nucleation mechanism
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The text is a contemporary continuation of an earlier publication, Kratochvíl B.: Chem. Listy 101, 3 (2007). It describes mainly the nucleation process (two-step nuclea-tion of active substances in pharmacy) and crystallization control processes (seeded crystallization and sonocrystalli-zation). The focus of the work is the description of the nucleation process monitoring by modern analytical tech-nologies, i.e., Focused Beam Reflectance Measurement (FBRM) and the BlazeMetrics system. Both methods pres-ently provide the best available information for a deeper understanding of the nucleation process mechanism in crystallizing active substances. The work is documented by high quality and original photographic attachments of the crystallizing material.Full text English translation is available in the on-line version.
- Klíčová slova
- systém Blaze, nukleace,
- MeSH
- farmaceutická technologie klasifikace metody přístrojové vybavení MeSH
- krystalizace * klasifikace metody přístrojové vybavení MeSH
- lasery MeSH
- výzkumný projekt MeSH
- Publikační typ
- přehledy MeSH
Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Neurons rely on the microtubule cytoskeleton to create and maintain their sophisticated cellular architectures. Advances in cryogenic electron microscopy, expansion microscopy, live imaging, and gene editing have enabled novel insights into mechanisms of centrosomal and acentrosomal microtubule nucleation, the key process generating new microtubules. This has paved the way for the functional dissection of distinct microtubule networks that regulate various processes during neuronal development, including neuronal delamination, polarization, migration, maturation, and synapse function. We review recent progress in understanding the molecular concepts of microtubule nucleation, how these concepts underlie neurodevelopmental processes, and pinpoint the open questions. Since microtubules play a pivotal role in axon regeneration within the adult central nervous system, understanding the processes of microtubule nucleation could inform strategies to enhance the regenerative capabilities of neurons in the future.
- MeSH
- centrozom * metabolismus fyziologie MeSH
- lidé MeSH
- mikrotubuly * metabolismus fyziologie MeSH
- neurogeneze * fyziologie MeSH
- neurony * fyziologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- centrozom metabolismus MeSH
- faktory zaměňující Rho guanin nukleotidy genetika metabolismus MeSH
- fluorescenční mikroskopie MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- imunoblotting MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- p21 aktivované kinasy genetika metabolismus MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- signální transdukce MeSH
- transformované buněčné linie MeSH
- tubulin metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The text is a contemporary continuation of an earlier publication, Kratochvíl B.: Chem. Listy 101, 3 (2007). It describes mainly the nucleation process (two-step nuclea-tion of active substances in pharmacy) and crystallization control processes (seeded crystallization and sonocrystalli-zation). The focus of the work is the description of the nucleation process monitoring by modern analytical tech-nologies, i.e., Focused Beam Reflectance Measurement (FBRM) and the BlazeMetrics system. Both methods pres-ently provide the best available information for a deeper understanding of the nucleation process mechanism in crystallizing active substances. The work is documented by high quality and original photographic attachments of the crystallizing material.
- Klíčová slova
- systém Blaze, nukleace,
- MeSH
- farmaceutická technologie klasifikace metody přístrojové vybavení MeSH
- krystalizace * klasifikace metody přístrojové vybavení MeSH
- lasery MeSH
- výzkumný projekt MeSH
- Publikační typ
- přehledy MeSH
Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.
- MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- jednovláknová DNA * metabolismus genetika MeSH
- lidé MeSH
- multiproteinové komplexy MeSH
- mutace MeSH
- rekombinasa Rad51 * metabolismus genetika MeSH
- replikace DNA * genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nucleation is a core scientific concept that describes the formation of new phases and materials. While classical nucleation theory is applied across wide-ranging fields, nucleation energy landscapes have never been directly measured at the atomic level, and experiments suggest that nucleation rates often greatly exceed the predictions of classical nucleation theory. Multistep nucleation via metastable states could explain unexpectedly rapid nucleation in many contexts, yet experimental energy landscapes supporting such mechanisms are scarce, particularly at nanoscale dimensions. In this work, we measured the nucleation energy landscape of diamond during chemical vapor deposition, using a series of diamondoid molecules as atomically defined protonuclei. We find that 26-carbon atom clusters, which do not contain a single bulk atom, are postcritical nuclei and measure the nucleation barrier to be more than four orders of magnitude smaller than prior bulk estimations. These data support both classical and nonclassical concepts for multistep nucleation and growth during the gas-phase synthesis of diamond and other semiconductors. More broadly, these measurements provide experimental evidence that agrees with recent conceptual proposals of multistep nucleation pathways with metastable molecular precursors in diverse processes, ranging from cloud formation to protein crystallization, and nanoparticle synthesis.
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.
Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.
- MeSH
- buňky kostní dřeně cytologie MeSH
- faktory zaměňující Rho guanin nukleotidy metabolismus MeSH
- mastocyty cytologie MeSH
- mikrotubuly metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- proteiny aktivující GTPasu metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of gamma-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- fosfatidylinositol-3-kinasy genetika metabolismus MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- myši MeSH
- podjednotky proteinů genetika metabolismus MeSH
- protoonkogenní proteiny c-fyn genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- tubulin genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
