target compound set was prepared by synthetic sequences based on chemoselective metal-mediated couplings, including assembly of the furo[3,2-b]pyridine scaffold by copper-mediated oxidative cyclization. Optimization of the subseries containing 3,5-disubstituted furo[3,2-b]pyridines afforded potent, cell-active, and highly selective inhibitors of CLKs. Profiling of the kinase-inactive subset of 3,5,7-trisubstituted furo[3,2-b]pyridines revealed sub-micromolar modulators of the Hedgehog pathway.
Assuntos
Furanos/química , Proteínas Hedgehog/química , Inibidores de Proteínas Quinases/síntese química , Piridinas/química , Bibliotecas de Moléculas Pequenas/síntese química , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Células MCF-7 , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
3,4-Substituted-5-aminopyrazoles and 4-substituted-2-aminothiazoles are frequently used intermediates in medicinal chemistry and drug discovery projects. We report an expedient flexible synthesis of 3,4-substituted-5-aminopyrazoles (35 examples), based on palladium-mediated α-arylation of ß-ketonitriles with aryl bromides. A library of 4-substituted-2-aminothiazoles (21 examples) was assembled by a sequence employing Suzuki coupling of newly prepared, properly protected pinacol ester and MIDA ester of 4-boronic acid-2-aminothiazole with (hetero)aryl halides.
RESUMO
A broad spectrum of tumors develop resistance to classic chemotherapy, necessitating the discovery of new therapies. One successful strategy exploits the synthetic lethality between poly(ADP-ribose) polymerase 1/2 proteins and DNA damage response genes, including BRCA1, a factor involved in homologous recombination-mediated DNA repair, and CDK12, a transcriptional kinase known to regulate the expression of DDR genes. CHK1 inhibitors have been shown to enhance the anti-cancer effect of DNA-damaging compounds. Since loss of BRCA1 increases replication stress and leads to DNA damage, we tested a hypothesis that CDK12- or BRCA1-depleted cells rely extensively on S-phase-related CHK1 functions for survival. The silencing of BRCA1 or CDK12 sensitized tumor cells to CHK1 inhibitors in vitro and in vivo. BRCA1 downregulation combined with CHK1 inhibition induced excessive amounts of DNA damage, resulting in an inability to complete the S-phase. Therefore, we suggest CHK1 inhibition as a strategy for targeting BRCA1- or CDK12-deficient tumors.
Assuntos
Proteína BRCA1/genética , Quinase 1 do Ponto de Checagem/genética , Neoplasias Colorretais/genética , Quinases Ciclina-Dependentes/genética , Animais , Proteína BRCA1/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Células HCT116 , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1/genética , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The structural basis for the interaction of roscovitine and analogues containing 13 different bioisosteric central heterocycles with the enzyme cyclin-dependent kinaseâ 2 (CDK2) is elucidated. Although all the central scaffolds are very similar to the purine core of roscovitine, the experimentally determined IC50 values of the inhibitors span three orders of magnitude. By using an extensive computational chemistry approach, the affinities of the inhibitors to CDK2 are determined as calculated binding scores of complexes of the inhibitors with the protein. The interactions of the inhibitors with CDK2 are computationally described by using a hybrid quantum mechanics/semi-empirical quantum mechanics method (QM/SQM), which combines the DFT-D method for the QM part and the PM6-D3H4X method for the SQM part. The solvent effect is described by the COSMO implicit solvation model at the SQM level for the whole system. The contributions of the scaffolds and the individual substituents, quantified and evaluated in relation to conformations of optimized protein-inhibitor complexes, are found not to be simply additive. The inhibitory activity of the selected candidates, including two newly prepared compounds, is tested against CDK2. The results of the calculations are in close agreement with the experimental data.
Assuntos
Quinase 2 Dependente de Ciclina/química , Compostos Heterocíclicos/química , Inibidores de Proteínas Quinases/química , Purinas/química , Teoria Quântica , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Compostos Heterocíclicos/farmacologia , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Roscovitina , Eletricidade Estática , TermodinâmicaRESUMO
Carbocyclic C-nucleosides are quite rare. Our route enables flexible preparation of three classes of these nucleoside analogs from common precursors-properly substituted cyclopentanones, which can be prepared racemic (in six steps) or optically pure (in ten steps) from inexpensive norbornadiene. The methodology allows flexible manipulation of individual positions around the cyclopentane ring, namely highly diastereoselective installation of carbo- and heterocyclic substituents at position 1', orthogonal functionalization of position 5', and efficient inversion of stereochemistry at position 2'. Newly prepared carbocyclic C-analog of tubercidine, profiled in MCF7 (breast cancer) and HFF1 (human foreskin fibroblasts) cell cultures, is less potent than tubercidine itself, but more selectively toxic toward the tumorigenic cells.
Assuntos
Ciclopentanos/farmacologia , Nucleosídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclopentanos/síntese química , Ciclopentanos/química , Humanos , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , EstereoisomerismoRESUMO
A 24-step synthesis of (±)-forskolin is presented, which delivered hundred milligram quantities of this complex diterpene in one pass. Transformations key to our approach include: a)â a strategic allylic transposition, b)â stepwise assembly of a sterically encumbered isoxazole ring, and c)â citric acid-modified Upjohn dihydroxylation of a resilient tetrasubstituted olefin. The developed route has exciting potential for the preparation of new forskolin analogues inaccessible by semisynthesis.
RESUMO
Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.
Assuntos
Aminoácidos/química , Fluorescência , Proteínas/química , Água/análise , Simulação de Dinâmica Molecular , Estrutura Molecular , Água/químicaRESUMO
Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.
Assuntos
Receptores de Activinas Tipo II , Animais , Humanos , Camundongos , Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo II/antagonistas & inibidores , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacos , Descoberta de Drogas , Sondas Moleculares/química , Proteínas Morfogenéticas Ósseas/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese químicaRESUMO
The synthesis and hit-to-lead SAR development from a pyrazolo[1,5-a]pyrimidine-derived hit 5 to the identification of a series of potent, pan-Pim inhibitors such as 11j are described.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-pim-1/química , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/síntese química , Relação Estrutura-AtividadeRESUMO
KEY MESSAGE: ClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds. Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , Plantas , Sementes/metabolismo , Xilitol/metabolismoRESUMO
Many dynamic interactions within the cell microenvironment modulate cell behavior and cell fate. However, the pathways and mechanisms behind cell-cell or cell-extracellular matrix interactions remain understudied, as they occur at a nanoscale level. Recent progress in nanotechnology allows for mimicking of the microenvironment at nanoscale in vitro; electron-beam lithography (EBL) is currently the most promising technique. Although this nanopatterning technique can generate nanostructures of good quality and resolution, it has resulted, thus far, in the production of only simple shapes (e.g., rectangles) over a relatively small area (100 × 100 µm), leaving its potential in biological applications unfulfilled. Here, we used EBL for cell-interaction studies by coating cell-culture-relevant material with electron-conductive indium tin oxide, which formed nanopatterns of complex nanohexagonal structures over a large area (500 × 500 µm). We confirmed the potential of EBL for use in cell-interaction studies by analyzing specific cell responses toward differentially distributed nanohexagons spaced at 1000, 500, and 250 nm. We found that our optimized technique of EBL with HaloTags enabled the investigation of broad changes to a cell-culture-relevant surface and can provide an understanding of cellular signaling mechanisms at a single-molecule level.