scholarly journals Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells

2018 ◽  
Vol 29 (9) ◽  
pp. 1003-1011 ◽  
Author(s):  
Jared Hennen ◽  
Cosmo A. Saunders ◽  
Joachim D. Mueller ◽  
G. W. Gant Luxton
Keyword(s):  
Nuclear Envelope ◽  
Living Cells ◽  
Protein Oligomerization ◽  
Linc Complex ◽  
Force Transmission ◽  
Fluorescence Fluctuation Spectroscopy ◽  
The Core ◽  
First Time

Linker-of-nucleoskeleton-and-cytoskeleton (LINC) complexes are conserved molecular bridges within the nuclear envelope that mediate mechanical force transmission into the nucleoplasm. The core of a LINC complex is formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. Recently, KASH proteins were shown to bind to the domain interfaces of trimeric SUN2 proteins in vitro. However, neither the existence of SUN2 trimers in living cells nor the extent to which other SUN proteins conform to this assembly state have been tested experimentally. Here we extend the application of fluorescence fluctuation spectroscopy to quantify SUN protein oligomerization in the nuclear envelopes of living cells. Using this approach, we demonstrate for the first time that SUN2 trimerizes in vivo and we demonstrate that the in vivo oligomerization of SUN1 is not limited to a trimer. In addition, we provide evidence to support the existence of potential regulators of SUN protein oligomerization in the nuclear envelope. The differential SUN protein oligomerization illustrated here suggests that SUN proteins may have evolved to form different assembly states in order to participate in diverse mechanotransduction events.

scholarly journals Identifying hetero-protein complexes in the nuclear envelope

2019 ◽  
Author(s):  
J. Hennen ◽  
K.H. Hur ◽  
J. Kohler ◽  
S.R. Karuka ◽  
I. Angert ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Protein Complexes ◽  
Living Cells ◽  
Model Systems ◽  
Linc Complex ◽  
Oligomeric State ◽  
Subcellular Compartment ◽  
Dual Color

AbstractThe nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ~40 nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes which are mediated by protein complexes within this subcellular compartment. While fluorescence fluctuation spectroscopy (FFS) is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional FFS methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton (LINC) complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE hetero-protein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies hetero-protein complexes and their stoichiometry in the NE by accounting for spectral crosstalk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the LINC complex, a hetero-protein complex composed of Klarsicht/ANC-1/SYNE homology (KASH) and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the KASH protein nesprin-2 to form a hetero-complex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE hetero-protein complexes.Statement of SignificanceProtein complexes found within the nuclear envelope (NE) play a vital role in regulating cellular functions ranging from gene expression to cellular movement. However, the assembly state of these complexes within their native environment remains poorly understood, which is compounded by a general lack of fluorescence techniques suitable for quantifying the oligomeric state of NE protein complexes. This study aims at addressing this issue by introducing dual-color time-shifted mean-segmented Q analysis as a fluorescence fluctuation method specifically designed to identify the average oligomeric state of hetero-protein complexes within the NE of living cells.

Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

2021 ◽  
Author(s):  
Lijuan Liu ◽  
Shengting Zhang ◽  
Xiaodan Zheng ◽  
Hongmei Li ◽  
Qi Chen ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Fusobacterium Nucleatum ◽  
Living Cells ◽  
First Time ◽  
Fluorescent Carbon Dots ◽  
Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

BAF is required for emerin assembly into the reforming nuclear envelope

2001 ◽  
Vol 114 (24) ◽  
pp. 4575-4585 ◽  
Author(s):  
Tokuko Haraguchi ◽  
Takako Koujin ◽  
Miriam Segura-Totten ◽  
Kenneth K. Lee ◽  
Yosuke Matsuoka ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Core Region ◽  
Lamin B ◽  
The Core ◽  
Double Stranded Dna ◽  
Recessive Form ◽  
Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

scholarly journals The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

2020 ◽  
Author(s):  
Emma Carley ◽  
Rachel K. Stewart ◽  
Abigail Zieman ◽  
Iman Jalilian ◽  
Diane. E. King ◽  
...  
Keyword(s):  
Cell Fate ◽  
Control Cell ◽  
Nuclear Lamina ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Fate Decisions

AbstractWhile the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

scholarly journals A Glance at the Nuclear Envelope Spectrin Repeat Protein 3

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Liwei Liao ◽  
Rongmei Qu ◽  
Jun Ouang ◽  
Jingxing Dai
Keyword(s):  
Nuclear Envelope ◽  
Active Role ◽  
Structural Protein ◽  
Linc Complex ◽  
Repeat Protein ◽  
Spectrin Repeat ◽  
Physiological Importance ◽  
Functional Perspective

Nuclear envelope spectrin repeat protein 3 (nesprin-3) is an evolutionarily-conserved structural protein, widely-expressed in vertebrate cells. Along with other nesprin family members, nesprin-3 acts as an essential component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Naturally, nesprin-3 shares many functions with LINC, including the localization of various cellular structures and bridging of the nucleoskeleton and cytoskeleton, observed in vitro. When nesprin-3 was knocked down in vivo, using zebrafish and mouse models, however, the animals were minimally affected. This paradoxical observation should not limit the physiological importance of nesprin-3, as recently, nesprin-3 has reignited the interest of the research community in studies on cancer cells migration. Moreover, nesprin-3 also plays an active role in certain developmental conditions such as adipogenesis and spermatogenesis, although more studies are needed. Meanwhile, the various protein binding partners of nesprin-3 should also be emphasized, as they are necessary for maintaining the structure of nesprin-3 and enabling it to carry out its various physiological and pathological functions. Nesprin-3 promises to further our understanding of these complex cellular events. Therefore, this review will focus on nesprin-3, examining it from a genetic, structural, and functional perspective. The final part of the review will in turn address the limitations of existing research and the future perspectives for the study of nesprin-3.

scholarly journals A Sequential Therapeutic Hydrogel With Injectability and Antibacterial Activity for Deep Burn Wounds’ Cleaning and Healing

2021 ◽  
Vol 9 ◽  
Author(s):  
Sizhen Wang ◽  
Jie Li ◽  
Zhiqiang Ma ◽  
Linhong Sun ◽  
Lei Hou ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Wound Dressing ◽  
Sequential Therapy ◽  
Burn Injuries ◽  
Burn Wounds ◽  
The Core ◽  
Clinical Challenge ◽  
First Time

As a severe clinical challenge, escharotomy and infection are always the core concerns of deep burn injuries. However, a usual dressing without multifunctionality leads to intractable treatment on deep burn wounds. Herein, we fabricated a sequential therapeutic hydrogel to solve this problem. Cross-linked by modified polyvinyl alcohol (PVA-SH/ε-PL) and benzaldehyde-terminated F127 triblock copolymers (PF127-CHO), the hydrogel demonstrated excellent mechanical properties, injectability, tissue adhesiveness, antibacterial activity, biocompatibility, and satisfactory wound cleaning through both in vitro and in vivo assays. Additionally, based on the conception of “sequential therapy,” we proposed for the first time to load bromelain and EGF into the same hydrogel in stages for wound cleaning and healing. This work provides a strategy to fabricate a promising wound dressing for the treatment of deep burn wounds with injectability and improved patients’ compliance as it simplified the process of treatment due to its “three in one” characteristic (antibacterial activity, wound cleaning, and healing effects); therefore, it has great potential in wound dressing development and clinical application.

scholarly journals The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Emma Carley ◽  
Rachel Stewart ◽  
Abigail G Zieman ◽  
Iman Jalilian ◽  
Diane E King ◽  
...  
Keyword(s):  
Cell Fate ◽  
Control Cell ◽  
Nuclear Lamina ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Fate Decisions

While the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

scholarly journals Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

2014 ◽  
Vol 13 (9) ◽  
pp. 1222-1231 ◽  
Author(s):  
Patrick C. Thiaville ◽  
Basma El Yacoubi ◽  
Ludovic Perrochia ◽  
Arnaud Hecker ◽  
Magali Prigent ◽  
...  
Keyword(s):  
Mitochondrial Pathway ◽  
Stem Loop ◽  
Content Type ◽  
E Coli ◽  
Functional Conservation ◽  
The Core ◽  
First Time ◽  
Keops Complex

ABSTRACT Threonylcarbamoyladenosine (t 6 A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t 6 A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t 6 A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, http://dx.doi.org/10.1093/nar/gkt322 ). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t 6 A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t 6 A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli . The data revealed a potential for TC-AMP channeling in the t 6 A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t 6 A and bring additional advancement in our understanding of the reaction mechanism.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

scholarly journals Fe3O4@Pt nanoparticles to enable combinational electrodynamic/chemodynamic therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tong Chen ◽  
Qiang Chu ◽  
Mengyang Li ◽  
Gaorong Han ◽  
Xiang Li
Keyword(s):  
Fenton Reaction ◽  
Pt Nanoparticles ◽  
Therapeutic Modality ◽  
Ros Generation ◽  
Tumor Treatment ◽  
Superior Efficacy ◽  
Platinum Nanocrystals ◽  
First Time

AbstractElectrodynamic therapy (EDT) has recently emerged as a potential external field responsive approach for tumor treatment. While it presents a number of clear superiorities, EDT inherits the intrinsic challenges of current reactive oxygen species (ROS) based therapeutic treatments owing to the complex tumor microenvironment, including glutathione (GSH) overexpression, acidity and others. Herein for the first time, iron oxide nanoparticles are decorated using platinum nanocrystals (Fe3O4@Pt NPs) to integrate the current EDT with chemodynamic phenomenon and GSH depletion. Fe3O4@Pt NPs can effectively induce ROS generation based on the catalytic reaction on the surface of Pt nanoparticles triggered by electric field (E), and meanwhile it may catalyze intracellular H2O2 into ROS via Fenton reaction. In addition, Fe3+ ions released from Fe3O4@Pt NPs under the acidic condition in tumor cells consume GSH in a rapid fashion, inhibiting ROS clearance to enhance its antitumor efficacy. As a result, considerable in vitro and in vivo tumor inhibition phenomena are observed. This study has demonstrated an alternative concept of combinational therapeutic modality with superior efficacy.

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