scholarly journals Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jan N Hansen ◽  
Fabian Kaiser ◽  
Christina Klausen ◽  
Birthe Stüven ◽  
Raymond Chong ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Protein Function ◽  
Mammalian Cells ◽  
Camp Signaling ◽  
Subcellular Compartment ◽  
Cellular Behavior ◽  
Health And Disease ◽  
And Function ◽  
Ciliary Signaling

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools to investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylyl cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

scholarly journals Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

2020 ◽  
Author(s):  
Jan N. Hansen ◽  
Fabian Kaiser ◽  
Christina Klausen ◽  
Birthe Stüven ◽  
Raymond Chong ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Protein Function ◽  
Camp Signaling ◽  
Subcellular Compartment ◽  
Cellular Behavior ◽  
Health And Disease ◽  
And Function ◽  
Ciliary Signaling

SummaryCompartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling and function has been challenging due to the lack of tools to temporarily manipulate and analyze ciliary signaling. Here, we describe a nanobodybased targeting approach for optogenetic tools that is applicable in vitro and in vivo and allows to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after direct fusion to a ciliary targeting sequence. We functionally localized modifiers of cAMP signaling, i.e. the photo-activated adenylate cyclase bPAC and the light-activated phosphodiesterase LAPD, as well as the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

scholarly journals In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

1997 ◽  
Vol 139 (5) ◽  
pp. 1197-1207 ◽  
Author(s):  
N. Doane Chilcoat ◽  
Aaron P. Turkewitz
Keyword(s):  
Membrane Fusion ◽  
Protein Function ◽  
Potential Role ◽  
Secretory Granules ◽  
Rabbit Muscle ◽  
Paramecium Tetraurelia ◽  
Post Translational Modifications ◽  
In Vivo Analysis ◽  
And Function

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

scholarly journals Primary Cilium in Cancer Hallmarks

2019 ◽  
Vol 20 (6) ◽  
pp. 1336 ◽  
Author(s):  
Lucilla Fabbri ◽  
Frédéric Bost ◽  
Nathalie Mazure
Keyword(s):  
Cancer Progression ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Wide Spectrum ◽  
Cancer Hallmarks ◽  
Mutual Regulation ◽  
Ultrastructure And Function ◽  
And Function ◽  
External Stimuli

The primary cilium is a solitary, nonmotile and transitory appendage that is present in virtually all mammalian cells. Our knowledge of its ultrastructure and function is the result of more than fifty years of research that has dramatically changed our perspectives on the primary cilium. The mutual regulation between ciliogenesis and the cell cycle is now well-recognized, as well as the function of the primary cilium as a cellular “antenna” for perceiving external stimuli, such as light, odorants, and fluids. By displaying receptors and signaling molecules, the primary cilium is also a key coordinator of signaling pathways that converts extracellular cues into cellular responses. Given its critical tasks, any defects in primary cilium formation or function lead to a wide spectrum of diseases collectively called “ciliopathies”. An emerging role of primary cilium is in the regulation of cancer development. In this review, we seek to describe the current knowledge about the influence of the primary cilium in cancer progression, with a focus on some of the events that cancers need to face to sustain survival and growth in hypoxic microenvironment: the cancer hallmarks.

Surprising roles for phospholipid binding proteins revealed by high throughput geneticsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 565-574 ◽  
Author(s):  
Marissa A. LeBlanc ◽  
Christopher R. McMaster
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Essential Gene ◽  
Peer Review Process ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
And Function ◽  
Lipid Binding Proteins

Saccharomyces cerevisiae remains an ideal organism for studying the cell biological roles of lipids in vivo, as yeast has phospholipid metabolic pathways similar to mammalian cells, is easy and economical to manipulate, and is genetically tractable. The availability of isogenic strains containing specific genetic inactivation of each non-essential gene allowed for the development of a high-throughput method, called synthetic genetic analysis (SGA), to identify and describe precise pathways or functions associated with specific genes. This review describes the use of SGA to aid in elucidating the function of two lipid-binding proteins that regulate vesicular transport, Sec14 and Kes1. Sec14 was first identified as a phosphatidylcholine (PC) – phosphatidylinositol (PI) transfer protein required for viability, with reduced Sec14 function resulting in diminished vesicular transport out of the trans-Golgi. Although Sec14 is required for cell viability, inactivating the KES1 gene that encodes for a member of the oxysterol binding protein family in cells lacking Sec14 function results in restoration of vesicular transport and cell growth. SGA analysis identified a role for Kes1 and Sec14 in regulating the level and function of Golgi PI-4-phosphate (PI-4-P). SGA also determined that Sec14 not only regulates vesicular transport out of the trans-Golgi, but also transport from endosomes to the trans-Golgi. Comparing SGA screens in databases, coupled with genetic and cell biological analyses, further determined that the PI-4-P pool affected by Kes1 is generated by the PI 4-kinase Pik1. An important biological role for Sec14 and Kes1 revealed by SGA is coordinate regulation of the Pik1-generated Golgi PI-4-P pool that in turn is essential for vesicular transport into and out of the trans-Golgi.

Chromatin structure and function: the heretical path to an RNA transcription factor

1996 ◽  
Vol 74 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Margarida O. Krause
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Myc Gene ◽  
And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

Electrical, Chemical, and Topological Addressing of Mammalian Cells With Microfabricated Systems

1999 ◽  
Vol 121 (1) ◽  
pp. 65-72 ◽  
Author(s):  
R. Kapur ◽  
J. M. Calvert ◽  
A. S. Rudolph
Keyword(s):  
Mammalian Cells ◽  
Model Systems ◽  
Cellular Behavior ◽  
Practical Applications ◽  
Form And Function ◽  
Factorial Model ◽  
Cell Substrate ◽  
And Function ◽  
Tools And Techniques ◽  
Fundamental Forces

This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell–substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling “communication” with living cells/tissues at the cellular and subcellular levels.

scholarly journals Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

2022 ◽  
Author(s):  
Lewis A Macdonald ◽  
Gillian C A Taylor ◽  
Jennifer M Brisbane ◽  
Ersi Christodoulou ◽  
Lucy Scott ◽  
...  
Keyword(s):  
Mouse Models ◽  
Protein Function ◽  
Mammalian Cells ◽  
Degradation Kinetics ◽  
Cell Types ◽  
Mouse Genetics ◽  
Chemical Genetic ◽  
Endogenous Proteins ◽  
Specific Degradation

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II, two essential regulators of mitotic chromosome structure, revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

scholarly journals Drosophila MICOS knockdown impairs mitochondrial structure and function and promotes mitophagy in muscle tissue

Biology Open ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. bio054262
Author(s):  
Li-jie Wang ◽  
Tian Hsu ◽  
Hsiang-ling Lin ◽  
Chi-yu Fu
Keyword(s):  
Muscle Tissue ◽  
Mammalian Cells ◽  
Structure And Function ◽  
Tissue Homeostasis ◽  
Mitochondrial Morphology ◽  
Mitochondrial Structure ◽  
And Function ◽  
Nucleoid Structure

ABSTRACTThe mitochondrial contact site and cristae organizing system (MICOS) is a multi-protein interaction hub that helps define mitochondrial ultrastructure. While the functional importance of MICOS is mostly characterized in yeast and mammalian cells in culture, the contributions of MICOS to tissue homeostasis in vivo remain further elucidation. In this study, we examined how knocking down expression of Drosophila MICOS genes affects mitochondrial function and muscle tissue homeostasis. We found that CG5903/MIC26-MIC27 colocalizes and functions with Mitofilin/MIC60 and QIL1/MIC13 as a Drosophila MICOS component; knocking down expression of any of these three genes predictably altered mitochondrial morphology, causing loss of cristae junctions, and disruption of cristae packing. Furthermore, the knockdown flies exhibited low mitochondrial membrane potential, fusion/fission imbalances, increased mitophagy, and limited cell death. Reductions in climbing ability indicated deficits in muscle function. Knocking down MICOS genes also caused reduced mtDNA content and fragmented mitochondrial nucleoid structure in Drosophila. Together, our data demonstrate an essential role of Drosophila MICOS in maintaining proper homeostasis of mitochondrial structure and function to promote the function of muscle tissue.

scholarly journals The basement membrane as a structured surface – role in vascular health and disease

2020 ◽  
Vol 133 (18) ◽  
pp. jcs239889
Author(s):  
Claire Leclech ◽  
Carlo F. Natale ◽  
Abdul I. Barakat
Keyword(s):  
Basement Membrane ◽  
Biochemical Composition ◽  
Vascular Tissue ◽  
Cell Structure ◽  
Mechanical Stiffness ◽  
Cellular Behavior ◽  
Structured Surface ◽  
Health And Disease ◽  
And Function ◽  
Biophysical Attributes

ABSTRACTThe basement membrane (BM) is a thin specialized extracellular matrix that functions as a cellular anchorage site, a physical barrier and a signaling hub. While the literature on the biochemical composition and biological activity of the BM is extensive, the central importance of the physical properties of the BM, most notably its mechanical stiffness and topographical features, in regulating cellular function has only recently been recognized. In this Review, we focus on the biophysical attributes of the BM and their influence on cellular behavior. After a brief overview of the biochemical composition, assembly and function of the BM, we describe the mechanical properties and topographical structure of various BMs. We then focus specifically on the vascular BM as a nano- and micro-scale structured surface and review how its architecture can modulate endothelial cell structure and function. Finally, we discuss the pathological ramifications of the biophysical properties of the vascular BM and highlight the potential of mimicking BM topography to improve the design of implantable endovascular devices and advance the burgeoning field of vascular tissue engineering.

Identification of a common deletion in the spike protein of SARS-CoV-2

2020 ◽  
Author(s):  
Zhe Liu ◽  
Huanying Zheng ◽  
Runyu Yuan ◽  
Mingyue Li ◽  
Huifang Lin ◽  
...  
Keyword(s):  
Protein Function ◽  
Cleavage Site ◽  
Vaccine Development ◽  
Mechanistic Explanation ◽  
Spike Protein ◽  
Clinical Samples ◽  
Genomic Changes ◽  
And Function

AbstractTwo notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

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