scholarly journals Augmin regulates kinetochore tension and spatial arrangement of spindle microtubules by nucleating bridging fibers

2020 ◽  
Author(s):  
Martina Manenica ◽  
Valentina Štimac ◽  
Isabella Koprivec ◽  
Juraj Simunić ◽  
Iva M. Tolić
Keyword(s):  
Critical Role ◽  
Spatial Arrangement ◽  
Knock Out ◽  
Daughter Cells ◽  
Sister Kinetochore ◽  
Division Spindle ◽  
Spindle Microtubules ◽  
Bridging Fibers ◽  
Poleward Flux

ABSTRACTThe mitotic spindle functions as a molecular micromachine that evenly distributes chromosomes into two daughter cells during cell division. Spindle microtubules in human cells are mainly nucleated at the centrosome and on the lateral surface of existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects functionally distinct microtubule bundles and consequently the forces within the spindle. Here we show, by using siRNA depletion and CRISPR knock-out of the augmin complex subunits HAUS6 or HAUS8, that augmin is crucial for the nucleation of bridging microtubules, which laterally link sister kinetochore fibers. Augmin depletion resulted in a reduction in the number of microtubules within bridging fibers by around 80% and in kinetochore fibers by 40%, suggesting that the bridging microtubules are mainly nucleated at the surface of present microtubules. In augmin-depleted cells, the interkinetochore distance decreased preferentially for kinetochores that lack a bridging fiber, independently of the thickness of their k-fibers, implying that augmin affects forces on kinetochores largely via bridging fibers. Without augmin the number of bridging fibers decreased, with the remaining ones mostly confined to the spindle periphery with an increased overlap length. A slower poleward flux of microtubules after augmin depletion is indicative of slower sliding within the bridging fiber. Our results demonstrate a critical role of augmin in the formation of bridging microtubules and proper architecture of the metaphase spindle, suggesting a model where sliding of augmin-nucleated bridging microtubules promotes poleward flux of k-fibers and thus tension on kinetochores.

Colloidal magnetic nanocrystal clusters: variable length-scale interaction mechanisms, synergetic functionalities and technological advantages

2015 ◽  
Vol 4 (6) ◽  
Author(s):  
Athanasia Kostopoulou ◽  
Alexandros Lappas
Keyword(s):  
Magnetic Particles ◽  
Critical Role ◽  
Exchange Interactions ◽  
Magnetic Behavior ◽  
Building Blocks ◽  
Spatial Arrangement ◽  
Magnetic Characteristics ◽  
Surface Functionality ◽  
Magnetic Nanocrystal

AbstractMagnetic particles of optimized nanoscale dimensions can be utilized as building blocks to generate colloidal nanocrystal assemblies with controlled size, well-defined morphology, and tailored properties. Recent advances in the state-of-the-art surfactant-assisted approaches for the directed aggregation of inorganic nanocrystals into cluster-like entities are discussed, and the synthesis parameters that determine their geometrical arrangement are highlighted. This review pays attention to the enhanced physical properties of iron oxide nanoclusters, while it also points to their emerging collective magnetic response. The current progress in experiment and theory for evaluating the strength and the role of intra- and inter-cluster interactions is analyzed in view of the spatial arrangement of the component nanocrystals. Numerous approaches have been proposed for the critical role of dipole-dipole and exchange interactions in establishing the nature of the nanoclusters’ cooperative magnetic behavior (be it ferromagnetic or spin-glass like). Finally, we point out why the purposeful engineering of the nanoclusters’ magnetic characteristics, including their surface functionality, may facilitate their use in diverse technological sectors ranging from nanomedicine and photonics to catalysis.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

1996 ◽  
Vol 109 (3) ◽  
pp. 561-567 ◽  
Author(s):  
F.J. McNally ◽  
K. Okawa ◽  
A. Iwamatsu ◽  
R.D. Vale
Keyword(s):  
Mitotic Spindle ◽  
Dynamic Properties ◽  
Mitotic Spindles ◽  
Microtubule Severing ◽  
Spindle Microtubules ◽  
Gamma Tubulin ◽  
And Function ◽  
Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

Abstract 420: Mrs2 is the Authentic Mammalian Mitochondrial Mg 2+ Channel

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Prema Velusamy ◽  
Shanmughapriya Santhanam
Keyword(s):  
Copy Number ◽  
Mitochondrial Permeability Transition ◽  
Critical Role ◽  
Permeability Transition ◽  
Inner Mitochondrial Membrane ◽  
Metabolic Switch ◽  
Knock Out ◽  
Mitochondrial Copy Number

Magnesium (Mg 2+ ) is an important cation critical for cellular functions and tissue integrity. Mitochondria have been demonstrated to be capable of both accumulate and release Mg 2+ . However, the exact molecular machinery associated with mitochondrial Mg 2+ (mMg 2+ ) influx has not yet been delineated. In the present study we characterized the mammalian mMg 2+ channel, Mrs2 and comprehensively studied its role in energy metabolism. Protein flux, membrane fractionation and STED microscopy studies revealed Mrs2 to localize on the inner mitochondrial membrane with its N and C-terminus in the matrix. Western blot and qPCR analysis confirmed the ubiquitous distribution of Mrs2 in all metabolically active tissues. We adopted lentiviral based strategy to stably knock down (KD) Mrs2 in vitro . Primarily, the use of FRET-based mMg 2+ sensor, MitoMario showed a decreased influx of Mg 2+ into mitochondria in Mrs2 KD cells. This was further confirmed by patch clamping the mitoplasts of the control and Mrs2 KD cells. Because Mg 2+ is an important co-factor in the machineries that replicate, we next assessed the mitochondrial copy number. The decreased influx of mMg 2+ impacted the mitochondrial copy number and electron transport chain (ETC) complex assembly. The defective ETC assembly was marked by increased generation of mitochondrial reactive oxygen species, increased proton leak, decreased ATP levels, and also prompted a metabolic switch from mitochondrial oxidative phosphorylation to glucose oxidation in Mrs2 KD cells. Additionally, Mrs2 KD cells had an increased sensitivity to mROS-induced mitochondrial permeability transition pore opening. To further study the role of Mrs2 in cardiac mitochondrial metabolism and cellular energetics, we have successfully adopted the CRISPR/Cas9 mediated gene targeting strategy to generate the cardiac-specific Mrs2 knock out mouse model. Our study is the first of its kind to characterize the mitochondrial Mg 2+ channel and its impact on mitochondrial copy number and cell viability. Our findings not only identify Mrs2 as an authentic mitochondrial Mg 2+ channel, but also validates the critical role of mMg 2+ in maintaining the bioenergetic state of the cell.

scholarly journals Midkine-a regulates the formation of a fibrotic scar during zebrafish heart regeneration

2021 ◽  
Author(s):  
Dimitrios Grivas ◽  
Álvaro González-Rajal ◽  
José Luis de la Pompa
Keyword(s):  
Critical Role ◽  
Cardiac Injury ◽  
Transcriptional Analysis ◽  
Endothelial Cell Proliferation ◽  
Heart Regeneration ◽  
Knock Out ◽  
Tgfβ Signalling ◽  
Fibrotic Scar ◽  
Zebrafish Heart

AbstractThe adult zebrafish heart regenerates after injury, unlike the hearts of mammals. Heart cryoinjury triggers the formation of a fibrotic scar that gradually degrades, leading to regeneration. Midkine-a (Mdka) is a multifunctional cytokine that is activated after cardiac injury. Here, we investigated the role of mdka in zebrafish heart regeneration. We show that mdka expression is strongly induced at 1-day post cryoinjury (dpci) throughout the epicardium, whereas by 7 dpci expression has become restricted to epicardial cells covering the injured area. To study the role of mdka in heart regeneration, we generated mdka-knock out (KO) zebrafish strains. Analysis of injured hearts showed that loss of mdka decreased endothelial cell proliferation and resulted in a blockade of heart regeneration characterized by retention of a collagenous scar. Transcriptional analysis revealed increases in collagen transcription and TGFβ signalling activity. These results reveal a critical role for mdka in fibrosis regulation during heart regeneration.

scholarly journals Midkine-a Regulates the Formation of a Fibrotic Scar During Zebrafish Heart Regeneration

2021 ◽  
Vol 9 ◽  
Author(s):  
Dimitrios Grivas ◽  
Álvaro González-Rajal ◽  
José Luis de la Pompa
Keyword(s):  
Critical Role ◽  
Cardiac Injury ◽  
Transcriptional Analysis ◽  
Endothelial Cell Proliferation ◽  
Heart Regeneration ◽  
Knock Out ◽  
Injured Area ◽  
Fibrotic Scar ◽  
Zebrafish Heart

Unlike the hearts of mammals, the adult zebrafish heart regenerates after injury. Heart cryoinjury in zebrafish triggers the formation of a fibrotic scar that gradually degrades, leading to regeneration. Midkine-a (Mdka) is a multifunctional cytokine that is activated after cardiac injury. Here, we investigated the role of mdka in zebrafish heart regeneration. We show that mdka expression was induced at 1-day post-cryoinjury (dpci) throughout the epicardial layer, whereas by 7 dpci expression had become restricted to the epicardial cells covering the injured area. To study the role of mdka in heart regeneration, we generated mdka-knock out (KO) zebrafish strains. Analysis of injured hearts showed that loss of mdka decreased endothelial cell proliferation and resulted in an arrest in heart regeneration characterized by retention of a collagenous scar. Transcriptional analysis revealed increases in collagen transcription and intense TGFβ signaling activity. These results reveal a critical role for mdka in fibrosis regulation during heart regeneration.

scholarly journals Complement Factor C7 Contributes to Lung Immunopathology Caused byMycobacterium tuberculosis

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Kerry J. Welsh ◽  
Cole T. Lewis ◽  
Sydney Boyd ◽  
Michael C. Braun ◽  
Jeffrey K. Actor
Keyword(s):  
Post Infection ◽  
Critical Role ◽  
Complement Factor ◽  
Wild Type ◽  
Knock Out ◽  
Host Protection ◽  
Colony Forming Units ◽  
Knock Out Mice ◽  
The Complement System

Mycobacterium tuberculosis(MTB) remains a significant global health burden despite the availability of antimicrobial chemotherapy. Increasing evidence indicates a critical role of the complement system in the development of host protection against the bacillus, but few studies have specifically explored the function of the terminal complement factors. Mice deficient in complement C7 and wild-type C57BL/6 mice were aerosol challenged with MTB Erdman and assessed for bacterial burden, histopathology, and lung cytokine responses at days 30 and 60 post-infection. Macrophages isolated from C7 −/− and wild-type mice were evaluated for MTB proliferation and cytokine production. C7 −/− mice had significantly less liver colony forming units (CFUs) at day 30; no differences were noted in lung CFUs. The C7 deficient mice had markedly reduced lung occlusion with significantly increased total lymphocytes, decreased macrophages, and increased numbers of CD4+ cells 60 days post-infection. Expression of lung IFN-γand TNF-αwas increased at day 60 compared to wild-type mice. There were no differences in MTB-proliferation in macrophages isolated from wild-type and knock-out mice. These results indicate a role for complement C7 in the development of MTB induced immunopathology which warrants further investigation.

scholarly journals Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mihaela Jagrić ◽  
Patrik Risteski ◽  
Jelena Martinčić ◽  
Ana Milas ◽  
Iva M Tolić
Keyword(s):  
Metaphase Chromosome ◽  
Mechanical Coupling ◽  
Chromosome Position ◽  
Sister Kinetochore ◽  
Chromosome Alignment ◽  
Bridging Fibers ◽  
Optogenetic Control

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

scholarly journals Double knock-out of Hmga1 and Hipk2 genes causes perinatal death associated to respiratory distress and thyroid abnormalities in mice

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Raffaele Gerlini ◽  
Elena Amendola ◽  
Andrea Conte ◽  
Valeria Valente ◽  
Mara Tornincasa ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Perinatal Death ◽  
Critical Role ◽  
Surfactant Proteins ◽  
Drastic Reduction ◽  
Differentiation Markers ◽  
Altered Expression ◽  
Knock Out

Abstract The serine–threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.

Abstract W P365: Critical Role of Smooth Muscle Cell Phenotypic Modulation in Cerebral Aneurysm Formation and Rupture

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Robert M Starke ◽  
Muhammad S Ali ◽  
Nohra Chalouhi ◽  
Pascal M Jabbour ◽  
Stavropoula I Tjoumakaris ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Carotid Arteries ◽  
Critical Role ◽  
Phenotypic Modulation ◽  
Knock Out ◽  
Aneurysm Formation ◽  
Tnf Α ◽  
Knock Out Mice

Objective: Little is known about smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. TNF-α has been associated with aneurysms, but a direct role has not been established. Methods: Cultured cerebral SMC were treated with TNF-α for PCR, western blot, chromatin immune-precipitation (CHIP), and adenovirus promoter transfection. In vivo experiments were carried out in the following models: application of TNF-α to the surface of carotid arteries, cerebral model of hypertension and hemodynamic stress, and cerebral model of aneurysm formation and rupture. The TNF-α inhibitor 3,6’dithiothalidomide (DTH) was synthesized. Results: Cultured cerebral SMC over-expressing myocardin induced expression of key SMC contractile genes (SM-α-actin, SM-22α, SM-MHC), while dominant negative suppressed expression. TNF-α treatment inhibited this contractile phenotype and induced pro-inflammatory genes (MCP-1, MMPs, VCAM-1, IL-1β). TNF-α increased expression of KLF4 and KLF4 siRNA abrogated TNF-α induced phentotypic modulation. These mechanisms were confirmed in vivo following exposure of rat carotid arteries to TNF-α and early in a model of cerebral hypertension and hemodynamic stress prior to cerebral aneurysm formation. Treatment with DTH reversed these pathological vessel wall alterations. TNF-α knock-out mice and DTH pre-treatment decreased the incidence of aneurysm formation and rupture. As compared with sham mice, TNF-α expression was not significantly different in TNF-α knock-out mice or those pre-treated with DTH, but was elevated in unruptured and ruptured aneurysms. Initiation of DTH 7 days after aneurysm induction did not alter aneurysm incidence, but resulted in stabilization and decreased rupture. CHIP assays in vivo and in vitro demonstrated that TNF-α promotes epigenetic changes through KLF4 dependent alterations in promoter regions of myocardin, SMC’s, and inflammatory genes. Conclusion: TNF-α induces phenotypic modulation of cerebral SMC through myocardin and KLF4 regulated pathways. These results demonstrate a novel role for TNF-α in promoting a pro-inflammatory phenotype. These data suggests a critical role of TNF-α in the formation and rupture of aneurysms.

scholarly journals Development of opioid-induced hyperalgesia depends on reactive astrocytes controlled by Wnt5a signaling

2021 ◽  
Author(s):  
XIN LIU ◽  
Chilman Bae ◽  
Bolong Liu ◽  
Yongmei Zhang ◽  
Xiangfu Zhou ◽  
...  
Keyword(s):  
Neural Circuit ◽  
Critical Role ◽  
Wnt Signaling Pathway ◽  
Opioid Analgesics ◽  
Reactive Astrocytes ◽  
Opioid Overdose ◽  
Knock Out ◽  
Genetic Ablation ◽  
Opioid Induced Hyperalgesia

Opioid analgesics are the frontline pain medicine for managing various types of pain. Paradoxically, repeated use of opioid analgesics may cause an exacerbated pain state known as opioid-induced hyperalgesia (OIH). OIH significantly contributes to dose escalation and consequently opioid overdose. In addition to neuronal malplasticity, emerging evidence suggests a critical role of reactive glia in OIH development. A potential astrocytic underpinning of OIH pathogenesis is indicated by their prominently activation in OIH animal models. However, this hypothesis has not been conclusively tested and the mechanism underlying the astrocyte activation remains unclear. Here, we show that reactive astrocytes (a.k.a. astrogliosis) are critical for OIH development in mice. Genetic ablation of astrogliosis inhibited the expression of OIH and morphine-induced neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We also found that Wnt5a is a neuron-to-astrocyte signal that is required for morphine-induced astrogliosis. Conditional knock-out of Wnt5a in neurons or its co-receptor ROR2 in astrocytes blocked not only morphine-induced astrogliosis but also OIH and NCP. Furthermore, we showed that the Wnt5a-ROR2 signaling-dependent astrogliosis contributes to OIH via inflammasome-regulated IL-1β. Our results reveal an important role of morphine-induced astrogliosis in OIH pathogenesis and elucidate a neuron-to-astrocyte intercellular Wnt signaling pathway that controls the astrogliosis.

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