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PLoS Biology Mar 2024Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many...
Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. We need to therefore gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the large amount of time required to age mammalian or vertebrate animal models. Here, we introduce a new cellular model within the Drosophila brain, in which we report classical ageing hallmarks previously observed in the primate brain. These hallmarks include axonal swellings, cytoskeletal decay, a reduction in axonal calibre, and morphological changes arising at synaptic terminals. In the fly brain, these changes begin to occur within a few weeks, ideal to study the underlying mechanisms of ageing. We discovered that the decay of the neuronal microtubule (MT) cytoskeleton precedes the onset of other ageing hallmarks. We showed that the MT-binding factors Tau, EB1, and Shot/MACF1, are necessary for MT maintenance in axons and synapses, and that their functional loss during ageing triggers MT bundle decay, followed by a decline in axons and synaptic terminals. Furthermore, genetic manipulations that improve MT networks slowed down the onset of neuronal ageing hallmarks and confer aged specimens the ability to outperform age-matched controls. Our work suggests that MT networks are a key lesion site in ageing neurons and therefore the MT cytoskeleton offers a promising target to improve neuronal decay in advanced age.
Topics: Animals; Quality of Life; Cytoskeleton; Neurons; Drosophila; Microtubules; Aging; Mammals; Microtubule-Associated Proteins; Drosophila Proteins
PubMed: 38478582
DOI: 10.1371/journal.pbio.3002504 -
The Journal of Neuroscience : the... Nov 2023Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral...
Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage both male and female mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of Layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and overexpression (OE) manipulations in an candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating Layer II/III cortical projection neuron (CPN) collateral axon branching LOF experiments show that Dbn1 is necessary to suppress the elongation of Layer II/III CPN collateral axon branches within Layer IV, where axon branching by Layer II/III CPNs is normally absent. Conversely, OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation. Laminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory Layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons , we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in and in may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in autism spectrum disorder and epilepsy.
Topics: Animals; Female; Male; Mice; Autism Spectrum Disorder; Axons; Cytoskeletal Proteins; Neurons; Neuropeptides
PubMed: 37798130
DOI: 10.1523/JNEUROSCI.0553-23.2023 -
Frontiers in Neuroscience 2023Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell... (Review)
Review
Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery-all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the 'dependency cycle of local axon homeostasis' as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases.
PubMed: 37564364
DOI: 10.3389/fnins.2023.1236815 -
Biomolecules Sep 2023Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to...
Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a "cargo recognition α-helix" upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development.
Topics: Dyneins; Microtubule-Associated Proteins; Microtubules; Biological Transport; Models, Structural
PubMed: 37892127
DOI: 10.3390/biom13101445 -
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 -
Nature Communications Feb 2024Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as...
Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E β-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.
Topics: Insulin Secretion; Actins; Glucose; Electron Microscope Tomography; Insulin; Insulin-Secreting Cells; Actin Cytoskeleton
PubMed: 38346988
DOI: 10.1038/s41467-024-45648-7 -
Cellular and Molecular Life Sciences :... Mar 2024Tektins are microtubule inner proteins (MIPs) and localize at the inside lumen of doublet microtubules (DMTs) of cilia/flagella. TEKTIP1, a newly identified protein by...
Tektins are microtubule inner proteins (MIPs) and localize at the inside lumen of doublet microtubules (DMTs) of cilia/flagella. TEKTIP1, a newly identified protein by cryo-electron microscopy (cryo-EM), is proposed to be localized at the center of the tektin bundle and hypothesized to recruit tektins or stabilize the bundle. However, the physiological role of TEKTIP1 is unknown. In this study, we generated Tektip1-knockout (Tektip1) mice and showed that they were male subfertile primarily due to reduced sperm motility. A high percentage of sperm from Tektip1 mice showed moderately disorganized axoneme structures and abnormal flagellar waveforms. TEKTIP1 predominately interacted with TEKT3 among tektins. Loss of TEKTIP1 partially disturbed the organization of tektin bundle by mainly affecting the native status of TEKT3 and its interaction with other tektins. Collectively, our study reveals the physiological role and potential molecular mechanism of TEKTIP1 in axonemal structure and sperm motility, highlights the importance of MIPs in stabilizing DMTs, and suggests a potential relevance of TEKTIP1 deficiency to human asthenospermia. Tektip1 mice will be an excellent animal model to study the DMT organization of sperm flagella using cryo-EM in future.
Topics: Humans; Male; Animals; Mice; Female; Axoneme; Cryoelectron Microscopy; Semen; Sperm Motility; Spermatozoa; Flagella; Microtubule Proteins
PubMed: 38448737
DOI: 10.1007/s00018-023-05081-3 -
Cell Discovery Nov 2023The propulsion for mammalian sperm swimming is generated by flagella beating. Microtubule doublets (DMTs) along with microtubule inner proteins (MIPs) are essential...
The propulsion for mammalian sperm swimming is generated by flagella beating. Microtubule doublets (DMTs) along with microtubule inner proteins (MIPs) are essential structural blocks of flagella. However, the intricate molecular architecture of intact sperm DMT remains elusive. Here, by in situ cryo-electron tomography, we solved the in-cell structure of mouse sperm DMT at 4.5-7.5 Å resolutions, and built its model with 36 kinds of MIPs in 48 nm periodicity. We identified multiple copies of Tektin5 that reinforce Tektin bundle, and multiple MIPs with different periodicities that anchor the Tektin bundle to tubulin wall. This architecture contributes to a superior stability of A-tubule than B-tubule of DMT, which was revealed by structural comparison of DMTs from the intact and deformed axonemes. Our work provides an overall molecular picture of intact sperm DMT in 48 nm periodicity that is essential to understand the molecular mechanism of sperm motility as well as the related ciliopathies.
PubMed: 37989994
DOI: 10.1038/s41421-023-00606-3 -
Molecular and Cellular Biochemistry Sep 2023β-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression,...
β-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42 °C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of β-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37 °C for 24 h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of β-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and β-III tubulin isotype remain unaltered. Upon depletion of β-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with β-III tubulin isotype shRNA. This features the role of the β-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of β-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of β-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of β-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of β-II tubulin isotype as a biomarker for cellular stiffness.
Topics: Humans; Tubulin; Neuroblastoma; Microtubules; Cell Differentiation; RNA, Small Interfering
PubMed: 36585545
DOI: 10.1007/s11010-022-04649-0 -
The FEBS Journal Oct 2023FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of...
FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.
Topics: Mice; Animals; Mice, Inbred mdx; Muscle, Skeletal; Muscle Fibers, Skeletal; Myoblasts; Cell Differentiation; Peptidylprolyl Isomerase
PubMed: 37345229
DOI: 10.1111/febs.16894