Although actin monomers polymerize into filaments in the cytoplasm, the form of actin in the nucleus remains elusive. We searched for the form and function of β-actin fused to nuclear localization signal and to enhanced yellow fluorescent protein (EN-actin). Our results reveal that EN-actin is either dispersed in the nucleoplasm (homogenous EN-actin) or forms bundled filaments in the nucleus (EN-actin filaments). Formation of such filaments was not connected with increased EN-actin levels. Among numerous actin-binding proteins tested, only cofilin is recruited to the EN-actin filaments. Overexpression of EN-actin causes increase in the nuclear levels of actin-related protein 3 (Arp3). Although Arp3, a member of actin nucleation complex Arp2/3, is responsible for EN-actin filament nucleation and bundling, the way cofilin affects nuclear EN-actin filaments dynamics is not clear. While cells with homogenous EN-actin maintained unaffected mitosis during which EN-actin re-localizes to the plasma membrane, generation of nuclear EN-actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-actin manifests in two mitotic-inborn defects-formation of binucleic cells and generation of micronuclei-suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis.

• MeSH
• aktiny metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• faktory depolymerizující aktin MeSH
• genetická transkripce MeSH
• HEK293 buňky MeSH
• lidé MeSH
• luminescentní proteiny metabolismus   MeSH
• mikrofilamenta metabolismus   MeSH
• mitóza genetika   MeSH
• nádorové buněčné linie MeSH
• protein 3 související s aktinem biosyntéza   metabolismus   MeSH
• restrukturace chromatinu MeSH
• RNA-polymerasa I genetika   MeSH
• RNA-polymerasa II genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

• MeSH
• aktiny metabolismus   MeSH
• buněčná adheze MeSH
• buněčné kultury MeSH
• cytoskelet metabolismus   MeSH
• fetální proteiny metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fokální adheze metabolismus   MeSH
• HEK293 buňky MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• melanom experimentální MeSH
• mikrofilamenta metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• pohyb buněk fyziologie   MeSH
• profiliny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Actin networks drive many essential cellular processes, including cell migration, cytokinesis and tissue morphogenesis. However, how cells organize and regulate dynamic actin networks that consist of long, unbranched actin filaments is only poorly understood. This study in mouse oocytes reveals that cells can use vesicles as adaptable, motorized network nodes to regulate the dynamics and density of intracellular actin networks. In particular, Rab11a-positive vesicles drive the network dynamics in a myosin-Vb-dependent manner, and modulate the network density by sequestering and clustering the network's actin nucleators. We also report a simple way by which networks of different densities can be generated, namely by adjusting the number and volume of vesicles in the cell. This vesicle-based mechanism of actin network modulation is essential for asymmetric positioning of the meiotic spindle in mouse oocytes, a vital step in the development of a fertilizable egg in mammals.

• MeSH
• aktiny * metabolismus   MeSH
• aparát dělícího vřeténka * metabolismus   MeSH
• biologické modely MeSH
• cytoplazmatické vezikuly * metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• myosin typu V metabolismus   MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• rab proteiny vázající GTP * metabolismus   MeSH
• těžké řetězce myosinu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

• MeSH
• aktiny metabolismus   MeSH
• Caco-2 buňky MeSH
• centrozom metabolismus   MeSH
• forminy metabolismus   MeSH
• genový knockout MeSH
• lidé MeSH
• melanom experimentální metabolismus   patologie   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• nádory kůže metabolismus   patologie   MeSH
• polymerizace MeSH
• profiliny genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transfekce MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Plant cell growth and morphogenesis depend on remodelling of both actin and microtubule cytoskeletons. AtFH1 (At5g25500), the main housekeeping Arabidopsis formin, is targeted to membranes and known to nucleate and bundle actin. The effect of mutations in AtFH1 on root development and cytoskeletal dynamics was examined. Consistent with primarily actin-related formin function, fh1 mutants showed increased sensitivity to the actin polymerization inhibitor latrunculin B (LatB). LatB-treated mutants had thicker, shorter roots than wild-type plants. Reduced cell elongation and morphological abnormalities were observed in both trichoblasts and atrichoblasts. Fluorescently tagged cytoskeletal markers were used to follow cytoskeletal dynamics in wild-type and mutant plants using confocal microscopy and VAEM (variable-angle epifluorescence microscopy). Mutants exhibited more abundant but less dynamic F-actin bundles and more dynamic microtubules than wild-type seedlings. Treatment of wild-type seedlings with a formin inhibitor, SMIFH2, mimicked the root growth and cell expansion phenotypes and cytoskeletal structure alterations observed in fh1 mutants. The results suggest that besides direct effects on actin organization, the in vivo role of AtFH1 also includes modulation of microtubule dynamics, possibly mediated by actin-microtubule cross-talk.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mikrofilamenta genetika   metabolismus   MeSH
• mikrotubuly genetika   metabolismus   MeSH
• mutace * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system.

• MeSH
• intracelulární membrány metabolismus   MeSH
• mikrofilamenta metabolismus   MeSH
• proteiny asociované s mikrotubuly chemie   genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• rostlinné buňky metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.

• MeSH
• Arabidopsis chemie   genetika   MeSH
• cytoskelet chemie   genetika   MeSH
• mikrofilamenta chemie   genetika   ultrastruktura   MeSH
• mikrotubuly chemie   genetika   MeSH
• mitóza genetika   MeSH
• polymerizace MeSH
• proteinové agregáty genetika   MeSH
• proteiny asociované s mikrotubuly chemie   genetika   MeSH
• tubulin chemie   genetika   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. Here, we report that the actin-binding protein filamin A (FlnA) physically interacts with the actin-nucleating protein formin 2 (Fmn2). Loss of FlnA and Fmn2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FlnA interacts with the Wnt co-receptor Lrp6. Loss of FlnA and Fmn2 impairs Lrp6 endocytosis, downstream Gsk3β activity, and β-catenin accumulation in the nucleus. The proliferative defect in Flna and Fmn2 null neural progenitors is rescued by inhibiting Gsk3β activity. Our findings thus reveal a novel mechanism whereby actin-associated proteins regulate proliferation by mediating the endocytosis and transportation of components in the canonical Wnt pathway. Moreover, the Fmn2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology protein Daam. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation.

• MeSH
• beta-katenin metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčná membrána fyziologie   MeSH
• buněčné linie MeSH
• endocytóza fyziologie   MeSH
• filaminy genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• kinasa glykogensynthasy 3beta antagonisté a inhibitory   metabolismus   MeSH
• LDL receptor related protein 6 metabolismus   MeSH
• lidé MeSH
• mikrocefalie genetika   MeSH
• mikrofilamentové proteiny genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• proliferace buněk genetika   fyziologie   MeSH
• proteiny Wnt metabolismus   MeSH
• signální dráha Wnt fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Filamin B (FlnB) is an actin-binding protein thought to transduce signals from various membrane receptors and intracellular proteins onto the actin cytoskeleton. Formin1 (Fmn1) is an actin-nucleating protein, implicated in actin assembly and intracellular signaling. Human mutations in FLNB cause several skeletal disorders associated with dwarfism and early bone fusion. Mouse mutations in Fmn1 cause aberrant fusion of carpal digits. We report here that FlnB and Fmn1 physically interact, are co-expressed in chondrocytes in the growth plate and share overlapping expression in the cell cytoplasm and nucleus. Loss of FlnB leads to a dramatic decrease in Fmn1 expression at the hypertrophic-to-ossification border. Loss of Fmn1-FlnB in mice leads to a more severe reduction in body size, weight and growth plate length, than observed in mice following knockout of either gene alone. Shortening of the long bone is associated with a decrease in chondrocyte proliferation and an overall delay in ossification in the double-knockout mice. In contrast to FlnB null, Fmn1 loss results in a decrease in the width of the prehypertrophic zone. Loss of both proteins, however, causes an overall decrease in the width of the proliferation zone and an increase in the differentiated hypertrophic zone. The current findings suggest that Fmn1 and FlnB have shared and independent functions. FlnB loss promotes prehypertrophic differentiation whereas Fmn1 leads to a delay. Both proteins, however, regulate chondrocyte proliferation, and FlnB may regulate Fmn1 function at the hypertrophic-to-ossification border, thereby explaining the overall delay in ossification.

• MeSH
• buněčná diferenciace * MeSH
• chondrocyty metabolismus   patologie   MeSH
• fetální proteiny nedostatek   metabolismus   MeSH
• filaminy nedostatek   metabolismus   MeSH
• fyziologická kalcifikace MeSH
• hypertrofie MeSH
• jaderné proteiny nedostatek   metabolismus   MeSH
• lidé MeSH
• mikrofilamentové proteiny nedostatek   metabolismus   MeSH
• myši knockoutované MeSH
• proliferace buněk MeSH
• receptor parathormonu typ 1 metabolismus   MeSH
• růstová ploténka metabolismus   patologie   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• komplex proteinů 2-3 souvisejících s aktinem genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH