Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

• MeSH
• aktinin MeSH
• aktiny analýza   MeSH
• buněčné jádro chemie   ultrastruktura   MeSH
• HeLa buňky MeSH
• jaderné proteiny analýza   MeSH
• lidé MeSH
• lymfocyty chemie   ultrastruktura   MeSH
• mikrofilamentové proteiny analýza   MeSH
• paxilin MeSH
• proteiny aktivující GTPasu MeSH
• spektrin MeSH
• tropomyosin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktinin MeSH
• aktiny MeSH
• jaderné proteiny MeSH
• mikrofilamentové proteiny MeSH
• paxilin MeSH
• proteiny aktivující GTPasu MeSH
• rho GTPase-activating protein MeSH Prohlížeč
• spektrin MeSH
• tropomyosin MeSH

BACKGROUND: Antibodies to human sperm are useful diagnostic reagents for detection of changes in sperm protein expression and their relationship with sperm defects and male infertility. The specificity of Hs-16 monoclonal antibody (mAb) and the localization and frequency of the occurrence of Hs-16-recognized protein on human spermatozoa were investigated. METHODS: Samples from 30 fertile men with normal spermiograms and 30 men with pathological spermiograms were studied. The specificity of Hs-16 mAb was analysed by the western blotting technique and matrix-assisted laser desorption/ionization mass spectrometry. Indirect immunofluorescence with Hs-16 antibody was used to test sperm ejaculates. RESULTS: The Hs-16 antibody detected a human sperm and seminal plasma protein, which was determined to be secretory actin-binding protein (SABP). This specificity was also verified by co-localization of SABP and actin on spermatozoa with Hs-16 and anti-actin antibodies, and partial co-localization of these proteins was found. SABP was localized on the sperm tail, mainly in the midpiece of the tail. Other parts of spermatozoa were labelled with lower frequency. A significant difference was found in SABP labelling between men with normal spermiograms and donors with asthenozoospermia or oligoasthenoteratozoospermia (both P < 0.01), and asthenozoospermia versus oligoasthenoteratozoospermia (P < 0.05). Increased expression of SABP was observed in men with pathological spermiograms. CONCLUSIONS: Hs-16 antibody reacts specifically with SABP. SABP can serve as a marker of defective sperm and may be associated with fertility failure.

• MeSH
• aktiny analýza   MeSH
• dospělí MeSH
• fluorescenční protilátková technika nepřímá MeSH
• lidé MeSH
• mikrofilamentové proteiny biosyntéza   metabolismus   MeSH
• monoklonální protilátky MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• spermie imunologie   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• mikrofilamentové proteiny MeSH
• monoklonální protilátky MeSH

Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.

• Klíčová slova
• MPRIP, PIP2, actin, nucleus, phase separation,
• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• aktiny metabolismus   MeSH
• buněčné jádro účinky léků   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• glykoly farmakologie   MeSH
• lidé MeSH
• myosin typu I metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• RNA-polymerasa II metabolismus   MeSH
• subcelulární frakce metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• aktiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• glykoly MeSH
• hexamethylene glycol MeSH Prohlížeč
• MPRIP protein, human MeSH Prohlížeč
• MYO1C protein, human MeSH Prohlížeč
• myosin typu I MeSH
• RNA-polymerasa II MeSH
• zelené fluorescenční proteiny MeSH

In many eukaryotic lineages, the RHO clade of small GTPases controls microfilament dynamics by direct binding to formin family actin nucleators. A new study in plants reveals that formin activity can also be regulated by a RHO cofactor rather than the GTPase itself.

• MeSH
• aktiny metabolismus   MeSH
• forminy * metabolismus   genetika   MeSH
• mikrofilamenta metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   genetika   MeSH
• rho proteiny vázající GTP metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH
• forminy * MeSH
• mikrofilamentové proteiny MeSH
• rho proteiny vázající GTP MeSH

The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.

• MeSH
• adaptorové proteiny signální transdukční MeSH
• aktiny genetika   imunologie   metabolismus   MeSH
• antigeny imunologie   MeSH
• buňky kostní dřeně cytologie   imunologie   metabolismus   MeSH
• cytoskelet genetika   imunologie   metabolismus   MeSH
• mastocyty cytologie   imunologie   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• proteiny genetika   imunologie   metabolismus   MeSH
• rac proteiny vázající GTP genetika   imunologie   metabolismus   MeSH
• rho proteiny vázající GTP genetika   imunologie   metabolismus   MeSH
• rhoA protein vázající GTP MeSH
• signální transdukce genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• aktiny MeSH
• antigeny MeSH
• LAT2 protein, mouse MeSH Prohlížeč
• proteiny MeSH
• rac proteiny vázající GTP MeSH
• rho proteiny vázající GTP MeSH
• rhoA protein vázající GTP MeSH
• RhoA protein, mouse MeSH Prohlížeč

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

• MeSH
• aktin zastřešující proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• fosfatidylinositolfosfáty chemie   metabolismus   MeSH
• fylogeneze MeSH
• hydrofobní a hydrofilní interakce MeSH
• kur domácí MeSH
• kyseliny fosfatidové chemie   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• proteiny huseníčku antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• ptačí proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• vazba proteinů MeSH
• výpočetní biologie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• aktin zastřešující proteiny MeSH
• fosfatidylinositolfosfáty MeSH
• kyseliny fosfatidové MeSH
• proteiny huseníčku MeSH
• ptačí proteiny MeSH

Microtubule (MT) and F-actin cytoskeletal cross-talk and organization are important aspects of axon guidance mechanisms, but how associated proteins facilitate this function remains largely unknown. While the MT-associated protein, CKAP5 (XMAP215/ch-TOG), has been best characterized as a MT polymerase, we have recently highlighted a novel role for CKAP5 in facilitating interactions between MT and F-actin in vitro and in embryonic Xenopus laevis neuronal growth cones. However, the mechanism by which it does so is unclear. Here, using in vitro reconstitution assays coupled with total internal reflection fluorescence microscopy, we report that the TOG5 domain of CKAP5 is necessary for its ability to bind to and bundle actin filaments, as well as to cross-link MTs and F-actin in vitro. Additionally, we show that this novel MT/F-actin cross-linking function of CKAP5 is possible even in MT polymerase-incompetent mutants of CKAP5 in vivo. Indeed, CKAP5 requires both MT and F-actin binding, but not MT polymerization, to promote MT-F-actin alignment in growth cones and axon outgrowth. Taken together, our findings provide mechanistic insights into how MT populations penetrate the growth cone periphery through CKAP5-facilitated interaction with F-actin during axon outgrowth and guidance.

• MeSH
• aktiny * metabolismus   MeSH
• axony metabolismus   MeSH
• mikrofilamenta * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• neurony * metabolismus   MeSH
• proteinové domény MeSH
• proteiny asociované s mikrotubuly * metabolismus   MeSH
• proteiny Xenopus * metabolismus   MeSH
• růstové kužele * metabolismus   MeSH
• vazba proteinů MeSH
• Xenopus laevis * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny * MeSH
• CKAP5 protein, Xenopus MeSH Prohlížeč
• proteiny asociované s mikrotubuly * MeSH
• proteiny Xenopus * MeSH

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.

• Klíčová slova
• actin, microtubules, mitosis, mitotic spindle, protein kinase,
• MeSH
• aktiny metabolismus   MeSH
• aparát dělícího vřeténka * metabolismus   MeSH
• HeLa buňky MeSH
• kasein kinasa I metabolismus   genetika   MeSH
• lidé MeSH
• mikrofilamenta * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• mitóza * MeSH
• proteiny buněčného cyklu metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH
• FAM110A protein, human MeSH Prohlížeč
• kasein kinasa I MeSH
• proteiny buněčného cyklu MeSH

MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.

• MeSH
• aktiny metabolismus   chemie   MeSH
• alosterická regulace MeSH
• calponiny MeSH
• elektronová kryomikroskopie * MeSH
• lidé MeSH
• mikrofilamenta metabolismus   ultrastruktura   MeSH
• mikrofilamentové proteiny metabolismus   chemie   ultrastruktura   MeSH
• molekulární modely MeSH
• oxygenasy se smíšenou funkcí MeSH
• proteinové domény MeSH
• proteiny s doménou LIM metabolismus   chemie   genetika   MeSH
• vazba proteinů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH
• calponiny MeSH
• MICAL1 protein, human MeSH Prohlížeč
• mikrofilamentové proteiny MeSH
• oxygenasy se smíšenou funkcí MeSH
• proteiny s doménou LIM MeSH

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• biologický transport genetika   fyziologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• mikrofilamenta metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny vázající takrolimus genetika   metabolismus   MeSH
• regulace genové exprese u rostlin genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• proteiny vázající takrolimus MeSH
• TWD1 protein, Arabidopsis MeSH Prohlížeč