scholarly journals Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells

2021 ◽  
Vol 4 (12) ◽  
pp. e202101222
Author(s):  
Jayant Asthana ◽  
Nicholas I Cade ◽  
Davide Normanno ◽  
Wei Ming Lim ◽  
Thomas Surrey
Keyword(s):  
Gene Editing ◽  
Morphological Changes ◽  
Dynamic Properties ◽  
Human Cells ◽  
Dynamic Changes ◽  
Central Spindle ◽  
Slowing Down ◽  
Microtubule Growth ◽  
Late Mitosis ◽  
Endogenous Locus

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent “bundling, sliding, and compaction” model of antiparallel midzone bundle formation in the central spindle during late mitosis.

scholarly journals PRC1 and EB1 Binding Dynamics Reveal a Solidifying Central Spindle during Anaphase Compaction in Human Cells

2020 ◽  
Author(s):  
Jayant Asthana ◽  
Nicholas I. Cade ◽  
Wei Ming Lim ◽  
Thomas Surrey
Keyword(s):  
Chromosome Segregation ◽  
Morphological Changes ◽  
Dynamic Properties ◽  
Binding Mode ◽  
Human Cells ◽  
Dependent Manner ◽  
Dynamic Changes ◽  
Central Spindle ◽  
Cytoskeletal Network ◽  
Endogenous Locus

ABSTRACTDuring mitosis the spindle undergoes considerable morphological and dynamic changes. Particularly the central spindle reorganizes drastically at the onset of anaphase when the antiparallel microtubule bundler PRC1 starts to accumulate and recruit a subset of spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using CRISPR/Cas9 gene editing, we generated human RPE1 cells that express from their endogenous locus fluorescently tagged versions of the two cytoskeletal network hub proteins PRC1 and the end binding protein EB1 to be able to quantify their spindle distribution and binding/unbinding turnover under native conditions. We find that throughout mitosis EB1 binds central spindle microtubule bundles in a PRC1-dependent manner using a binding mode different from EB1 at growing microtubule ends. Both proteins, PRC1 and EB1, progressively accumulate and bind increasingly strongly to compacting central antiparallel microtubule overlaps. These results show that the central spindle gradually ‘solidifies’ during mitosis, suggesting that the two protein interaction networks around PRC1 and EB1 cooperate to stabilize the shortening central spindle, explaining the importance of both proteins for correct chromosome segregation and cytokinesis.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Eman A. Ageely ◽  
Ramadevi Chilamkurthy ◽  
Sunit Jana ◽  
Leonora Abdullahu ◽  
Daniel O’Reilly ◽  
...  
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Model Organisms ◽  
Enzyme Properties ◽  
Chemical Compatibility ◽  
Modified Nucleotides ◽  
Chemical Modifications ◽  
Pseudoknot Structure ◽  
Therapeutic Development

AbstractCRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics.

Morphological Aspects of the Traumatic Chronic Subdural Hematoma Capsule: SEM Studies

2007 ◽  
Vol 13 (3) ◽  
pp. 211-219 ◽  
Author(s):  
Marek Moskała ◽  
Igor Gościński ◽  
Józef Kałuża ◽  
Jarosław Polak ◽  
Mariusz Krupa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Vessels ◽  
Morphological Changes ◽  
Chronic Subdural Hematoma ◽  
Dynamic Changes ◽  
Inflammatory Reactions ◽  
Morphological Aspects ◽  
Thin Walled ◽  
Scanning Electron

The morphology of the outer and inner membranes of traumatic chronic subdural hematomas (CSDHs) surgically removed from eight patients was investigated by scanning electron microscopy (SEM). Hematomas were divided into three groups based on time that had passed from the initiation of trauma to surgery. Structure of the CSDHs showed gradual morphological changes of the developing hematoma capsule. They initially included angiogenic and aseptic inflammatory reactions followed by progressive involvement of fibroblasts—proliferating and producing collagen fibrils. Numerous capillaries suggesting formation of new blood vessels were observed mainly in young hematomas removed between 15 and 21 days after trauma. In “older” hematomas (40 days after trauma), more numerous capillaries and thin-walled sinusoids were accompanied by patent, larger diameter blood vessels. Within the fibrotic outer membrane of the “oldest” hematoma capsules (60 or more days after trauma), especially in the area over the hematoma cavity, blood vessels were frequently occluded by clots. The results suggest dynamic changes in cellular and vascular organization of traumatic CSDH capsules paralleling the progression in hematoma age.

scholarly journals Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis

2006 ◽  
Vol 174 (4) ◽  
pp. 491-497 ◽  
Author(s):  
Carlos G. Reverte ◽  
Angela Benware ◽  
Christopher W. Jones ◽  
Susan E. LaFlamme
Keyword(s):  
Mitotic Spindle ◽  
Cell Types ◽  
Cellular Systems ◽  
Integrin Β1 ◽  
Dynamic Changes ◽  
Matrix Adhesion ◽  
Cell Matrix ◽  
Microtubule Growth ◽  
Cell Matrix Adhesion

In many mammalian cell types, integrin-mediated cell-matrix adhesion is required for the G1–S transition of the cell cycle. As cells approach mitosis, a dramatic remodeling of their cytoskeleton accompanies dynamic changes in matrix adhesion, suggesting a mechanistic link. However, the role of integrins in cell division remains mostly unexplored. Using two cellular systems, we demonstrate that a point mutation in the β1 cytoplasmic domain (β1 tail) known to decrease integrin activity supports entry into mitosis but inhibits the assembly of a radial microtubule array focused at the centrosome during interphase, the formation of a bipolar spindle at mitosis and cytokinesis. These events are restored by externally activating the mutant integrin with specific antibodies. This is the first demonstration that the integrin β1 tail can regulate centrosome function, the assembly of the mitotic spindle, and cytokinesis.

scholarly journals Study of the Mechanics and Micro-Structure of Wheat Straw Returned to Soil in Relation to Different Tillage Methods

Agronomy ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 894
Author(s):  
Yanpeng Wang ◽  
Adnan Abbas ◽  
Xiaochan Wang ◽  
Sijun Yang ◽  
Morice R. O. Odhiambo ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Wheat Straw ◽  
Decomposition Rate ◽  
Morphological Changes ◽  
Micro Structure ◽  
Dynamic Changes ◽  
Straw Decomposition ◽  
Nylon Mesh ◽  
Mesh Bag ◽  
Tillage Methods

A field experiment was conducted to study the effects of different tillage methods, and their interaction on the dynamic changes of straw decomposition rate, mechanical properties, and micro-structure of the stalk. A nylon mesh bag technique was used. An obvious change was observed in the decomposition rate of straw, and its mechanical, and micro-structural properties. The decomposition rate of straw was increased in all tillage treatments. Specifically, it increased consistently in conventional and dry rotary tillage, and sharply in wet rotary tillage. Furthermore, for all tillage, the mechanical properties like shear and bending strengths decreased sharply while compressive strength first decreased linearly and then increased, whereas the micro-structure of wheat straw showed a fluctuating trend, i.e., it changed neither regularly nor consistently over time. Moreover, the micro-structure of the stalk explained the morphological changes to the straw that returned to the field, which may impact the mechanical properties. However, these changes could not explain the degradation trend of straw directly. The findings of the study could be used as a theoretical reference for the design of tillage and harvesting machinery keeping in view soil solidification and compaction dynamics.

Structure-function analysis of anaphase spindle elongation using isolated diatom spindles

1991 ◽  
Vol 49 ◽  
pp. 206-207
Author(s):  
W. Z. Cande ◽  
C.J. Hogan ◽  
M. Lee
Keyword(s):  
Morphological Changes ◽  
Function Analysis ◽  
Light And Electron Microscopy ◽  
Near Neighbor ◽  
Model Systems ◽  
Central Spindle ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Anaphase B

Diatom spindles are important model systems for describing the morphological changes associated with anaphase chromosome movement because the fibrous systems responsible for anaphase A (chromosome-to-pole movement) and anaphase B (spindle elongation) are spatially separate and the central spindle is a paracrystalline array of microtubules. The diatom central spindle, which is responsible for anaphase B, is constructed of two sets of interdigiting microtubules that originate from plate-like spindle poles and display specific near-neighbor interactions in the zone of microtubule overlap. The microtubules of each half-spindle are of relatively unifrom length such that the plus ends are clustered together in narrow zones at each edge of the zone of microtubule overlap. This has allowed us to monitor changes in extent of microtubule overlap in the light microscope with polarization optics. We have isolated spindles from synchronized populations of several species of dividing diatom cells to study the mechanochemistry of anaphase spindle elongation in vitro and to analyze the rearrangement of spindle components by light and electron microscopy during reactivation.

scholarly journals SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

2015 ◽  
Vol 89 ◽  
pp. 379-386 ◽  
Author(s):  
Kimberly Cramer-Morales ◽  
Collin D. Heer ◽  
Kranti A. Mapuskar ◽  
Frederick E. Domann
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Targeted Gene Editing

scholarly journals CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ashley J. Clark ◽  
Niamh Mullooly ◽  
Dewi Safitri ◽  
Matthew Harris ◽  
Tessa de Vries ◽  
...  
Keyword(s):  
Gene Editing ◽  
Human Cells ◽  
Calcitonin Receptor ◽  
Physiological Relevance ◽  
Starting Point ◽  
Calcitonin Gene ◽  
Ramp Function ◽  
Physiological Pathways ◽  
Cardiovascular Cells ◽  
Primary Human Cells

AbstractAgonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

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