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Structure (London, England : 1993) May 2024Dyneins are an AAA+ motor responsible for motility and force generation toward the minus end of microtubules. Dynein motility is powered by nucleotide-dependent...
Dyneins are an AAA+ motor responsible for motility and force generation toward the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we performed all-atom molecular dynamics simulations of human dynein-2 primed for its power stroke. Simulations revealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy barrier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence of the AAA+ ring, indicating that the linker stores energy as it bends and releases this energy during the powerstroke.
Topics: Molecular Dynamics Simulation; Humans; Dyneins; Thermodynamics; Protein Binding; Protein Conformation
PubMed: 38430911
DOI: 10.1016/j.str.2024.02.003 -
Journal of Medical Genetics May 2024Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked...
BACKGROUND
Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked , which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers.
METHODS
XCI patterns of six mothers of male patients with -related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated allele.
RESULTS
The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern.
CONCLUSION
This study highlights the importance of searching for heterozygous pathogenic mutations in all female relatives of male PCD patients with a defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.
Topics: Adolescent; Adult; Child; Child, Preschool; Female; Humans; Male; Cilia; Ciliary Motility Disorders; DNA Methylation; Dyneins; Kartagener Syndrome; Mutation; Phenotype; X Chromosome Inactivation
PubMed: 38408845
DOI: 10.1136/jmg-2023-109700 -
The Journal of Cell Biology May 2024Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell...
Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.
Topics: Humans; Axonal Transport; Axons; Dynactin Complex; Dyneins; Neurons; Neural Stem Cells
PubMed: 38407313
DOI: 10.1083/jcb.202309084 -
Cells Feb 2024Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved... (Review)
Review
Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.
Topics: Cytoplasmic Dyneins; Catalytic Domain; Dyneins
PubMed: 38391943
DOI: 10.3390/cells13040330 -
Journal of Biochemistry Apr 2024Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author... (Review)
Review
Mysterin is a large intracellular protein harboring a RING finger ubiquitin ligase domain and is also referred to as RING finger protein 213 (RNF213). The author performed the first molecular cloning of the mysterin gene as the final step in genetic exploration of cerebrovascular moyamoya disease (MMD) and initiated the next round of exploration to understand its molecular and cellular functions. Although much remains unknown, accumulating findings suggest that mysterin functions in cells by targeting massive intracellular structures, such as lipid droplets (LDs) and various invasive pathogens. In the latter case, mysterin appears to directly surround and ubiquitylate the surface of pathogens and stimulate cell-autonomous antimicrobial reactions, such as xenophagy and inflammatory response. To date, multiple mutations causing MMD have been identified within and near the RING finger domain of mysterin; however, their functional relevance remains largely unknown. Besides the RING finger, mysterin harbors a dynein-like ATPase core and an RZ finger, another ubiquitin ligase domain unique to mysterin, while functional exploration of these domains has also just commenced. In this review, the author attempts to summarize the core findings regarding the molecular structure and function of the mysterin protein, with an emphasis on the perspective of MMD research.
Topics: Humans; Ubiquitin-Protein Ligases; Animals; Moyamoya Disease; Adenosine Triphosphatases
PubMed: 38378744
DOI: 10.1093/jb/mvae020 -
Current Biology : CB Mar 2024The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona...
The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores. The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins. CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression, interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358). We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined. Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.
Topics: Humans; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Kinetochores; Microtubules; Mad2 Proteins; Mitosis; Dyneins; Spindle Apparatus; HeLa Cells
PubMed: 38354735
DOI: 10.1016/j.cub.2024.01.042 -
Journal of Cell Science Feb 2024Most of the vesicular transport pathways inside the cell are facilitated by molecular motors that move along cytoskeletal networks. Autophagy is a well-explored... (Review)
Review
Most of the vesicular transport pathways inside the cell are facilitated by molecular motors that move along cytoskeletal networks. Autophagy is a well-explored catabolic pathway that is initiated by the formation of an isolation membrane known as the phagophore, which expands to form a double-membraned structure that captures its cargo and eventually moves towards the lysosomes for fusion. Molecular motors and cytoskeletal elements have been suggested to participate at different stages of the process as the autophagic vesicles move along cytoskeletal tracks. Dynein and kinesins govern autophagosome trafficking on microtubules through the sequential recruitment of their effector proteins, post-translational modifications and interactions with LC3-interacting regions (LIRs). In contrast, myosins are actin-based motors that participate in various stages of the autophagic flux, as well as in selective autophagy pathways. However, several outstanding questions remain with regard to how the dominance of a particular motor protein over another is controlled, and to the molecular mechanisms that underlie specific disease variants in motor proteins. In this Review, we aim to provide an overview of the role of molecular motors in autophagic flux, as well as highlight their dysregulation in diseases, such as neurodegenerative disorders and pathogenic infections, and ageing.
Topics: Autophagy; Autophagosomes; Cytoskeleton; Actins; Dyneins; Kinesins
PubMed: 38329417
DOI: 10.1242/jcs.260481 -
The Journal of Cell Biology Mar 2024In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane...
In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR.
Topics: Humans; Actin Cytoskeleton; Autophagosomes; Dynactin Complex; Dyneins; GTPase-Activating Proteins; Lysosomes; rab GTP-Binding Proteins; Sorting Nexins
PubMed: 38323995
DOI: 10.1083/jcb.202306040 -
Nature Structural & Molecular Biology Mar 2024Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo...
Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors.
Topics: Animals; Kinesins; Drosophila Proteins; Dyneins; Tropomyosin; Drosophila; Microtubules; RNA, Messenger
PubMed: 38297086
DOI: 10.1038/s41594-024-01212-x -
Nature Communications Jan 2024Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The...
Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.
Topics: Adaptor Proteins, Signal Transducing; Protein Binding; rab GTP-Binding Proteins; Lysosomes; Autophagy; Dyneins
PubMed: 38296963
DOI: 10.1038/s41467-024-44957-1