Aggregation of high-affinity IgE receptors (FcεRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcεRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.
Microtubule dynamic is exceptionally sensitive to modulation by small-molecule ligands. Our previous work presented the preparation of microtubule-targeting estradiol dimer (ED) with anticancer activity. In the present study, we explore the effect of selected linkers on the biological activity of the dimer. The linkers were designed as five-atom chains with carbon, nitrogen or oxygen in their centre. In addition, the central nitrogen was modified by a benzyl group with hydroxy or methoxy substituents and one derivative possessed an extended linker length. Thirteen new dimers were subjected to cytotoxicity assay and cell cycle profiling. Dimers containing linker with benzyl moiety substituted with one or more methoxy groups and longer branched ones were found inactive, whereas other structures had comparable efficacy as the original ED (e.g. D1 with IC50 = 1.53 μM). Cell cycle analysis and immunofluorescence proved the interference of dimers with microtubule assembly and mitosis. The proposed in silico model and calculated binding free energy by the MM-PBSA method were closely correlated with in vitro tubulin assembly assay.
- MeSH
- antitumorózní látky * chemie farmakologie MeSH
- apoptóza MeSH
- ethinylestradiol * chemie farmakologie MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- mikrotubuly MeSH
- modulátory tubulinu * chemie farmakologie MeSH
- nádorové buněčné linie MeSH
- triazoly * chemie farmakologie MeSH
- tubulin * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: To investigate the structural bases of human oocytes' cytoplasmic abnormalities and the causative mechanism of their emergence. Knowledge of an abnormal oocyte's intracellular organization is vital to establishing reliable criteria for clinical evaluation of oocyte morphology. DESIGN: Laboratory-based study on experimental material provided by a private assisted reproduction clinic. SETTING: University laboratory and imaging center. PATIENTS: A total of 105 women undergoing hormonal stimulation for in vitro fertilization (IVF) donated their spare oocytes for this study. INTERVENTIONS: Transmission electron microscopy (TEM) was used to analyze the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely, refractile bodies; centrally located cytoplasmic granularity (CLCG); smooth endoplasmic reticulum (SER) disc; and vacuoles. A total of 133 immature oocytes were exposed to cytoskeleton-targeting compounds or matured in control conditions, and their morphology was examined using fluorescent and electron microscopy. MAIN OUTCOME MEASURES: The ultrastructural morphology of dysmorphic oocytes was analyzed. Drug-treated oocytes had their maturation efficiency, chromosome-microtubule configurations, and fine intracellular morphology examined. RESULTS: TEM revealed ultrastructural characteristics of common oocyte aberrations and indicated that excessive organelle clustering was the underlying cause of 2 of the studied morphotypes. Inhibition experiments showed that disruption of actin, not microtubules, allows for inordinate aggregation of subcellular structures, resembling the ultrastructural pattern seen in morphologically abnormal oocytes retrieved in IVF cycles. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. CONCLUSION: The ultrastructural analogy between dysmorphic oocytes and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to a more objective oocyte quality assessment in IVF practice.
- MeSH
- aktiny * MeSH
- cytoplazma MeSH
- cytoskelet MeSH
- lidé MeSH
- mikrotubuly MeSH
- oocyty * ultrastruktura MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.
- MeSH
- cilie metabolismus MeSH
- flagella * metabolismus MeSH
- mikrotubuly metabolismus MeSH
- myši MeSH
- protein-serin-threoninkinasy * metabolismus MeSH
- proteiny metabolismus MeSH
- savci metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.
- MeSH
- axony metabolismus MeSH
- centrozom MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mozek metabolismus MeSH
- neurony * fyziologie MeSH
- tubulin * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MAP/microtubule affinity-regulating kinases (MARKs) were recently identified as potential drug targets for Alzheimer's disease (AD) due to their role in pathological hyperphosphorylation of tau protein. Hyperphosphorylated tau has decreased affinity for microtubule binding, impairing their stability and associated functions. Destabilization of microtubules in neuronal cells leads to neurodegeneration, and microtubule-unbound tau forms neurofibrillary tangles, one of the primary hallmarks of AD. Many phosphorylation sites of tau protein have been identified, but phosphorylation at Ser262, which occurs in early stages of AD, plays a vital role in the pathological hyperphosphorylation of tau. It has been found that Ser262 is phosphorylated by MARK4, which is currently an intensively studied target for treating Alzheimer's disease and other neurodegenerative diseases. Our present study aimed to develop a high throughput compatible assay to directly detect MARK enzymatic activity using echoacoustic transfer and MALDI-TOF mass spectrometer. We optimized the assay for all four isoforms of MARK and validated its use for identifying potential inhibitors by the screening of 1280 compounds from the LOPAC®1280 International (Library Of Pharmacologically Active Compounds). Six MARK4 inhibitors with IC50 < 1 μM were identified. To demonstrate their therapeutic potential, active compounds were further tested for MARK4 selectivity and ability to cross the blood-brain barrier. Lastly, the molecular docking with the most active inhibitors to predict their interaction with MARK4 was performed.
- MeSH
- Alzheimerova nemoc farmakoterapie MeSH
- fosforylace fyziologie MeSH
- hematoencefalická bariéra metabolismus MeSH
- inhibiční koncentrace 50 MeSH
- mikrotubuly metabolismus MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory MeSH
- proteiny tau metabolismus MeSH
- simulace molekulového dockingu MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.
- MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- neurodegenerativní nemoci * metabolismus MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny tau * metabolismus MeSH
- proteiny metabolismus MeSH
- tubulin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.
- MeSH
- extracelulárním signálem regulované MAP kinasy * metabolismus MeSH
- fosforylace MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinkinasy řízené prolinem * metabolismus MeSH
- proteiny asociované s mikrotubuly * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microtubules composed of tubulin heterodimers represent highly dynamic structures. These structures are essential for basic cellular functions, such as cell division. Microtubules can grow or shrink in response to environmental signals, principally chemical cues. Here, we provide an alternative-physical-strategy to modulate tubulin properties and its self-assembly process. The conformation and electrical properties of tubulin subunits are modulated by nanosecond electropulse signals. The formed structures of electrically treated tubulin are tightly linked to the degree of conformational and electrical properties changes induced by nanosecond electropulses. This strategy opens a new way for controlling the self-assembly process in biomolecules as well as in bioinspired materials.
Intracellular trafficking of organelles driven by molecular motors underlies essential cellular processes. Mitochondria, the powerhouses of the cell, are one of the major cargoes of molecular motors. Efficient distribution of mitochondria ensures cellular fitness while defects in this process contribute to severe pathologies, such as neurodegenerative diseases. Reconstitution of the mitochondrial microtubule-based transport in vitro in a bottom-up approach provides a powerful tool to investigate the mitochondrial trafficking machinery in a controlled environment in the absence of complex intracellular interactions. In this chapter, we describe the procedures for achieving such reconstitution of mitochondrial transport.
- MeSH
- biologický transport MeSH
- kineziny * MeSH
- mikrotubuly * metabolismus MeSH
- mitochondrie metabolismus MeSH
- organely MeSH
- Publikační typ
- časopisecké články MeSH
