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Cell Structure and Function Jun 2024In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation...
In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.Key words: nuclear envelope reassembly, inner nuclear membrane protein, nuclear pore complex, semi-intact cell, in vitro reconstitution.
PubMed: 38839376
DOI: 10.1247/csf.24003 -
Plant Methods Jun 2024PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin...
PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.
PubMed: 38825682
DOI: 10.1186/s13007-024-01182-7 -
SLAS Discovery : Advancing Life... May 2024We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of...
We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC values between 100 nM and 1 μM in cells, which align with the EC values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.
PubMed: 38797286
DOI: 10.1016/j.slasd.2024.100165 -
Cells Apr 2024The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their...
The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.
Topics: Animals; Mice; NIH 3T3 Cells; Nanoparticles; RAW 264.7 Cells; Cell Survival; Holography; Quantitative Phase Imaging
PubMed: 38667312
DOI: 10.3390/cells13080697 -
ACS Applied Materials & Interfaces Apr 2024Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require...
Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.
PubMed: 38666624
DOI: 10.1021/acsami.4c03013 -
Methods in Molecular Biology (Clifton,... 2024Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is...
Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.
Topics: Immunohistochemistry; Antibodies; Cerebral Cortex; Growth Cones; Neurons
PubMed: 38630229
DOI: 10.1007/978-1-0716-3810-1_15 -
Veterinary Microbiology Apr 2024This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a...
This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) KKGKSK and nuclear export signal (NES) LIITSYL TI of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.
Topics: Animals; Active Transport, Cell Nucleus; Cell Nucleus; Chromosomes; Cytoplasm; Lamin Type A; Nuclear Localization Signals; Ephemeral Fever Virus, Bovine; Viral Structural Proteins
PubMed: 38364467
DOI: 10.1016/j.vetmic.2024.110026 -
STAR Protocols Mar 2024Cleavage under targets & release using nuclease (CUT&RUN) is a technique for identifying genomic sites where proteins or histone modifications are present in chromatin...
Cleavage under targets & release using nuclease (CUT&RUN) is a technique for identifying genomic sites where proteins or histone modifications are present in chromatin in permeabilized cells. Here, we present a fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components, for efficient cell permeabilization and retention of target epitopes on chromatin. We describe steps for capturing cells on concanavalin A beads and using a fluorescently labeled secondary antibody to titrate concentrations of digitonin and NaCl in CUT&RUN buffers. We then detail procedures for fluorescence imaging to identify optimal conditions. For complete details on the use and execution of this protocol, please refer to Lerner et al..
Topics: Chromatin; Antibodies; Endonucleases; Epitopes; Genomics
PubMed: 38329880
DOI: 10.1016/j.xpro.2024.102866 -
Toxicology Mechanisms and Methods Jun 2024In a previous study, diethylstilbestrol (DES) was shown to induce oocyte maturation in fish. In the present study, the interaction of DES on goldfish membrane...
BACKGROUND
In a previous study, diethylstilbestrol (DES) was shown to induce oocyte maturation in fish. In the present study, the interaction of DES on goldfish membrane progesterone receptor α (GmPRα) was investigated using a competitive binding assay with radiolabeled steroids. The results indicate that DES exerts its effects on membrane progesterone receptor alpha (mPRα) and induces oocyte maturation through nongenomic steroid mechanisms. This study provides empirical data that demonstrate the binding between DES and GmPRα.
METHODS
Binding of DES to GmPRα was achieved by using radiolabeled DES and recombinant GmPRα expressed in culture cells or purified GmPRα proteins that coupled to graphene quantum dots (GQDs). Additionally, the competitive binding of fluorescently labeled progesterone to GmPRα-expressing cells was evaluated.
RESULTS
Although significant nonspecific binding of radiolabeled DES to the cell membrane that expresses GmPRα has been observed, specific binding of DES to GmPRα has been successfully identified in the presence of digitonin. Furthermore, the specific binding of DES to GmPRα was confirmed by a binding assay using GQD-GmPRα. The radiolabeled DES was shown to bind to GQD-GmPRα. Additionally, the competition for the binding of fluorescently labeled progesterone to GmPRα-expressing cells was achieved with the DES.
CONCLUSIONS
The results of the experiments revealed that DES binds to GmPRα. Thus, it can be concluded that DES induces goldfish oocyte maturation by binding to GmPRα.
Topics: Animals; Binding, Competitive; Cell Membrane; Diethylstilbestrol; Fish Proteins; Goldfish; Oocytes; Progesterone; Protein Binding; Receptors, Progesterone
PubMed: 38317456
DOI: 10.1080/15376516.2024.2311185 -
Frontiers in Molecular Biosciences 2023TMEM16 family proteins are involved in a variety of functions, including ion transport, phospholipid scrambling, and the regulation of membrane proteins. Among them,...
TMEM16 family proteins are involved in a variety of functions, including ion transport, phospholipid scrambling, and the regulation of membrane proteins. Among them, TMEM16F has dual functions as a phospholipid scramblase and a nonselective ion channel. TMEM16F is widely expressed and functions in platelet activation during blood clotting, bone formation, and T cell activation. Despite the functional importance of TMEM16F, the modulators of TMEM16F function have not been sufficiently studied. In this study, we generated TMEM16F-specific affibodies by performing phage display with brain-specific TMEM16F (hTMEM16F) variant 1 purified from GnTi cells expressing this variant in the presence of digitonin as a detergent. Purified human TMEM16F protein, which was proficient in transporting phospholipids in a Ca-dependent manner in proteoliposomes, was coated onto plates and then the phage library was added to fish out TMEM16F-binding affibodies. For the validation of interaction between affibodies and TMEM16F proteins, ELISA, bio-layer interferometry, and size exclusion chromatography were conducted. As a result, the full sequences of 38 candidates were acquired from 98 binding candidates. Then, we selected 10 candidates and purified seven of them from expressing these candidates. Using various assays, we confirmed that two affibodies bound to human TMEM16F with high affinity. These affibodies can be useful for therapeutical and diagnostic applications of TMEM16F-related cancer and neurodegenerative diseases. Future studies will be required to investigate the effects of these affibodies on TMEM16F function.
PubMed: 38274091
DOI: 10.3389/fmolb.2023.1319251