scholarly journals Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag

2018 ◽  
Author(s):  
Adam Pickard ◽  
Antony Adamson ◽  
Yinhui Lu ◽  
Joan Chang ◽  
Richa Garva ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gold Standard ◽  
Half Life ◽  
Live Imaging ◽  
Collagen Fibrils ◽  
Collagen I ◽  
Live Cells ◽  
Dynamic Information ◽  
Imaging Methods ◽  
Intracellular Collagen

Electron microscopy has been the “gold standard” for studying collagen networks but dynamic information on how cells synthesise the networks has been lacking. Live imaging methods have been unable to distinguish newly-synthesised fibrils from pre-existing fibrils and intracellular collagen. Here, we tagged endogenous collagen-I using CRISPR-Cas9 with photoswitchable Dendra2 and demonstrate live cells synthesising, migrating on, and interacting with, collagen fibrils. This strategy is applicable for other long half-life proteins.

scholarly journals SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

1974 ◽  
Vol 60 (1) ◽  
pp. 92-127 ◽  
Author(s):  
Melvyn Weinstock ◽  
C. P. Leblond
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Phosphotungstic Acid ◽  
Secretory Granules ◽  
Low Ph ◽  
Collagen Fibrils ◽  
Dentin Collagen ◽  
Odontoblast Process ◽  
High Radioactivity

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

The morphogenesis of avian tendon

1956 ◽  
Vol 144 (917) ◽  
pp. 556-572 ◽  
Keyword(s):  
Electron Microscopy ◽  
Average Diameter ◽  
Relative Volume ◽  
Morphological Features ◽  
Collagen Fibrils ◽  
Intercellular Spaces ◽  
Intercellular Substance ◽  
Thin Sections ◽  
Cellular Components ◽  
Development Proceeds

Observations by electron microscopy on thin sections of the metatarsal tendon of embryonic fowls show that in the 8-day embryo the earliest definable collagen fibrils of 80 Å in diameter are intimately associated with the cytoplasm of the compact, apparently syncytial, cells of which the tendon rudiment is composed. As development proceeds, some intracytoplasmic groups of fibrils are distinguishable, but intercellular spaces also develop and these gradually become filled with fibrils; finally, bundles are formed and lie packed between the adjacent cells. Soon the extracellular organization predominates until at 20days the average diameter of the fibrils is 400 Å and the normal 640 Å periodicity of collagen has been achieved. The morphological features demonstrated have been correlated with histochemical data, and the possible function of the various cellular components in the formation of the intercellular substance has been discussed. By the use of sections in which fibrils have been cut exactly transverse to the bundle axis it has been shown that each fibril is invested by interfibrillar material. As the diameter of the fibrils increases with age the relative volume of interfibrillar material within a bundle diminishes; it is therefore concluded that this material must contain either collagen or the necessary precursors in order to account for the enlargement of the fibrils. Thus the interfibrillar material is of fundamental importance to the formation and growth of the collagen fibrils.

Collagen Phagocytosis by Fibroblasts in the Periodontal Ligament of the Mouse Molar During the Initial Phase of Hypofunction

1987 ◽  
Vol 66 (12) ◽  
pp. 1708-1712 ◽  
Author(s):  
W. Beertsen
Keyword(s):  
Electron Microscopy ◽  
Morphometric Analysis ◽  
Extracellular Space ◽  
Periodontal Ligament ◽  
Initial Phase ◽  
Volume Density ◽  
Collagen Fibrils ◽  
Fibrillar Collagen ◽  
Collagen Phagocytosis

This study was undertaken in order to determine whether hypofunction of teeth is associated with changes in collagen phagocytosis by fibroblasts of the periodontal ligament. In mice, the lower right molars were extracted and the animals killed one, two, three, four, or seven days later. The maxillary first molars with their surrounding periodontium were processed for electron microscopy and their periodontal ligament subjected to morphometric analysis. It was observed that, whereas the volume density of extracellular collagen in the ligament of the hypofunctional molars decreased from 50% to 30% during the course of the experiment the fraction of fibrillar collagen ingested by the cells increased over two-fold. This increase was already manifest very shortly after the onset of the experiment and offers an explanation for the net loss of collagen fibrils from the extracellular space.

scholarly journals Microfluidic Live-Imaging Technology to Perform Research Activities in 3D Models

2021 ◽  
Author(s):  
Arnaud Martino Capuzzo ◽  
Daniele Vigo
Keyword(s):  
Fluorescence Intensity ◽  
Regulatory Networks ◽  
3D Structure ◽  
Live Imaging ◽  
Two Dimensions ◽  
Live Cells ◽  
Straight Line Passing ◽  
3D Cultures ◽  
Research Activities

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-a-kind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber, and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, ImageJ software was used.

scholarly journals The precipitation of toroidal collagen fibrils

1969 ◽  
Vol 112 (4) ◽  
pp. 515-519 ◽  
Author(s):  
Alan Cooper
Keyword(s):  
Electron Microscopy ◽  
Free Energy ◽  
Lower Limit ◽  
Small Proportion ◽  
Helical Structure ◽  
Collagen Fibrils ◽  
Theoretical Considerations ◽  
Continuous Injection ◽  
Calf Skin

The morphology of aggregates of calf-skin tropocollagen, precipitated by continuous injection into neutral phosphate buffers at 35°, has been studied by electron microscopy. Although most of the collagen is precipitated as normal native fibrils, a small proportion forms closed toroidal structures having the usual native band–interband pattern. Theoretical considerations, based on elastic energies in a general microfibril model, predict that the toroids should have a simple super-helical structure, and this is not inconsistent with the observations. From the theoretical energies it was possible to estimate a crude lower limit of 3kcal./mole for the free energy of association of the tropocollagen macromolecules.

scholarly journals Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.

1984 ◽  
Vol 99 (6) ◽  
pp. 2024-2033 ◽  
Author(s):  
D E Birk ◽  
R L Trelstad
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
High Voltage ◽  
Collagen Fibril ◽  
Corneal Stroma ◽  
Collagen Fibrils ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Corneal Fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.

scholarly journals FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles

2015 ◽  
Vol 360 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Jeroen Kuipers ◽  
Tjakko J. van Ham ◽  
Ruby D. Kalicharan ◽  
Anneke Veenstra-Algra ◽  
Klaas A. Sjollema ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Live Imaging
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