DNA nanostructures (DNs) have gained popularity in various biomedical applications due to their unique properties, including structural programmability, ease of synthesis and functionalization, and low cytotoxicity. Effective utilization of DNs in biomedical applications requires a fundamental understanding of their interactions with living cells and the mechanics of cellular uptake. Current knowledge primarily focuses on how the physicochemical properties of DNs, such as mass, shape, size, and surface functionalization, affect uptake efficacy. However, the role of cellular mechanics and morphology in DN uptake remains largely unexplored. In this work, we show that cells subjected to geometric constraints remodel their actin cytoskeleton, resulting in differential mechanical force generation that facilitates DN uptake. The length, number, and orientation of F-actin fibers are influenced by these constraints, leading to distinct mechanophenotypes. Overall, DN uptake is governed by F-actin forces arising from filament reorganisation under geometric constraints. These results underscore the importance of actin dynamics in the cellular uptake of DNs and suggest that leveraging geometric constraints to induce specific cell morphology adaptations could enhance the uptake of therapeutically designed DNs.

• MeSH
• aktiny metabolismus   chemie   MeSH
• cytoskelet metabolismus   účinky léků   MeSH
• DNA * chemie   metabolismus   MeSH
• lidé MeSH
• mikrofilamenta metabolismus   účinky léků   MeSH
• nanostruktury * chemie   MeSH
• velikost částic MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aktiny MeSH
• DNA * 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

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

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

Actin cytoskeleton and reactive oxygen species are principal determinants of root hair polarity and tip growth. Loss of function in RESPIRATORY BURST OXIDASE HOMOLOG C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2), an NADPH oxidase emitting superoxide to the apoplast, and in ACTIN 2, a vegetative actin isovariant, in rhd2-1 and der1-3 mutants, respectively, lead to similar defects in root hair formation and elongation Since early endosome-mediated polar localization of AtRBOHC/RHD2 depends on actin cytoskeleton, comparing the proteome-wide consequences of both mutations might be of eminent interest. Therefore, we employed a differential proteomic analysis of Arabidopsis rhd2-1 and der1-3 mutants. Both mutants exhibited substantial alterations in abundances of stress-related proteins. Notably, plasma membrane (PM)-localized PIP aquaporins showed contrasting abundance patterns in the mutants compared to wild-types. Drought-responsive proteins were mostly downregulated in rhd2-1 but upregulated in der1-3. Proteomic data suggest that opposite to der1-3, altered vesicular transport in rhd2-1 mutant likely contributes to the deregulation of PM-localized proteins, including PIPs. Moreover, lattice light sheet microscopy revealed reduced actin dynamics in rhd2-1 roots, a finding contrasting with previous reports on der1-3 mutant. Phenotypic experiments demonstrated a drought stress susceptibility in rhd2-1 and resistance in der1-3. Thus, mutations in AtRBOHC/RHD2 and ACTIN2 cause similar root hair defects, but they differently affect the actin cytoskeleton and vesicular transport. Reduced actin dynamics in rhd2-1 mutant is accompanied by alteration of vesicular transport proteins abundance, likely leading to altered protein delivery to PM, including aquaporins, thereby significantly affecting drought stress responses.

• Klíčová slova
• ACTIN2, Aquaporin, Arabidopsis, Drought stress, NADPH oxidase, PIP1, Plasma membrane, Proteomics, RBOHC, actin,
• MeSH
• akvaporiny * metabolismus   genetika   MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• buněčná membrána * metabolismus   MeSH
• kořeny rostlin * metabolismus   genetika   MeSH
• mikrofilamenta * metabolismus   MeSH
• mutace MeSH
• období sucha * MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akvaporiny * MeSH
• proteiny huseníčku * 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
• kaseinkinasa 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
• kaseinkinasa I MeSH
• proteiny buněčného cyklu MeSH

Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule polymerase, CKAP5 (XMAP215 homolog), has been reported to play a role in mediating crosstalk between microtubules and actin filaments in the neuronal growth cones. However, the molecular mechanism of this process is unknown. Here, we demonstrate, in a reconstituted system, that CKAP5 enables the formation of persistent actin bundles templated by dynamically instable microtubules. We explain the templating by the difference in CKAP5 binding to microtubules and actin filaments. Binding to the microtubule lattice with higher affinity, CKAP5 enables the formation of actin bundles exclusively on the microtubule lattice, at CKAP5 concentrations insufficient to support any actin bundling in the absence of microtubules. Strikingly, when the microtubules depolymerize, actin bundles prevail at the positions predetermined by the microtubules. We propose that the local abundance of available CKAP5-binding sites in actin bundles allows the retention of CKAP5, resulting in persisting actin bundles. In line with our observations, we found that reducing CKAP5 levels in vivo results in a decrease in actin-microtubule co-localization in growth cones and specifically decreases actin intensity at microtubule plus ends. This readily suggests a mechanism explaining how exploratory microtubules set the positions of actin bundles, for example, in cytoskeleton-rich neuronal growth cones.

• Klíčová slova
• CKAP5, XMAP215, actin filaments, cytoskeleton, cytoskeleton-associated proteins, filament crosslinkers, in vitro reconstitution, microtubules, neuronal growth cones,
• MeSH
• aktiny * metabolismus   MeSH
• čípky retiny metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• mikrofilamenta metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aktiny * MeSH

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

• Klíčová slova
• Actin, Lipid, Micropattern, Microtubule, Reconstitution assays,
• MeSH
• aktiny * fyziologie   MeSH
• mikrofilamenta chemie   MeSH
• mikrotubuly * fyziologie   MeSH
• polarita buněk MeSH
• pseudopodia MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny * MeSH

Interaction between an antigen-presenting cell and a T cell, and their subsequent conjugation are a prerequisite for the formation of the immunological synapse and productive, antigen-dependent activation of T cells. This initial interaction is accompanied by recognition of the presented antigen by the T cell receptor, and by changes in the morphology of the interacting cells and in actin cytoskeleton structure in the site of interaction. The experimental protocol below describes a simple assay for quantitative assessment of antigen-presenting cells-T cell conjugation using confocal microscopy or flow cytometry.

• Klíčová slova
• B3Z, Confocal microscopy, Conjugation assay, Flow cytometry, Immunological synapse, MutuDC,
• MeSH
• aktivace lymfocytů MeSH
• antigen prezentující buňky * MeSH
• imunologické synapse fyziologie   MeSH
• mikrofilamenta MeSH
• receptory antigenů T-buněk MeSH
• T-lymfocyty * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH

BACKGROUND: FLNC is one of the few genes associated with all types of cardiomyopathies, but it also underlies neuromuscular phenotype. The combination of concomitant neuromuscular and cardiac involvement is not often observed in filaminopathies and the impact of this on the disease prognosis has hitherto not been analyzed. RESULTS: Here we provide a detailed clinical, genetic, and structural prediction analysis of distinct FLNC-associated phenotypes based on twelve pediatric cases. They include early-onset restrictive cardiomyopathy (RCM) in association with congenital myopathy. In all patients the initial diagnosis was established during the first year of life and in five out of twelve (41.7%) patients the first symptoms were observed at birth. RCM was present in all patients, often in combination with septal defects. No ventricular arrhythmias were noted in any of the patients presented here. Myopathy was confirmed by neurological examination, electromyography, and morphological studies. Arthrogryposes was diagnosed in six patients and remained clinically meaningful with increasing age in three of them. One patient underwent successful heart transplantation at the age of 18 years and two patients are currently included in the waiting list for heart transplantation. Two died due to congestive heart failure. One patient had ICD instally as primary prevention of SCD. In ten out of twelve patients the disease was associated with missense variants and only in two cases loss of function variants were detected. In half of the described cases, an amino acid substitution A1186V, altering the structure of IgFLNc10, was found. CONCLUSIONS: The present description of twelve cases of early-onset restrictive cardiomyopathy with congenital myopathy and FLNC mutation, underlines a distinct unique phenotype that can be suggested as a separate clinical form of filaminopathies. Amino acid substitution A1186V, which was observed in half of the cases, defines a mutational hotspot for the reported combination of myopathy and cardiomyopathy. Several independent molecular mechanisms of FLNC mutations linked to filamin structure and function can explain the broad spectrum of FLNC-associated phenotypes. Early disease presentation and unfavorable prognosis of heart failure demanding heart transplantation make awareness of this clinical form of filaminopathy of great clinical importance.

• Klíčová slova
• Childhood, Congenital myopathy, FLNC-associated phenotype, Genes, Mutation, Rare clinical phenotype, Restrictive cardiomyopathy, Unfavourable prognosis,
• MeSH
• fenotyp MeSH
• filaminy chemie   genetika   metabolismus   MeSH
• kardiomyopatie * genetika   metabolismus   MeSH
• lidé MeSH
• nemoci svalů * MeSH
• restriktivní kardiomyopatie * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• filaminy MeSH