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European Journal of Cell Biology Jun 2024Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical...
Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called 'ACTIF' that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.
Topics: Vimentin; Actins; Humans; Intermediate Filaments; Actin Cytoskeleton; Animals; Protein Binding
PubMed: 38503131
DOI: 10.1016/j.ejcb.2024.151403 -
Frontiers in Cell and Developmental... 2023Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved...
Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin , in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.
PubMed: 37484914
DOI: 10.3389/fcell.2023.981425 -
Non-coding RNA Research Sep 2024Cervical cancer, a leading global cause of female mortality, exhibits diverse molecular aberrations influencing gene expression and signaling pathways. Epigenetic...
Cervical cancer, a leading global cause of female mortality, exhibits diverse molecular aberrations influencing gene expression and signaling pathways. Epigenetic factors, including histone deacetylases (HDACs) such as HDAC8 and HDAC6, along with microRNAs (miRNAs), play pivotal roles in cervical cancer progression. Recent investigations have unveiled miRNAs as potential regulators of HDACs, offering a promising therapeutic avenue. This study employed in-silico miRNA prediction, qRT-PCR co-expression studies, and Dual-Luciferase reporter assays to identify miRNAs governing HDAC8 and HDAC6 in HeLa, cervical cancer cells. Results pinpointed miR-497-3p and miR-324-3p as novel negative regulators of HDAC8 and HDAC6, respectively. Functional assays demonstrated that miR-497-3p overexpression in HeLa cells suppressed HDAC8, leading to increased acetylation of downstream targets p53 and α-tubulin. Similarly, miR-324-3p overexpression inhibited HDAC6 mRNA and protein expression, enhancing acetylation of Hsp90 and α-tubulin. Notably, inhibiting HDAC8 via miRNA overexpression correlated with reduced cell viability, diminished epithelial-to-mesenchymal transition (EMT), and increased microtubule bundle formation in HeLa cells. In conclusion, miR-497-3p and miR-324-3p emerge as novel negative regulators of HDAC8 and HDAC6, respectively, with potential therapeutic implications. Elevated expression of these miRNAs in cervical cancer cells holds promise for inhibiting metastasis, offering a targeted approach for intervention in cervical malignancy.
PubMed: 38577018
DOI: 10.1016/j.ncrna.2024.02.009 -
Microscopy Research and Technique May 2024Teleost fish exhibit the most pronounced and widespread adult neurogenesis. Recently, functional development and the fate of newborn neurons have been reported in the...
Teleost fish exhibit the most pronounced and widespread adult neurogenesis. Recently, functional development and the fate of newborn neurons have been reported in the optic tectum (OT) of fish. To determine the role of neurogenesis in the OT, this study used histological, immunohistochemical, and electron microscopic investigations on 18 adult Molly fish specimens (Poecilia sphenops). The OT of the Molly fish was a bilateral lobed structure located in the dorsal part of the mesencephalon. It exhibited a laminated structure made up of alternating fiber and cellular layers, which were organized into six main layers. The stratum opticum (SO) was supplied by optic nerve fibers, in which the neuropil was the main component. Radial bipolar neurons that possessed bundles of microtubules were observed in the stratum fibrosum et griseum superficiale (SFGS). Furthermore, oligodendrocytes with their processes wrapped around the nerve fibers could be observed. The stratum album centrale (SAC) consisted mainly of the axons of the stratum griseum centrale (SGC) and the large tectal, pyriform, and horizontal neurons. The neuronal cells of the SO and large tectal cells of the SAC expressed autophagy-related protein-5 (APG5). Interleukin-1β (IL-1β) was expressed in both neurons and glia cells of SGC. Additionally, inducible nitric oxide synthase (iNOS) was expressed in the neuropil of the SAC synaptic layer and granule cells of the stratum periventriculare (SPV). Also, transforming growth factor beta (TGF-β), SRY-box transcription factor 9 (SOX9), and myostatin were clearly expressed in the proliferative neurons. In all strata, S100 protein and Oligodendrocyte Lineage Transcription Factor 2 (Olig2) were expressed by microglia, oligodendrocytes, and astrocytes. In conclusion, it was possible to identify different varieties of neurons in the optic tectum, each with a distinct role. The existence of astrocytes, proliferative neurons, and stem cells highlights the regenerative capacity of OT. RESEARCH HIGHLIGHTS: The OT of the Molly fish exhibited a laminated structure made up of alternating fiber and cellular layers, which were organized into six main layers. Radial bipolar neurons that possessed bundles of microtubules were observed in the stratum fibrosum et griseum superficiale (SFGS). The stratum album central (SAC) consisted mainly of the axons of the stratum griseum centrale (SGC) and the large tectal, pyriform, and horizontal neurons. Inducible nitric oxide synthase (iNOS) was expressed in the neuropil of the SAC synaptic layer and granule cells of the stratum periventricular (SPV). Also, transforming growth factor beta (TGF-β), SRY-box transcription factor 9 (SOX9), and myostatin were clearly expressed in the proliferative neurons. The existence of astrocytes, proliferative neurons, and stem cells highlights the regenerative capacity of OT.
PubMed: 38778562
DOI: 10.1002/jemt.24617 -
ELife Jul 2023At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers...
At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the pole's contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.
Topics: Animals; Kinetochores; Microtubules; Metaphase; Cell Division; Mammals; Spindle Apparatus
PubMed: 37395732
DOI: 10.7554/eLife.85208 -
Current Biology : CB Jan 2024Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule...
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.
Topics: Actins; Microtubules; Cytoskeleton; Actin Cytoskeleton; Retinal Cone Photoreceptor Cells
PubMed: 38086388
DOI: 10.1016/j.cub.2023.11.031 -
Journal of Anatomy Apr 2024Auditory sensitivity and frequency resolution depend on the optimal transfer of sound-induced vibrations from the basilar membrane (BM) to the inner hair cells (IHCs),...
Auditory sensitivity and frequency resolution depend on the optimal transfer of sound-induced vibrations from the basilar membrane (BM) to the inner hair cells (IHCs), the principal auditory receptors. There remains a paucity of information on how this is accomplished along the frequency range in the human cochlea. Most of the current knowledge is derived either from animal experiments or human tissue processed after death, offering limited structural preservation and optical resolution. In our study, we analyzed the cytoarchitecture of the human cochlear partition at different frequency locations using high-resolution microscopy of uniquely preserved normal human tissue. The results may have clinical implications and increase our understanding of how frequency-dependent acoustic vibrations are carried to human IHCs. A 1-micron-thick plastic-embedded section (mid-modiolar) from a normal human cochlea uniquely preserved at lateral skull base surgery was analyzed using light and transmission electron microscopy (LM, TEM). Frequency locations were estimated using synchrotron radiation phase-contrast imaging (SR-PCI). Archival human tissue prepared for scanning electron microscopy (SEM) and super-resolution structured illumination microscopy (SR-SIM) were also used and compared in this study. Microscopy demonstrated great variations in the dimension and architecture of the human cochlear partition along the frequency range. Pillar cell geometry was closely regulated and depended on the reticular lamina slope and tympanic lip angle. A type II collagen-expressing lamina extended medially from the tympanic lip under the inner sulcus, here named "accessory basilar membrane." It was linked to the tympanic lip and inner pillar foot, and it may contribute to the overall compliance of the cochlear partition. Based on the findings, we speculate on the remarkable microanatomic inflections and geometric relationships which relay different sound-induced vibrations to the IHCs, including their relevance for the evolution of human speech reception and electric stimulation with auditory implants. The inner pillar transcellular microtubule/actin system's role of directly converting vibration energy to the IHC cuticular plate and ciliary bundle is highlighted.
PubMed: 38613211
DOI: 10.1111/joa.14045 -
Development (Cambridge, England) Jul 2024Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila...
Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.
Topics: Animals; Drosophila Proteins; Adherens Junctions; Embryonic Development; Microtubules; Drosophila melanogaster; Embryo, Nonmammalian; alpha Catenin; Actomyosin; Morphogenesis; Drosophila; Cell Shape; Intracellular Signaling Peptides and Proteins
PubMed: 38864272
DOI: 10.1242/dev.202577 -
Frontiers in Molecular Neuroscience 2023Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing...
INTRODUCTION
Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In , anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. We previously isolated the () mutant in a mutagenesis screen for auditory mutants. Although many mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology.
RESULTS
Here we molecularly characterize the gene and demonstrate that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. We also describe morphological changes in Johnston's organ as flies age to 30 days, and we find that morphological and electrophysiological phenotypes in this ch organ of mutants become more severe with age. We show that NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, mutants show a 50% reduction in mechanoreceptor potentials.
DISCUSSION
Thus, the -encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs.
PubMed: 37808471
DOI: 10.3389/fnmol.2023.1263411 -
Biomedicine & Pharmacotherapy =... Jun 2024Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and...
Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.
Topics: Humans; Colorectal Neoplasms; Microfilament Proteins; Carrier Proteins; Neoplasm Invasiveness; Kinesins; Animals; Cell Line, Tumor; Cell Movement; Neoplasm Metastasis; Pyrimidines; Signal Transduction; Thiones; Antineoplastic Agents
PubMed: 38781869
DOI: 10.1016/j.biopha.2024.116785