Whole-ExM: Expansion microscopy imaging of all anatomical structures of whole larval zebrafish

Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the proteins of the larvae with fluorophores and expanding them four-fold. We first optimize the fluorophore selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an expansion protocol for zebrafish larvae having calcified body parts. Through this process, we visualize the nanoscale details of diverse larvae organs, which have corresponding organ counterparts in humans, over the intact larvae. We show that whole-ExM retains the fluorescence signals of fluorescent proteins, and its resolution is high enough to visualize various structures that can be imaged only with electron microscopy. Whole-ExM would enable the nanoscale study of the molecular mechanisms of human diseases.

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Orange Fluorescent Proteins: Filling the Spectral Gap

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083296 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1670-C1670

Author(s):

Sergei Pletnev ◽

Daria Shcherbakova ◽

Oksana Subach ◽

Vladimir Malashkevich ◽

Steven Almo ◽

...

Keyword(s):

In Vivo Imaging ◽

Spectral Properties ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Stokes Shift ◽

Emission Wavelength ◽

Microscopy Imaging ◽

Large Stokes Shift

Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.

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Live cell imaging of mechanotransduction

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0042.focus ◽

2010 ◽

Vol 7 (suppl_3) ◽

Cited By ~ 18

Author(s):

Bo Liu ◽

Tae-Jin Kim ◽

Yingxiao Wang

Keyword(s):

Mechanical Stimulation ◽

Molecular Mechanisms ◽

Fluorescent Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Stem Cell Differentiation ◽

Cellular Functions ◽

Signalling Molecules ◽

Signalling Transduction ◽

Systematic Understanding

Mechanical forces play important roles in the regulation of cellular functions, including polarization, migration and stem cell differentiation. Tremendous advancement in our understanding of mechanotransduction has been achieved with the recent development of imaging technologies and molecular biosensors. In particular, genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) technology have been widely developed and applied in the field of mechanobiology. In this article, we will provide an overview of the recent progress of FRET application in mechanobiology, specifically mechanotransduction. We first introduce fluorescent proteins and FRET technology. We then discuss the mechanotranduction processes in different cells including stem cells, with a special emphasis on the important signalling molecules involved in mechanotransduction. Finally, we discuss methods that can allow the integration of simultaneous FRET imaging and mechanical stimulation to trigger signalling transduction. In summary, FRET technology has provided a powerful tool for the study of mechanotransduction to advance our systematic understanding of the molecular mechanisms by which cells respond to mechanical stimulation.

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Molecular mechanisms regulating cytotoxic lymphocyte development and function, and their associations to human diseases

10.3389/978-2-88919-279-3 ◽

2015 ◽

Keyword(s):

Molecular Mechanisms ◽

Human Diseases ◽

Lymphocyte Development ◽

Cytotoxic Lymphocyte ◽

And Function

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Ultrastructural and molecular analysis of the origin and differentiation of cells mediating brittle star skeletal regeneration

BMC Biology ◽

10.1186/s12915-020-00937-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Laura Piovani ◽

Anna Czarkwiani ◽

Cinzia Ferrario ◽

Michela Sugni ◽

Paola Oliveri

Keyword(s):

Molecular Mechanisms ◽

Close Contact ◽

Skeletal Development ◽

Morphological Differentiation ◽

Brittle Star ◽

Marker Genes ◽

Body Parts ◽

Coelomic Cavity ◽

Expression Of Genes ◽

Skeletal Regeneration

Abstract Background Regeneration is the ability to re-grow body parts or tissues after trauma, and it is widespread across metazoans. Cells involved in regeneration can arise from a pool of undifferentiated proliferative cells or be recruited from pre-existing differentiated tissues. Both mechanisms have been described in different phyla; however, the cellular and molecular mechanisms employed by different animals to restore lost tissues as well as the source of cells involved in regeneration remain largely unknown. Echinoderms are a clade of deuterostome invertebrates that show striking larval and adult regenerative abilities in all extant classes. Here, we use the brittle star Amphiura filiformis to investigate the origin and differentiation of cells involved in skeletal regeneration using a combination of microscopy techniques and molecular markers. Results Our ultrastructural analyses at different regenerative stages identify a population of morphologically undifferentiated cells which appear in close contact with the proliferating epithelium of the regenerating aboral coelomic cavity. These cells express skeletogenic marker genes, such as the transcription factor alx1 and the differentiation genes c-lectin and msp130L, and display a gradient of morphological differentiation from the aboral coelomic cavity towards the epidermis. Cells closer to the epidermis, which are in contact with developing spicules, have the morphology of mature skeletal cells (sclerocytes), and express several skeletogenic transcription factors and differentiation genes. Moreover, as regeneration progresses, sclerocytes show a different combinatorial expression of genes in various skeletal elements. Conclusions We hypothesize that sclerocyte precursors originate from the epithelium of the proliferating aboral coelomic cavity. As these cells migrate towards the epidermis, they differentiate and start secreting spicules. Moreover, our study shows that molecular and cellular processes involved in skeletal regeneration resemble those used during skeletal development, hinting at a possible conservation of developmental programmes during adult regeneration. Finally, we highlight that many genes involved in echinoderm skeletogenesis also play a role in vertebrate skeleton formation, suggesting a possible common origin of the deuterostome endoskeleton pathway.

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The impact of Traditional Chinese Medicine on mitophagy in disease models

Current Pharmaceutical Design ◽

10.2174/1381612827666211006150410 ◽

2021 ◽

Vol 27 ◽

Author(s):

Li-Ping Yu ◽

Ting-Ting Shi ◽

Yan-Qin Li ◽

Jian-Kang Mu ◽

Ya-Qin Yang ◽

...

Keyword(s):

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Molecular Mechanisms ◽

Human Diseases ◽

Regulatory Mechanisms ◽

Disease Models ◽

Relationship Of ◽

The Impact ◽

The Relationship

: Mitophagy plays an important role in maintaining mitochondrial quality and cell homeostasis through the degradation of damaged, aged, and dysfunctional mitochondria and misfolded proteins. Many human diseases, particularly neurodegenerative diseases, are related to disorders of mitochondrial phagocytosis. Exploring the regulatory mechanisms of mitophagy is of great significance for revealing the molecular mechanisms underlying the related diseases. Herein, we summarize the major mechanisms of mitophagy, the relationship of mitophagy with human diseases, and the role of traditional Chinese medicine (TCM) in mitophagy. These discussions enhance our knowledge of mitophagy and its potential therapeutic targets using TCM.

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The Sea Urchin Embryo: A Model for Studying Molecular Mechanisms Involved in Human Diseases and for Testing Bioactive Compounds

Sea Urchin - From Environment to Aquaculture and Biomedicine ◽

10.5772/intechopen.70301 ◽

2017 ◽

Cited By ~ 1

Author(s):

Maria Di Bernardo ◽

Marta Di Carlo

Keyword(s):

Bioactive Compounds ◽

Sea Urchin ◽

Molecular Mechanisms ◽

Human Diseases ◽

Sea Urchin Embryo

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Current and Emerging Biomarkers of Cell Death in Human Disease

BioMed Research International ◽

10.1155/2014/690103 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 7

Author(s):

Kongning Li ◽

Deng Wu ◽

Xi Chen ◽

Ting Zhang ◽

Lu Zhang ◽

...

Keyword(s):

Cell Death ◽

Molecular Mechanisms ◽

Research Area ◽

Human Diseases ◽

Mitotic Catastrophe ◽

Regulated Necrosis ◽

Early Diagnose ◽

Multicellular Organisms ◽

The Relationship ◽

Made In

Cell death is a critical biological process, serving many important functions within multicellular organisms. Aberrations in cell death can contribute to the pathology of human diseases. Significant progress made in the research area enormously speeds up our understanding of the biochemical and molecular mechanisms of cell death. According to the distinct morphological and biochemical characteristics, cell death can be triggered by extrinsic or intrinsic apoptosis, regulated necrosis, autophagic cell death, and mitotic catastrophe. Nevertheless, the realization that all of these efforts seek to pursue an effective treatment and cure for the disease has spurred a significant interest in the development of promising biomarkers of cell death to early diagnose disease and accurately predict disease progression and outcome. In this review, we summarize recent knowledge about cell death, survey current and emerging biomarkers of cell death, and discuss the relationship with human diseases.

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Brick Strex: a robust device built of LEGO bricks for mechanical manipulation of cells

Scientific Reports ◽

10.1038/s41598-021-97900-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Elina Mäntylä ◽

Teemu O. Ihalainen

Keyword(s):

Molecular Mechanisms ◽

Compressive Force ◽

Tissue Mechanics ◽

Cell Junctions ◽

Physical Factors ◽

Force Transmission ◽

Cell Nuclei ◽

Microscopy Imaging ◽

Robust Solution ◽

Mechanical Manipulation

AbstractCellular forces, mechanics and other physical factors are important co-regulators of normal cell and tissue physiology. These cues are often misregulated in diseases such as cancer, where altered tissue mechanics contribute to the disease progression. Furthermore, intercellular tensile and compressive force-related signaling is highlighted in collective cell behavior during development. However, the mechanistic understanding on the role of physical forces in regulation of cellular physiology, including gene expression and signaling, is still lacking. This is partly because studies on the molecular mechanisms of force transmission require easily controllable experimental designs. These approaches should enable both easy mechanical manipulation of cells and, importantly, readouts ranging from microscopy imaging to biochemical assays. To achieve a robust solution for mechanical manipulation of cells, we developed devices built of LEGO bricks allowing manual, motorized and/or cyclic cell stretching and compression studies. By using these devices, we show that $$\upbeta$$ β -catenin responds differentially to epithelial monolayer stretching and lateral compression, either localizing more to the cell nuclei or cell–cell junctions, respectively. In addition, we show that epithelial compression drives cytoplasmic retention and phosphorylation of transcription coregulator YAP1. We provide a complete part listing and video assembly instructions, allowing other researchers to build and use the devices in cellular mechanics-related studies.

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A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

10.1101/2021.01.07.425778 ◽

2021 ◽

Author(s):

Eftychios Frangedakis ◽

Manuel Waller ◽

Tomoaki Nishiyama ◽

Hirokazu Tsukaya ◽

Xia Xu ◽

...

Keyword(s):

Molecular Mechanisms ◽

Genetic Manipulation ◽

Fluorescent Proteins ◽

Land Plant ◽

Plant Evolution ◽

Stable Transformation ◽

35S Promoter ◽

Low Light ◽

Transformation Technique ◽

High Transformation

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the β-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

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