scholarly journals Molecular requirements for transition from lateral to end-on microtubule binding and dynamic coupling

2018 ◽  
Author(s):  
Manas Chakraborty ◽  
Ekaterina Tarasovetc ◽  
Anatoly V. Zaytsev ◽  
Maxim Godzi ◽  
Ana C. Figueiredo ◽  
...  
Keyword(s):  
Brownian Dynamics ◽  
Primary Role ◽  
Dynamics Model ◽  
Dynamic Coupling ◽  
Microtubule Binding ◽  
Microtubule Attachment ◽  
Close Proximity ◽  
Dynamic Microtubule

ABSTRACTAccurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are ill-understood. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

scholarly journals Depletion of Centromeric MCAK Leads to Chromosome Congression and Segregation Defects Due to Improper Kinetochore Attachments

2004 ◽  
Vol 15 (3) ◽  
pp. 1146-1159 ◽  
Author(s):  
Susan L. Kline-Smith ◽  
Alexey Khodjakov ◽  
Polla Hergert ◽  
Claire E. Walczak
Keyword(s):  
Essential Role ◽  
Dominant Negative ◽  
Primary Role ◽  
Complex Behavior ◽  
Chromosome Movement ◽  
Immunofluorescence Localization ◽  
Microtubule Attachment ◽  
Chromosome Congression

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore–microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore–microtubule attachments.

scholarly journals RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

2022 ◽  
Author(s):  
Shan Qi ◽  
Javier Mota ◽  
Siu-Hong Chan ◽  
Johanna Villarreal ◽  
Nan Dai ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rna Binding ◽  
Methyl Group ◽  
High Affinity ◽  
Methylation Of Dna ◽  
Dna And Rna ◽  
Close Proximity ◽  
Methylation Activity

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

scholarly journals On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Affinity of Deaza-Epothilone C

Chemistry ◽  
2020 ◽  
Vol 2 (2) ◽  
pp. 499-509
Author(s):  
Adriana Edenharter ◽  
Lucie Ryckewaert ◽  
Daniela Cintulová ◽  
Juan Estévez-Gallego ◽  
José Fernando Díaz ◽  
...  
Keyword(s):  
Decisive Role ◽  
Ring Closure ◽  
Side Chain ◽  
Microtubule Binding ◽  
Acid Fragment ◽  
Epothilone A ◽  
Thiophene Moiety ◽  
Free Energy Of Binding

Deaza-epothilone C, which incorporates a thiophene moiety in place of the thiazole heterocycle in the natural epothilone side chain, has been prepared by semisynthesis from epothilone A, in order to assess the contribution of the thiazole nitrogen to microtubule binding. The synthesis was based on the esterification of a known epothilone A-derived carboxylic acid fragment and a fully synthetic alcohol building block incorporating the modified side chain segment and subsequent ring-closure by ring-closing olefin metathesis. The latter proceeded with unfavorable selectivity and in low yield. Distinct differences in chemical behavior were unveiled between the thiophene-derived advanced intermediates and what has been reported for the corresponding thiazole-based congeners. Compared to natural epothilone C, the free energy of binding of deaza-epothilone C to microtubules was reduced by ca. 1 kcal/mol or less, thus indicating a distinct but non-decisive role of the thiazole nitrogen in the interaction of epothilones with the tubulin/microtubule system. In contrast to natural epothilone C, deaza-epothilone C was devoid of antiproliferative activity in vitro up to a concentration of 10 μM, presumably due to an insufficient stability in the cell culture medium.

Role of fatty acid oxidation in mechanism of action of gastric secretagogues

1980 ◽  
Vol 238 (3) ◽  
pp. G255-G262
Author(s):  
J. Chacin ◽  
G. Martinez ◽  
E. Severin
Keyword(s):  
Fatty Acid ◽  
Gastric Mucosa ◽  
Acid Secretion ◽  
Fatty Acid Oxidation ◽  
Acid Oxidation ◽  
Primary Role ◽  
Dependent Manner ◽  
Beta Oxidation

The role of beta-oxidation in the mechanism of stimulation of acid secretion was examined in toad gastric mucosa in vitro. The incubation with 4-pentenoate selectively inhibited in a dose-dependent manner the rate of 14CO2 formation from [1-14C]octanoate. Pretreatment with 20 mM 4-pentenoate sharply reduced the respiratory and secretory responses to theophylline and histamine. Tracer studies showed a major utilization of exogenous octanoate over glucose and pyruvate by the in vitro toad gastric mucosa. Theophylline and histamine stimulated by 69% the rate of octanoate oxidation. Over 60% of the increments in oxygen uptake produced by theophylline and histamine accounted for the increments in octanoate oxidation, whereas glucose and pyruvate together accounted for less than 25%. Octanoate-dependent respiration was shown to correlate with octanoate oxidation under both inhibition with 4-pentenoate and stimulation with theophylline. Theophylline stimulated by 25% the rate of octanoate oxidation in Cl--free glucuronate-nutrient solutions. The present work provides further evidence for the primary role of fatty acid oxidation in the mechanism of acid secretion in amphibian.

scholarly journals Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection.

1994 ◽  
Vol 180 (4) ◽  
pp. 1273-1282 ◽  
Author(s):  
M B Graham ◽  
V L Braciale ◽  
T J Braciale
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Lymphocytes ◽  
Cd4 T Cells ◽  
Primary Role ◽  
T Helper ◽  
Helper Type

T lymphocytes play a primary role in recovery from viral infections and in antiviral immunity. Although viral-specific CD8+ and CD4+ T cells have been shown to be able to lyse virally infected targets in vitro and promote recovery from lethal infection in vivo, the role of CD4+ T lymphocytes and their mechanism(s) of action in viral immunity are not well understood. The ability to further dissect the role that CD4+ T cells play in the immune response to a number of pathogens has been greatly enhanced by evidence for more extensive heterogeneity among the CD4+ T lymphocytes. To further examine the role of CD4+ T cells in the immune response to influenza infection, we have generated influenza virus-specific CD4+ T cell clones from influenza-primed BALB/c mice with differential cytokine secretion profiles that are defined as T helper type 1 (Th1) clones by the production of interleukin 2 (IL-2) and interferon gamma (IFN-gamma), or as Th2 clones by the production of IL-4, IL-5, and IL-10. Our studies have revealed that Th1 clones are cytolytic in vitro and protective against lethal challenge with virus in vivo, whereas Th2 clones are noncytolytic and not protective. Upon further evaluation of these clonal populations we have shown that not only are the Th2 clones nonprotective, but that pulmonary pathology is exacerbated as compared with control mice as evidenced by delayed viral clearance and massive pulmonary eosinophilia. These data suggest that virus-specific CD4+ T cells of the Th2 subset may not play a primary role in virus clearance and recovery and may lead to immune mediated potentiation of injury.

Possible Role of Each Repeat Structure of the Microtubule-Binding Domain of the Tau Protein in In Vitro Aggregation

2005 ◽  
Vol 138 (4) ◽  
pp. 413-423 ◽  
Author(s):  
Koji Tomoo ◽  
Tian-Ming Yao ◽  
Katsuhiko Minoura ◽  
Shuko Hiraoka ◽  
Miho Sumida ◽  
...  
Keyword(s):  
Tau Protein ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Repeat Structure ◽  
Microtubule Binding Domain

AlphaVBeta3 (αvβ3) Integrin Drives the Osteoclastogenesis through a Osteoclast-Like Functional Differentiation of Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 814-814 ◽  
Author(s):  
Marco Tucci ◽  
Lucia Lombardi ◽  
Steve Reshkin ◽  
Rosa A. Cardone ◽  
Silvestris Franco
Keyword(s):  
Bone Erosion ◽  
Bone Lesions ◽  
Αvβ3 Integrin ◽  
Primary Role ◽  
Myeloma Cells ◽  
Group A ◽  
Group B ◽  
Rpmi 8226

Abstract Accelerated osteoclastogenesis is the major event promoting the skeletal impairment in multiple myeloma (MM). Osteoclasts (OC) are directly activated by myeloma cells (MC), although these cells themselves may apparently undergo to OC-like morphologic transformation and produce bone erosion in vitro. Since OCs exert their function and promote osteoclastogenesis through activation of several adhesion molecules, including avb3, we investigated the role of this integrin expressed by MCs in their OC-like activity in vitro. Bone marrow MCs were purified from eight patients with severe skeletal disease (group A) and from two patients without bone lesions (group B). U266 and RPMI-8226 MC lines were the controls. Semi-nested PCR assessed the CDR3 immunoglobulin (Ig) gene rearrangement, whereas OC markers including TRAcP, cathepsin-k, calcitonin-receptor, carbonic anhydrase and vATPase were evaluated by RT-PCR. The cytoskeletal rearrangement of F-actin was analyzed by immunofluorescence. av and b3 expression on MCs was evaluated by flow-cytometry, whereas bone erosion on calcium phosphate discs and number of pits was measured by dedicated software. The effect of avb3 stimulation on the activation of osteoclastogenic function was investigated by exploring the phosphorilation of transcriptional kinases and downstream molecules, as ERK1/2 and cFos, respectively. The primary role of avb3 in OC-like functional transdifferentiation was explored in MCs by siRNA silencing for both chains. Ontogenetic derivation from the B-cell lineage was confirmed by the monoclonal CDR3 rearrangement, CD138/CD38 and Pax-5 expression. Cells from patients of group A expressed OC markers, in contrast with those of group B or U266 and RPMI-8226. Formation of the F-actin ring confirmed the differentiation of MCs toward the OC-like phenotype. Cells from group A expressed av and b3 (80±7% and 75±9%) similarly to U266 and RPMI-8226 (>90% in both instances), whereas a minimal expression was demonstrated in group B (av:6±2%; b3:8±3%). avb3+ cells produced a high number of erosive pits, at variance from avb3− cells (35±8 vs. 4±1 pits/cm2 ). The highest phosphorilation of ERK1/2 and expression of cFos was revealed in patients of group A as compared to B (840±110 OD and 905±210 OD vs. 270±35 OD and 315±80 OD, p<0.0001 in both instances). Finally, the silencing of av and/or b3 chain inhibited the erosion by avb3+ cells, resulting in a reduced number of pits (7±2 pits/cm2) similar to the pattern obtained with avb3− cells. Similarly, an inhibition of both ERK1/2 and cFos was demonstrated in silenced cells from group A with values similar to those of group B. Since avb3 drives both adhesion of Ocs to extracellular matrix and activation of osteoclastogenesis, it is conceivable that avb3+ MCs may functionally transdifferentiate to OCs in response to integrin activation induced by the contact with stromal cells within the marrow environment, thus resulting in a typical OC-like behaviour. In addition, our data suggest that MCs induce this effect by avb3, since its silencing reduces the OC-like activity in vitro.

scholarly journals Influence of fibrinolitic potential reduction in ischemic brain disease development

2008 ◽  
Vol 61 (5-6) ◽  
pp. 247-251 ◽  
Author(s):  
Biljana Vuckovic ◽  
Mirjana Djeric ◽  
Marija Zarkov
Keyword(s):  
Plasminogen Activator ◽  
Brain Disease ◽  
Fibrinolytic System ◽  
Brain Diseases ◽  
Primary Role ◽  
Plasminogen Activator Inhibitor 1 ◽  
Ischemic Brain ◽  
Pai 1

Introduction Vascular brain diseases are the third leading cause of morbidity and mortality today. Among them ischemic brain disease caused mostly by cerebral atherosclerosis makes almost 80%. The main risk factors for this disease are arterial hypertension, dislipoproteinemia and diabetes but, recently hemostatic system disorders have also been underlined, especially fibrinolytic disorders. Physiology of fibrinolysis The primary role of fibrinolytic system is to make blood vessels passable by prevention of thromb forming or by removing the existing ones. This role is enabled by constant dynamic balance between activators and inhibitors of fibrinolysis. The leading activators are tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) and the most important inhibitors are 2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1) and thrombin activated fibrinolytic inhibitor (TAFI). Fibrinolysis and thrombosis In patients with atherosclerosis there is clear insufficiency of fibrinolysis caused mainly by the increased activity of fibrinolytic inhibitors. There is evidence that the level of PAI-1 connected to fibrin directly affects thromb resistance to fibrinolysis and that velocity of thromb lysis in vitro predominantly depends on TAFI concentration. Factor XIII also has influence on thromb structure and stability and Lp(a) lipoprotein is one more factor that is responsible for resistency of thromb to fibrinolysis. Fibrinolysis in ischemic brain disease Reduced fibrinolytic capacity is noticed in patients with ischemic brain disease resulting from the increased PAI-1 activity. Higher risk for ischemic brain disease is verified among people with 4G/4G PAI-1 genotype. Raised level of t-PA antigene can also induce reduction of endogenous fibrinolysis. Conclusion In conclusion we want to underline that a possible pathophysiological significance of fibrinolytic system is assumed in a high percentage of patients with ischemic brain disease. Because of that we need further investigations to establish precise role of fibrinolytic mechanisms in genesis of this disease.

scholarly journals Direct observation of microtubule-f-actin interaction in cell free lysates

1999 ◽  
Vol 112 (12) ◽  
pp. 1947-1956 ◽  
Author(s):  
J.R. Sider ◽  
C.A. Mandato ◽  
K.L. Weber ◽  
A.J. Zandy ◽  
D. Beach ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Microtubule Binding ◽  
Cellular Processes ◽  
Close Proximity ◽  
Mutual Regulation ◽  
Egg Extracts ◽  
Xenopus Egg Extracts ◽  
In Vitro Interaction

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.

scholarly journals The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Thibaut Laboute ◽  
Jorge Gandía ◽  
Lucie P Pellissier ◽  
Yannick Corde ◽  
Florian Rebeillard ◽  
...  
Keyword(s):  
G Protein ◽  
Opioid Receptors ◽  
Orphan Receptor ◽  
Locomotor Sensitization ◽  
Promising Therapeutic Target ◽  
Close Proximity ◽  
The Impact ◽  
G Protein Coupled

GPR88 is an orphan G protein-coupled receptor (GPCR) considered as a promising therapeutic target for neuropsychiatric disorders; its pharmacology, however, remains scarcely understood. Based on our previous report of increased delta opioid receptor activity in Gpr88 null mice, we investigated the impact of GPR88 co-expression on the signaling of opioid receptors in vitro and revealed that GPR88 inhibits the activation of both their G protein- and β-arrestin-dependent signaling pathways. In Gpr88 knockout mice, morphine-induced locomotor sensitization, withdrawal and supra-spinal analgesia were facilitated, consistent with a tonic inhibitory action of GPR88 on µOR signaling. We then explored GPR88 interactions with more striatal versus non-neuronal GPCRs, and revealed that GPR88 can decrease the G protein-dependent signaling of most receptors in close proximity, but impedes β-arrestin recruitment by all receptors tested. Our study unravels an unsuspected buffering role of GPR88 expression on GPCR signaling, with intriguing consequences for opioid and striatal functions.

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