scholarly journals Sorting Nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes

Haematologica ◽  
2022 ◽  
Author(s):  
Joanne Lacey ◽  
Simon J. Webster ◽  
Paul R. Heath ◽  
Chris J. Hill ◽  
Lucinda Nicholson-Goult ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Transport ◽  
Microscopic Analysis ◽  
Intermediate Stage ◽  
Regulatory Control ◽  
Electron Microscopic ◽  
Sorting Nexin ◽  
Significant Enrichment ◽  
Proplatelet Formation ◽  
Granule Maturation

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterised by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harbouring a DNA-binding variant of FLI1. Our analysis identified 2276 transcripts that were differentially expressed in FLI1- deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an iPSC-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (p=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localisation studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

scholarly journals Characteristics of endoplasmic reticulum-derived transport vesicles.

1994 ◽  
Vol 126 (5) ◽  
pp. 1133-1148 ◽  
Author(s):  
M F Rexach ◽  
M Latterich ◽  
R W Schekman
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
De Novo ◽  
Microscopic Analysis ◽  
Equilibrium Density ◽  
Protein Markers ◽  
Electron Microscopic ◽  
Golgi Membranes ◽  
Transport Vesicles

We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane proteins in the ER are sorted prior to transport vesicle scission. Inhibition of Ypt1p-function, which prevents newly formed vesicles from docking to cis-Golgi membranes, was used to block transport. Vesicles that accumulate are competent for fusion with cis-Golgi membranes, but not with ER membranes, and thus are functionally committed to vectorial transport. A 900-fold enrichment was developed using differential centrifugation and a series of velocity and equilibrium density gradients. Electron microscopic analysis shows a uniform population of 60 nm vesicles that lack peripheral protein coats. Quantitative Western blot analysis indicates that protein markers of cytosol and cellular membranes are depleted throughout the purification, whereas the synaptobrevin-like Bet1, Sec22, and Bos1 proteins are highly enriched. Uncoated ER-derived transport vesicles (ERV) contain twelve major proteins that associate tightly with the membrane. The ERV proteins may represent abundant cargo and additional targeting molecules.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Electron Microscopic Analysis of Myonecrosis Induced by a Pure Component Isolated From Rattlesnake Venom

1976 ◽  
Vol 34 ◽  
pp. 292-293
Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu
Keyword(s):  
Gel Filtration ◽  
Microscopic Analysis ◽  
The United States ◽  
Exchange Chromatography ◽  
Pure Component ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Mouse Muscle ◽  
Major Health Problem ◽  
Rattlesnake Venom

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.

Correlative video enhanced differential interference contrast light microscopy (VDIC)-LM), low-voltage high-resolution SEM (LV-HR-SEM), and high-voltage electron microscopy (HVEM) in the observation of gold-labelled surface antigens and the subjacent cytoskeletal matrix

1989 ◽  
Vol 47 ◽  
pp. 904-905
Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
Marek Malecki
Keyword(s):  
High Resolution ◽  
Light Microscopy ◽  
Low Voltage ◽  
Microscopic Analysis ◽  
Cell Labelling ◽  
Video Technology ◽  
Electron Microscopic ◽  
Image Capture ◽  
High Voltage Electron Microscopy ◽  
Fluorescence Studies

The development of video-enhanced light microscopy (LM) as well as associated image processing and analysis have significantly broadened the scope of investigations which can be undertaken using (LM). Interference/polarization based microscopies can provide high resolution and higher levels of “detectability” especially in unstained living systems. Confocal light microscopy also holds the promise of further improvements in resolution, fluorescence studies, and 3 dimensional reconstruction. Video technology now provides, among other things, a means to detect differences in contrast difficult to detect with the human eye; furthermore, computerized image capture, processing, and analysis can be used to enhance features of interest, average images, subtract background, and provide a quantitative basis to studies of cells, cell features, cell labelling, and so forth. Improvements in video technology, image capture, and cost-effective computer image analysis/processing have contributed to the utility and potential of the various interference and confocal microscopic instrumentation.Electron microscopic technology has made advances as well. Microprocessor control and improved design have contributed to high resolution SEMs which have imaging capability at the molecular level and can operate at a range of accelerating voltages starting at 1KV. Improvements have also been seen in the HVEM and IVEM transmission instruments. As a whole, these advances in LM and EM microscopic technology provide the biologist with an array of information on structure, composition, and function which can be obtained from a single specimen. Corrrelative light microscopic analysis permits examination of living specimens and is critical where the “history” of a cell, cellular components, or labels needs to be known up to the time of chemical or physical fixation. Features such as cytoskeletal elements or gold label as small as 0.01 μm, well below the 0.2 μm limits of LM resolution, can be “detected” and their movement followed by VDIC-LM. Appropriate identification and preparation can then lead to the examination of surface detail and surface label with stereo LV-HR-SEM. Increasing the KV in the HR-SEM while viewing uncoated or thinly coated specimens can provide information from beneath the surface as well as increasing Z contrast so that positive identification of surface and subsurface colloidal gold or other heavy metal labelled/stained material is possible. Further examination of the same cells using stereo HVEM or IVEM provides information on internal ultrastructure and on the relationship of labelled material to cytoskeletal or organellar distribution, A wide variety of investigations can benefit from this correlative approach and a number of instrumentational configurations and preparative pathways can be tailored for the particular study. For a surprisingly small investment in time and technique, it is often possible to clear ambiguities or questions that arise when a finding is presented in the context of only one modality.

scholarly journals Mechanistic Study on Gold-Like Luster Development of Solution-Cast Oligo(3-methoxythiophene) Film

Coatings ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 861
Author(s):  
Minako Kubo ◽  
Minako Tachiki ◽  
Terumasa Mitogawa ◽  
Kota Saito ◽  
Ryota Saito ◽  
...  
Keyword(s):  
Microscopic Analysis ◽  
Mechanistic Study ◽  
Free Electrons ◽  
Coating Films ◽  
Electron Microscopic ◽  
Regular Structures ◽  
Scanning Electron Microscopic ◽  
Solution Cast ◽  
Scanning Electron Microscopic Analysis ◽  
Reflectance Measurements

Solution-cast coating films of perchlorate-doped oligo(3-methoxythiophene) exhibited a gold-like luster similar to that of metallic gold despite the involvement of no metals. However, the development mechanism of the luster remains ambiguous. To understand the mechanism, we performed scanning electron microscopic analysis, variable-angle spectral reflectance measurements, and ellipsometry measurements on ClO4−-doped oligo(3-methoxythiophene) cast film with a gold-like luster. The results revealed that the lustrous color of the film was not induced by the submicron-sized regular structures (structural color), nor by the high-density free electrons (reflective response based on Drude model), but by the large optical constants (refractive index and extinction coefficient) of the film, as speculated previously.

A Bibliometric Analysis of the Top 100 Cited Papers in Retinal Detachment

2021 ◽  
pp. 247412642110073
Author(s):  
Masumi George Asahi ◽  
Haig Pakhchanian ◽  
Christine Doepker ◽  
Rahul Raiker ◽  
Ron P. Gallemore
Keyword(s):  
Retinal Detachment ◽  
Bibliometric Analysis ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Microscopic Analysis ◽  
The United States ◽  
Electron Microscopic ◽  
Research Areas ◽  
Science Index ◽  
Funding Agencies

Purpose: This work aimed to identify and analyze the most frequently cited articles in retinal detachment (RD). Methods: Institute for Scientific Information’s Web of Science index (Thomas Scientific) was used to identify the top 100 most cited articles on RD between 1900 and 2019. Data from the top 100 most cited articles that met inclusion criteria were analyzed based on title, citation frequency, authorship, institution, journal, year of publication, and country of origin. Results: The top 100 articles in RD were cited 88 to 480 times. Steven K. Fisher was the most cited individual, with the University of California system being the most cited organization. Sixty-four percent of the top 100 articles originated from the United States and were published in the American Journal of Ophthalmology, Ophthalmology, and Archives of Ophthalmology at frequencies of 36%, 24%, and 11%, respectively. The top funding agencies included the US Department of Health and Human Services, the National Institutes of Health, and the National Eye Institute at 29%, 28%, and 27%, respectively. The top-cited article, which assessed the role of the retinal pigment epithelium by histologic and electron microscopic analysis of RDs in eyes of owl monkeys, was by Machemer and Laqua in the American Journal of Ophthalmology. Conclusions: This bibliometric analysis provides researchers and clinicians with a detailed overview of the most cited manuscripts in RD. Such analyses may guide researchers and funding agencies on important research areas in the field.

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