scholarly journals Hypoxia elicits broad and systematic changes in protein subcellular localization

2011 ◽  
Vol 301 (4) ◽  
pp. C913-C928 ◽  
Author(s):  
Robert Michael Henke ◽  
Ranita Ghosh Dastidar ◽  
Ajit Shah ◽  
Daniela Cadinu ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein Localization ◽  
Cell Periphery ◽  
Protein Distribution ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Regulatory ◽  
Chromatin Remodeling Complexes ◽  
Oxygen Signaling ◽  
Cellular Compartments ◽  
Control Protein

Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae . We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans.

scholarly journals Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

2019 ◽  
Author(s):  
Maja Klevanski ◽  
Frank Herrmannsdoerfer ◽  
Varun Venkataramani ◽  
Steffen Sass ◽  
Mike Heilemann ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Localization ◽  
Super Resolution ◽  
Protein Distribution ◽  
Tissue Samples ◽  
Target Proteins ◽  
Presynaptic Nerve Terminal ◽  
Solution Exchange ◽  
Staining Protocol ◽  
Cellular Compartments

ABSTRACTUnderstanding the nano-architecture of protein machines in diverse sub-cellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While singlemolecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D dSTORM imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 target proteins in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision. This allowed us to define novel nano-architectural features of protein distribution within the presynaptic nerve terminal.

scholarly journals Global, quantitative and dynamic mapping of protein subcellular localization

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Daniel N Itzhak ◽  
Stefka Tyanova ◽  
Jürgen Cox ◽  
Georg HH Borner
Keyword(s):  
Subcellular Localization ◽  
Protein Function ◽  
Cell Biology ◽  
Dynamic Capabilities ◽  
Protein Translocation ◽  
Protein Localization ◽  
Quantitative Model ◽  
Dynamic Mapping ◽  
Global Mapping ◽  
Control Protein

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

scholarly journals Synthetic protein interactions reveal a functional map of the cell

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lisa K Berry ◽  
Guðjón Ólafsson ◽  
Elena Ledesma-Fernández ◽  
Peter H Thorpe
Keyword(s):  
Protein Interactions ◽  
Protein Localization ◽  
Eukaryotic Cells ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Synthetic Protein ◽  
Protein Functions ◽  
Functional Map ◽  
Cellular Compartments ◽  
New Protein

To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells.

scholarly journals Convolutional Neural Network-Based Artificial Intelligence for Classification of Protein Localization Patterns

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 264
Author(s):  
Kaisa Liimatainen ◽  
Riku Huttunen ◽  
Leena Latonen ◽  
Pekka Ruusuvuori
Keyword(s):  
Neural Network ◽  
Artificial Intelligence ◽  
Convolutional Neural Network ◽  
High Throughput ◽  
Protein Function ◽  
Protein Localization ◽  
Convolutional Network ◽  
High Throughput Analysis ◽  
Fully Convolutional Network

Identifying localization of proteins and their specific subpopulations associated with certain cellular compartments is crucial for understanding protein function and interactions with other macromolecules. Fluorescence microscopy is a powerful method to assess protein localizations, with increasing demand of automated high throughput analysis methods to supplement the technical advancements in high throughput imaging. Here, we study the applicability of deep neural network-based artificial intelligence in classification of protein localization in 13 cellular subcompartments. We use deep learning-based on convolutional neural network and fully convolutional network with similar architectures for the classification task, aiming at achieving accurate classification, but importantly, also comparison of the networks. Our results show that both types of convolutional neural networks perform well in protein localization classification tasks for major cellular organelles. Yet, in this study, the fully convolutional network outperforms the convolutional neural network in classification of images with multiple simultaneous protein localizations. We find that the fully convolutional network, using output visualizing the identified localizations, is a very useful tool for systematic protein localization assessment.

Optobiochemistry: Genetically Encoded Control of Protein Activity by Light

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Jihye Seong ◽  
Michael Z. Lin
Keyword(s):  
Protein Function ◽  
Living Cells ◽  
Optical Methods ◽  
Annual Review ◽  
Publication Date ◽  
Protein Activity ◽  
Spatiotemporal Resolution ◽  
Protein Functions ◽  
Protein Classes ◽  
Control Protein

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light.Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

2006 ◽  
Vol 80 (13) ◽  
pp. 6368-6377 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Nayak ◽  
You Zhou ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Protein Function ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
De Novo ◽  
Virus Production ◽  
Hinge Region ◽  
Cell Periphery ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

scholarly journals Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

1991 ◽  
Vol 39 (11) ◽  
pp. 1531-1538 ◽  
Author(s):  
M Haftek ◽  
G Serre ◽  
V Mils ◽  
J Thivolet
Keyword(s):  
Stratum Corneum ◽  
Cell Envelope ◽  
Ultrastructural Localization ◽  
Antigenic Determinants ◽  
Cell Periphery ◽  
Protein Distribution ◽  
Cell Structures ◽  
Physical Resistance ◽  
Cell Envelopes

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function

1987 ◽  
Vol 7 (7) ◽  
pp. 2344-2351
Author(s):  
R J Deschenes ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Membrane Fraction ◽  
Carbon Sources ◽  
Fatty Acyl ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fatty Acyl Moiety ◽  
Fatty Acylation ◽  
Absolute Requirement

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.

scholarly journals Trimethyllysine: From Carnitine Biosynthesis to Epigenetics

2020 ◽  
Vol 21 (24) ◽  
pp. 9451
Author(s):  
Marijn N. Maas ◽  
Jordi C. J. Hintzen ◽  
Miriam R. B. Porzberg ◽  
Jasmin Mecinović
Keyword(s):  
Protein Function ◽  
Genetic Disorders ◽  
Enzymatic Reactions ◽  
Lysine Methylation ◽  
Nucleosome Assembly ◽  
Cellular Processes ◽  
Subsequent Effect ◽  
Control Protein ◽  
Epigenetic Processes

Trimethyllysine is an important post-translationally modified amino acid with functions in the carnitine biosynthesis and regulation of key epigenetic processes. Protein lysine methyltransferases and demethylases dynamically control protein lysine methylation, with each state of methylation changing the biophysical properties of lysine and the subsequent effect on protein function, in particular histone proteins and their central role in epigenetics. Epigenetic reader domain proteins can distinguish between different lysine methylation states and initiate downstream cellular processes upon recognition. Dysregulation of protein methylation is linked to various diseases, including cancer, inflammation, and genetic disorders. In this review, we cover biomolecular studies on the role of trimethyllysine in carnitine biosynthesis, different enzymatic reactions involved in the synthesis and removal of trimethyllysine, trimethyllysine recognition by reader proteins, and the role of trimethyllysine on the nucleosome assembly.

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