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BioRxiv : the Preprint Server For... Jun 2024Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately...
UNLABELLED
Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a ∼50 µm window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.
SUMMARY
Single-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and co-aligned with microtubules.
PubMed: 38915582
DOI: 10.1101/2024.06.10.598346 -
Soft Matter Jun 2024Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data...
Connecting the large-scale emergent behaviors of active cytoskeletal materials to the microscopic properties of their constituents is a challenge due to a lack of data on the multiscale dynamics and structure of such systems. We approach this problem by studying the impact of depletion attraction on bundles of microtubules and kinesin-14 molecular motors. For all depletant concentrations, kinesin-14 bundles generate comparable extensile dynamics. However, this invariable mesoscopic behavior masks the transition in the microscopic motion of microtubules. Specifically, with increasing attraction, we observe a transition from bi-directional sliding with extension to pure extension with no sliding. Small-angle X-ray scattering shows that the transition in microtubule dynamics is concurrent with a structural rearrangement of microtubules from an open hexagonal to a compressed rectangular lattice. These results demonstrate that bundles of microtubules and molecular motors can display the same mesoscopic extensile behaviors despite having different internal structures and microscopic dynamics. They provide essential information for developing multiscale models of active matter.
PubMed: 38872426
DOI: 10.1039/d3sm01336g -
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 -
Combinatorial Chemistry & High... Jun 2024Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm that usually originates from liver cells and is one of the most common malignancies worldwide. To improve...
BACKGROUND
Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm that usually originates from liver cells and is one of the most common malignancies worldwide. To improve the survival rate of HCC patients, specific prognostic markers are essential to guide HCC therapy. CEP55 is a microtubule-bundling protein involved in critical cell functions, including cell growth, transformation, and cytokinesis.
AIMS
This study examined gene alterations in HCC tumor tissues through comprehensive analysis, aiming to elucidate their contribution to disease development.
METHODS
Bioinformatics tools were employed to investigate the expression, genetic variations, prognostic significance, and clinicopathological relevance of CEP55 across GEO and TCGA datasets. We further explored gene alterations, DNA methylation levels, and immune infiltration of CEP55. To elucidate the potential molecular mechanisms involved, GO and KEGG analysis was performed. Finally, RT-qPCR was also performed on a number of normal and tumoral cell lines in vitro, which demonstrated that the expression of the CEP55 was significantly higher in the tumor cell lines.
RESULTS
We observed that CEP55 was upregulated in 16 cancers compared to corresponding normal tissues. CEP55 was found to be related to T stages, pathologic stages, histologic grade, and levels of AFP. K-M analysis demonstrated that CEP55 expression was associated with a worse outcome. ROC curve analysis showed that CEP55 expression accurately distinguished HCC from normal tissue (AUC = 0.954). The area under 1-,3- and 5-year survival ROCs were above 0.6. The HSPA4 genetic alterations in HCC were 0.8%. Among the 15 DNA methylation CpG sites, 6 were related to the prognosis of HCC. HSPA4 was positively related to immune cell infiltration and immune checkpoints in HCC. The KEGG pathway analysis indicated that CEP55 was associated with the cell cycle and presented together with CDK1. HCC cell lines were demonstrated to express high levels of CEP55 compared to normal cells.
CONCLUSION
As a result of bioinformatic analyses and RT-qPCR validation in HCC, CEP55 increased in HCC tissues and was associated with the stage of the disease and survival rate.
PubMed: 38847172
DOI: 10.2174/0113862073298525240522104104 -
Journal of Cell Science Jun 2024Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies....
Centrosomal proteins play pivotal roles in orchestrating microtubule dynamics, and their dysregulation leads to disorders, including cancer and ciliopathies. Understanding the multifaceted roles of centrosomal proteins is vital to comprehend their involvement in disease development. Here, we report novel cellular functions of CEP41, a centrosomal and ciliary protein implicated in Joubert syndrome. We show that CEP41 is an essential microtubule-associated protein with microtubule-stabilizing activity. Purified CEP41 binds to preformed microtubules, promotes microtubule nucleation, and suppresses microtubule disassembly. When overexpressed in cultured cells, CEP41 localizes to microtubules and promotes microtubule bundling. Conversely, shRNA-mediated knockdown of CEP41 disrupts the interphase microtubule network and delays microtubule reassembly, emphasizing its role in microtubule organization. Further, we demonstrate that CEP41's association with microtubules relies on its conserved rhodanese homology domain (RHOD) and the N-terminal region. Interestingly, a disease-causing mutation in the RHOD domain impairs CEP41-microtubule interaction. Moreover, depletion of CEP41 inhibits cell proliferation and disrupts cell cycle progression, suggesting its potential involvement in cell cycle regulation. These insights into the cellular functions of CEP41 hold promise for unraveling the impact of its mutations in ciliopathies.
PubMed: 38841887
DOI: 10.1242/jcs.261927 -
Open Biology Jun 2024Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of... (Review)
Review
Platelets are blood cells derived from megakaryocytes that play a central role in regulating haemostasis and vascular integrity. The microtubule cytoskeleton of megakaryocytes undergoes a critical dynamic reorganization during cycles of endomitosis and platelet biogenesis. Quiescent platelets have a discoid shape maintained by a marginal band composed of microtubule bundles, which undergoes remarkable remodelling during platelet activation, driving shape change and platelet function. Disrupting or enhancing this process can cause platelet dysfunction such as bleeding disorders or thrombosis. However, little is known about the molecular mechanisms underlying the reorganization of the cytoskeleton in the platelet lineage. Recent studies indicate that the emergence of a unique platelet tubulin code and specific pathogenic tubulin mutations cause platelet defects and bleeding disorders. Frequently, these mutations exhibit dominant negative effects, offering valuable insights into both platelet disease mechanisms and the functioning of tubulins. This review will highlight our current understanding of the role of the microtubule cytoskeleton in the life and death of platelets, along with its relevance to platelet disorders.
Topics: Humans; Blood Platelets; Megakaryocytes; Cytoskeleton; Microtubules; Tubulin; Animals; Blood Platelet Disorders; Mutation
PubMed: 38835242
DOI: 10.1098/rsob.240041 -
BioRxiv : the Preprint Server For... May 2024One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by...
One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In , this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected γ-tubulin and γ-tubulin complex protein 4 (GCP4). While γ-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the γ-tubulin complex in tubulin scaffold formation .
PubMed: 38826480
DOI: 10.1101/2024.05.25.595886 -
BioRxiv : the Preprint Server For... May 2024Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite...
UNLABELLED
Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function , we examined TRIM46 knockout mice. Contrary to previous reports, we find that TRIM46 is dispensable for AIS formation and maintenance, and axon specification. TRIM46 knockout mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. We also show TRIM46 enrichment in the first ∼100 μm of axon occurs independently of ankyrinG (AnkG), although AnkG is required to restrict TRIM46 only to the AIS. Our results suggest an unidentified protein may compensate for loss of TRIM46 and highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function.
SIGNIFICANCE STATEMENT
A healthy nervous system requires the polarization of neurons into structurally and functionally distinct compartments, which depends on both the axon initial segment (AIS) and the microtubule cytoskeleton. In contrast to previous reports, we show that the microtubule-associated protein TRIM46 is not required for axon specification or AIS formation in mice. Our results emphasize the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function.
PubMed: 38826451
DOI: 10.1101/2024.05.23.595556 -
Science Advances May 2024In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the...
In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.
Topics: Robotics; DNA; Microtubules; Kinesins; Molecular Motor Proteins
PubMed: 38820146
DOI: 10.1126/sciadv.adn4490 -
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