scholarly journals Current problems and future avenues in proteoliposome research

2020 ◽  
Vol 48 (4) ◽  
pp. 1473-1492 ◽  
Author(s):  
Andrea Marco Amati ◽  
Simone Graf ◽  
Sabina Deutschmann ◽  
Nicolas Dolder ◽  
Christoph von Ballmoos
Keyword(s):  
Lipid Bilayers ◽  
Fluorescent Dyes ◽  
Experimental System ◽  
High Temporal Resolution ◽  
Cellular Functions ◽  
Functional Studies ◽  
Extraction And Purification ◽  
Vectorial Function ◽  
Enzymatic Cascades ◽  
Cellular Context

Membrane proteins (MPs) are the gatekeepers between different biological compartments separated by lipid bilayers. Being receptors, channels, transporters, or primary pumps, they fulfill a wide variety of cellular functions and their importance is reflected in the increasing number of drugs that target MPs. Functional studies of MPs within a native cellular context, however, is difficult due to the innate complexity of the densely packed membranes. Over the past decades, detergent-based extraction and purification of MPs and their reconstitution into lipid mimetic systems has been a very powerful tool to simplify the experimental system. In this review, we focus on proteoliposomes that have become an indispensable experimental system for enzymes with a vectorial function, including many of the here described energy transducing MPs. We first address long standing questions on the difficulty of successful reconstitution and controlled orientation of MPs into liposomes. A special emphasis is given on coreconstitution of several MPs into the same bilayer. Second, we discuss recent progress in the development of fluorescent dyes that offer sensitive detection with high temporal resolution. Finally, we briefly cover the use of giant unilamellar vesicles for the investigation of complex enzymatic cascades, a very promising experimental tool considering our increasing knowledge of the interplay of different cellular components.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
Vector System ◽  
In Planta ◽  
Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals Remote control of neural function by X-ray-induced scintillation

2019 ◽  
Author(s):  
Takanori Matsubara ◽  
Takayuki Yanagida ◽  
Noriaki Kawaguchi ◽  
Takashi Nakano ◽  
Junichiro Yoshimoto ◽  
...  
Keyword(s):  
Dopamine Neurons ◽  
Neural Function ◽  
X Rays ◽  
Cellular Functions ◽  
X Ray ◽  
Functional Studies ◽  
Clinical Dose ◽  
Visible Luminescence ◽  
Midbrain Dopamine ◽  
The Brain

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), could effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles were non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level was sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables less invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

scholarly journals A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

2011 ◽  
Vol 437 (2) ◽  
pp. 269-278 ◽  
Author(s):  
José R. Pérez-Castiñeira ◽  
Agustín Hernández ◽  
Rocío Drake ◽  
Aurelio Serrano
Keyword(s):  
Fluorescent Dyes ◽  
Experimental System ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Inorganic Pyrophosphatase ◽  
Proton Pumps ◽  
Specific Class ◽  
Ph Gradients ◽  
Atpase Subunit ◽  
Relative Contribution

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

scholarly journals New Tetrahymena basal body protein components identify basal body domain structure

2007 ◽  
Vol 178 (6) ◽  
pp. 905-912 ◽  
Author(s):  
Chandra L. Kilburn ◽  
Chad G. Pearson ◽  
Edwin P. Romijn ◽  
Janet B. Meehl ◽  
Thomas H. Giddings ◽  
...  
Keyword(s):  
Basal Body ◽  
Ultrastructural Level ◽  
Cellular Functions ◽  
Structural Domains ◽  
Body Protein ◽  
Functional Studies ◽  
Specific Localization ◽  
Body Components ◽  
Protein Components ◽  
Molecular Components

Basal bodies organize the nine doublet microtubules found in cilia. Cilia are required for a variety of cellular functions, including motility and sensing stimuli. Understanding this biochemically complex organelle requires an inventory of the molecular components and the contribution each makes to the overall structure. We define a basal body proteome and determine the specific localization of basal body components in the ciliated protozoan Tetrahymena thermophila. Using a biochemical, bioinformatic, and genetic approach, we identify 97 known and candidate basal body proteins. 24 novel T. thermophila basal body proteins were identified, 19 of which were localized to the ultrastructural level, as seen by immunoelectron microscopy. Importantly, we find proteins from several structural domains within the basal body, allowing us to reveal how each component contributes to the overall organization. Thus, we present a high resolution localization map of basal body structure highlighting important new components for future functional studies.

scholarly journals NAA10 p.(N101K) disrupts N-terminal acetyltransferase complex NatA and is associated with developmental delay and hemihypertrophy

2020 ◽  
Author(s):  
Nina McTiernan ◽  
◽  
Harinder Gill ◽  
Carlos E. Prada ◽  
Harry Pachajoa ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Developmental Delay ◽  
Genetic Disorders ◽  
Cellular Functions ◽  
Functional Studies ◽  
Phenotypic Spectrum ◽  
Independent Functions ◽  
Evolutionarily Conserved ◽  
Human Proteins ◽  
The Impact

Abstract Nearly half of all human proteins are acetylated at their N-termini by the NatA N-terminal acetyltransferase complex. NAA10 is evolutionarily conserved as the catalytic subunit of NatA in complex with NAA15, but may also have NatA-independent functions. Several NAA10 variants are associated with genetic disorders. The phenotypic spectrum includes developmental delay, intellectual disability, and cardiac abnormalities. Here, we have identified the previously undescribed NAA10 c.303C>A and c.303C>G p.(N101K) variants in two unrelated girls. These girls have developmental delay, but they both also display hemihypertrophy a feature normally not observed or registered among these cases. Functional studies revealed that NAA10 p.(N101K) is completely impaired in its ability to bind NAA15 and to form an enzymatically active NatA complex. In contrast, the integrity of NAA10 p.(N101K) as a monomeric acetyltransferase is intact. Thus, this NAA10 variant may represent the best example of the impact of NatA mediated N-terminal acetylation, isolated from other potential NAA10-mediated cellular functions and may provide important insights into the phenotypes observed in individuals expressing pathogenic NAA10 variants.

scholarly journals Transcriptomic, proteomic, and metabolomic landscape of positional memory in the caudal fin of zebrafish

2017 ◽  
Vol 114 (5) ◽  
pp. E717-E726 ◽  
Author(s):  
Jeremy S. Rabinowitz ◽  
Aaron M. Robitaille ◽  
Yuliang Wang ◽  
Catherine A. Ray ◽  
Ryan Thummel ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Amino Acid ◽  
Differential Expression ◽  
Spatial Information ◽  
Expression Patterns ◽  
Spatial Position ◽  
Adult Zebrafish ◽  
Cellular Functions ◽  
Caudal Fin ◽  
Functional Studies

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.

scholarly journals Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

2015 ◽  
Vol 112 (41) ◽  
pp. 12687-12692 ◽  
Author(s):  
Xiaorui Chen ◽  
Fengyun Ni ◽  
Elena Kondrashkina ◽  
Jianpeng Ma ◽  
Qinghua Wang
Keyword(s):  
Crystal Structure ◽  
Signal Transduction ◽  
Atp Hydrolysis ◽  
Muscle Cells ◽  
Nemaline Myopathy ◽  
Actin Binding ◽  
Mechanistic Studies ◽  
Cellular Functions ◽  
Functional Studies ◽  
High Level

Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2162–495 nucleus. The structure contains two actin subunits connected by one Lmod2162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators.

scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

2018 ◽  
Author(s):  
Ward Decaestecker ◽  
Rafael Andrade Buono ◽  
Marie L. Pfeiffer ◽  
Nick Vangheluwe ◽  
Joris Jourquin ◽  
...  
Keyword(s):  
Lateral Roots ◽  
Cell Types ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Functional Studies ◽  
Organ Specific ◽  
Plant Genes ◽  
Mutant Lines ◽  
Functional Analyses ◽  
Efficient Selection

AbstractDetailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.

The role of regulated CFTR trafficking in epithelial secretion

2003 ◽  
Vol 285 (1) ◽  
pp. C1-C18 ◽  
Author(s):  
Carol A. Bertrand ◽  
Raymond A. Frizzell
Keyword(s):  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Cellular Localization ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Experimental Manipulation ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Cellular Context ◽  
Polarized Epithelial Cells

The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.

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