scholarly journals Reconstitution defines the roles of p62, NBR1 and TAX1BP1 in ubiquitin condensate formation and autophagy initiation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Eleonora Turco ◽  
Adriana Savova ◽  
Flora Gere ◽  
Luca Ferrari ◽  
Julia Romanov ◽  
...  
Keyword(s):  
Cell Biology ◽  
Selective Autophagy ◽  
Ubiquitinated Proteins ◽  
High Affinity ◽  
In Vitro Reconstitution ◽  
Main Driver ◽  
Condensate Formation ◽  
Uba Domain ◽  
Autophagic Degradation

AbstractThe autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62–ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.

scholarly journals NBR1 directly promotes the formation of p62 – ubiquitin condensates via its PB1 and UBA domains

2020 ◽  
Author(s):  
Adriana Savova ◽  
Julia Romanov ◽  
Sascha Martens
Keyword(s):  
Phase Separation ◽  
Wild Type ◽  
Selective Autophagy ◽  
Ubiquitinated Proteins ◽  
Cargo Receptor ◽  
Condensate Formation ◽  
Uba Domain ◽  
Reconstituted System ◽  
Pb1 Domain

SummarySelective autophagy removes harmful intracellular structures such as ubiquitinated, aggregated proteins ensuring cellular homeostasis. This is achieved by the encapsulation of this cargo material within autophagosomes. The cargo receptor p62/SQSTM1 mediates the phase separation of ubiquitinated proteins into condensates, which subsequently become targets for the autophagy machinery. NBR1, another cargo receptor, is a crucial regulator of condensate formation. The mechanisms of the interplay between p62 and NBR1 are not well understood. Employing a fully reconstituted system we show that two domains of NBR1, the PB1 domain which binds to p62 and the UBA domain which binds to ubiquitin, are required to promote p62-ubiquitin condensate formation. In cells, acute depletion of endogenous NBR1 reduces formation of p62 condensates, a phenotype that can be rescued by re-expression of wild-type NBR1, but not PB1 or UBA domain mutants. Our results provide mechanistic insights into the role of NBR1 in selective autophagy.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

2022 ◽  
Author(s):  
Karim Labib ◽  
Ryo Fujisawa
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Eukaryotic Cell ◽  
Ubiquitin Chain ◽  
Ubiquitin Ligase Activity ◽  
Uba Domain ◽  
Quality Control Mechanism ◽  
Ubiquitin Receptors

The unfolding of ubiquitylated proteins by the p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 is essential in many areas of eukaryotic cell biology. Previous studies showed that yeast Cdc48-Ufd1-Npl4 is governed by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that substrate processing by mammalian p97-UFD1-NPL4 involves a complex interplay between ubiquitin chain length and additional p97 cofactors. Using disassembly of the ubiquitylated CMG helicase as a model in vitro system, we find that reconstituted p97-UFD1-NPL4 only unfolds substrates with very long ubiquitin chains. However, this high ubiquitin threshold is greatly reduced, to a level resembling yeast Cdc48-Ufd1-Npl4, by the UBXN7, FAF1 or FAF2 partners of mammalian p97-UFD1-NPL4. Stimulation by UBXN7/FAF1/FAF2 requires the UBX domain that connects each factor to p97, together with the ubiquitin-binding UBA domain of UBXN7 and a previously uncharacterised coiled-coil domain in FAF1/FAF2. Furthermore, we show that deletion of the UBXN7 and FAF1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

In vitro reconstitution of germ cell development†

2019 ◽  
Vol 101 (3) ◽  
pp. 567-578 ◽  
Author(s):  
Katsuhiko Hayashi
Keyword(s):  
Germ Cell ◽  
Cell Biology ◽  
New Technology ◽  
Rapid Development ◽  
Cell Development ◽  
Alternative Source ◽  
Germ Cell Development ◽  
In Vitro Reconstitution ◽  
Key Issues

Abstract Germ cell development is a series of highly specialized processes through which diploid pluripotent cells differentiate into haploid gametes. The processes include biologically important events such as epigenetic reprogramming, sex determination, and meiosis. The mechanisms underlying these events are key issues in reproductive and developmental biology, yet they still remain elusive. As a tool to elucidate these mechanisms, in vitro gametogenesis, which reproduces germ cell development in culture, has long been sought for decades. Recently, methods of in vitro gametogenesis have undergone rapid development in association with stem cell biology, opening many possibilities in this field. This new technology is considered an alternative source of gametes for the reproduction of animals and perhaps humans. This review summarizes current advances and problems in in vitro gametogenesis.

scholarly journals Using quantitative reconstitution to investigate multi-component condensates

RNA ◽  
2021 ◽  
pp. rna.079008.121
Author(s):  
Simon L Currie ◽  
Michael K Rosen
Keyword(s):  
Stress Granules ◽  
Lessons Learned ◽  
Sequencing Data ◽  
Processing Bodies ◽  
P Bodies ◽  
Multiple Components ◽  
In Vitro Reconstitution ◽  
Condensate Formation ◽  
Liquid Liquid Phase Separation

Many biomolecular condensates are thought to form via liquid-liquid phase separation (LLPS) of multivalent macromolecules. For those that form through this mechanism, our understanding has benefitted significantly from biochemical reconstitutions of key components and activities. Reconstitutions of RNA-based condensates to date have mostly been based on relatively simple collections of molecules. However, proteomics and sequencing data indicate that natural RNA-based condensates are enriched in hundreds to thousands of different components, and genetic data suggest multiple interactions can contribute to condensate formation to varying degrees. In this perspective we describe recent progress in understanding RNA-based condensates through different levels of biochemical reconstitutions, as a means to bridge the gap between simple in vitro reconstitution and cellular analyses. Complex reconstitutions provide insight into the formation, regulation, and functions of multi-component condensates. We focus on two RNA-protein condensate case studies: stress granules and RNA processing bodies (P bodies), and examine the evidence for cooperative interactions among multiple components promoting LLPS. An important concept emerging from these studies is that composition and stoichiometry regulate biochemical activities within condensates. Based on the lessons learned from stress granules and P bodies we discuss forward-looking approaches to understand the thermodynamic relationships between condensate components, with the goal of developing predictive models of composition and material properties, and their effects on biochemical activities. We anticipate that quantitative reconstitutions will facilitate understanding of the complex thermodynamics and functions of diverse RNA-protein condensates.

scholarly journals Fibrinogen anchors for micropatterning of active proteins and subcellular receptor relocalisation

2020 ◽  
Author(s):  
Joseph L. Watson ◽  
Samya Aich ◽  
Benjamí Oller Salvia ◽  
Andrew A. Drabek ◽  
Stephen C. Blacklow ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Cell Shape ◽  
Control Cell ◽  
High Specificity ◽  
Transmembrane Receptors ◽  
Current Technology ◽  
In Vitro Reconstitution ◽  
General Method

AbstractProtein micropatterning allows proteins to be precisely deposited onto a substrate of choice, and is now routinely used in cell biology and in vitro reconstitution. However, a drawback of current technology is that micropatterning efficiency can be variable between proteins, and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, Fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard to micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

scholarly journals High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Joseph L. Watson ◽  
Samya Aich ◽  
Benjamí Oller-Salvia ◽  
Andrew A. Drabek ◽  
Stephen C. Blacklow ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Cell Shape ◽  
Control Cell ◽  
High Specificity ◽  
High Efficacy ◽  
Transmembrane Receptors ◽  
In Vitro Reconstitution ◽  
General Method

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

Correlative microscopy in distrubuted (client/server) computing environments: tools for catalyzing the rapid dissemination of information about the cell biology of the human prostate

1995 ◽  
Vol 53 ◽  
pp. 682-683
Author(s):  
A. Hakam ◽  
J.T. Gau ◽  
M.L. Grove ◽  
B.A. Evans ◽  
M. Shuman ◽  
...  
Keyword(s):  
United States ◽  
Cell Biology ◽  
Tissue Bank ◽  
The United States ◽  
Prostate Adenocarcinoma ◽  
Correlative Microscopy ◽  
Client Server ◽  
Critical Elements ◽  
Transplant Organ

Prostate adenocarcinoma is the most common malignant tumor of men in the United States and is the third leading cause of death in men. Despite attempts at early detection, there will be 244,000 new cases and 44,000 deaths from the disease in the United States in 1995. Therapeutic progress against this disease is hindered by an incomplete understanding of prostate epithelial cell biology, the availability of human tissues for in vitro experimentation, slow dissemination of information between prostate cancer research teams and the increasing pressure to “ stretch” research dollars at the same time staff reductions are occurring.To meet these challenges, we have used the correlative microscopy (CM) and client/server (C/S) computing to increase productivity while decreasing costs. Critical elements of our program are as follows:1) Establishing the Western Pennsylvania Genitourinary (GU) Tissue Bank which includes >100 prostates from patients with prostate adenocarcinoma as well as >20 normal prostates from transplant organ donors.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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