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Journal of Cell Science Apr 2024Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis...
Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.
Topics: Cilia; Humans; Microtubule-Associated Proteins; Animals; Dyneins; Hedgehog Proteins; Signal Transduction; Mice; Flagella
PubMed: 38533689
DOI: 10.1242/jcs.261816 -
Trends in Parasitology May 2024Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton. The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms... (Review)
Review
Microtubules (MTs) play a vital role as key components of the eukaryotic cytoskeleton. The phylum Apicomplexa comprises eukaryotic unicellular parasitic organisms defined by the presence of an apical complex which consists of specialized secretory organelles and tubulin-based cytoskeletal elements. One apicomplexan parasite, Toxoplasma gondii, is an omnipresent opportunistic pathogen with significant medical and veterinary implications. To ensure successful infection and widespread dissemination, T. gondii heavily relies on the tubulin structures present in the apical complex. Recent advances in high-resolution imaging, coupled with reverse genetics, have offered deeper insights into the composition, functionality, and dynamics of these tubulin-based structures. The apicomplexan tubulins differ from those of their mammalian hosts, endowing them with unique attributes and susceptibility to specific classes of inhibitory compounds.
Topics: Toxoplasma; Tubulin; Cytoskeleton; Animals; Microtubules; Humans; Protozoan Proteins
PubMed: 38531711
DOI: 10.1016/j.pt.2024.02.010 -
Journal of Clinical Laboratory Analysis Apr 2024The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor...
BACKGROUND
The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions.
METHODS
Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex.
RESULTS
We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1).
CONCLUSIONS
The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.
Topics: Methylation; Arginine; Humans; Cytoplasmic Dyneins; Protein-Arginine N-Methyltransferases; Protein Processing, Post-Translational; Dyneins; Amino Acid Sequence; Repressor Proteins
PubMed: 38525916
DOI: 10.1002/jcla.25030 -
The American Journal of Case Reports Mar 2024BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease that can present at different ages with different phenotypes. Missed and delayed...
BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease that can present at different ages with different phenotypes. Missed and delayed diagnoses are fairly common. Many variants in the DNAH5 gene have been described that confirm the diagnosis of PCD. Advances in medicine, especially in molecular genetics, have led to increasingly early discoveries of such cases, especially in those with nonclassical presentations. CASE REPORT This report describes a patient with bronchiectasis, lung cysts, finger clubbing, and failure to thrive who was misdiagnosed for several years as having asthma. Many differentials were suspected and worked up, including a suspicion of PCD. Genetic tests with whole-exome sequencing (WES) and whole-genome sequencing (WGS) detected a heterozygous, likely pathogenic, variant in the DNAH5 gene associated with PCD. CONCLUSIONS Despite a thorough workup done for this case, including a genetic workup, a PCD diagnosis was not established. We plan to reanalyze the WGS in the future, and with advent of technology and better coverage of genes, a genetic answer for this challenging case may resolve this diagnostic quandary in the future.
Topics: Humans; Axonemal Dyneins; Genetic Testing; Kartagener Syndrome; Lung; Mutation
PubMed: 38521969
DOI: 10.12659/AJCR.942444 -
The Journal of Cell Biology Apr 2024Centrosomes are the primary microtubule organizer in eukaryotic cells. In addition to shaping the intracellular microtubule network and the mitotic spindle, centrosomes... (Review)
Review
Centrosomes are the primary microtubule organizer in eukaryotic cells. In addition to shaping the intracellular microtubule network and the mitotic spindle, centrosomes are responsible for positioning cilia and flagella. To fulfill these diverse functions, centrosomes must be properly located within cells, which requires that they undergo intracellular transport. Importantly, centrosome mispositioning has been linked to ciliopathies, cancer, and infertility. The mechanisms by which centrosomes migrate are diverse and context dependent. In many cells, centrosomes move via indirect motor transport, whereby centrosomal microtubules engage anchored motor proteins that exert forces on those microtubules, resulting in centrosome movement. However, in some cases, centrosomes move via direct motor transport, whereby the centrosome or centriole functions as cargo that directly binds molecular motors which then walk on stationary microtubules. In this review, we summarize the mechanisms of centrosome motility and the consequences of centrosome mispositioning and identify key questions that remain to be addressed.
Topics: Biological Transport; Centrioles; Centrosome; Microtubules; Spindle Apparatus; Cilia; Humans; Animals; Dyneins
PubMed: 38512059
DOI: 10.1083/jcb.202311140 -
PLoS Genetics Mar 2024Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans,...
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.
Topics: Humans; Chlamydomonas reinhardtii; Cilia; Mutation; Dyneins; Proteins; Chlamydomonas; Gene Dosage; Axoneme
PubMed: 38498551
DOI: 10.1371/journal.pgen.1011038 -
PloS One 2024In intracellular active transport, molecular motors are responsible for moving biological cargo along networks of microtubules that serve as scaffolds. Cargo dynamics...
In intracellular active transport, molecular motors are responsible for moving biological cargo along networks of microtubules that serve as scaffolds. Cargo dynamics can be modified by different features of microtubule networks such as geometry, density, orientation modifications. Also, the dynamical behaviour of the molecular motors is determined by the microtubule network and by the individual and/or collective action of the motors. For example, unlike single kinesins, the mechanistic behavior of multiple kinesins varies from one experiment to another. However, the reasons for this experimental variability are unknown. Here we show theoretically how non-radial and quasi-radial microtubule architectures modify the collective behavior of two kinesins attached on a cargo. We found out under which structural conditions transport is most efficient and the most likely way in which kinesins are organized in active transport. In addition, with motor activity, mean intermotor distance and motor organization, we determined the character of the collective interaction of the kinesins during transport. Our results demonstrate that two-dimensional microtubule structures promote branching due to crossovers that alter directionality in cargo movement and may provide insight into the collective organization of the motors. Our article offers a perspective to analyze how the two-dimensional network can modify the cargo-motor dynamics for the case in which multiple motors move in different directions as in the case of kinesin and dynein.
Topics: Kinesins; Biological Transport; Biological Transport, Active; Dyneins; Microtubules
PubMed: 38478520
DOI: 10.1371/journal.pone.0295652 -
Kidney360 Apr 2024AMP kinase senses diabetic stresses in podocytes, subsequently upregulates specificity protein 1–mediated dynein expression and promotes podocyte injury....
KEY POINTS
AMP kinase senses diabetic stresses in podocytes, subsequently upregulates specificity protein 1–mediated dynein expression and promotes podocyte injury. Pharmaceutical restoration of dynein expression by targeting specificity protein 1 represents an innovative therapeutic strategy for diabetic nephropathy.
BACKGROUND
Diabetic nephropathy (DN) is a major complication of diabetes. Injury to podocytes, epithelial cells that form the molecular sieve of a kidney, is a preclinical feature of DN. Protein trafficking mediated by dynein, a motor protein complex, is a newly recognized pathophysiology of diabetic podocytopathy and is believed to be derived from the hyperglycemia-induced expression of subunits crucial for the transportation activity of the dynein complex. However, the mechanism underlying this transcriptional signature remains unknown.
METHODS
Through promoter analysis, we identified binding sites for transcription factor specificity protein 1 (SP1) as the most shared motif among hyperglycemia-responsive dynein genes. We demonstrated the essential role of AMP-activated protein kinase (AMPK)–regulated SP1 in the transcription of dynein subunits and dynein-mediated trafficking in diabetic podocytopathy using chromatin immunoprecipitation quantitative PCR and live cell imaging. SP1-dependent dynein-driven pathogenesis of diabetic podocytopathy was demonstrated by pharmaceutical intervention with SP1 in a mouse model of streptozotocin-induced diabetes.
RESULTS
Hyperglycemic conditions enhance SP1 binding to dynein promoters, promoted dynein expression, and enhanced dynein-mediated mistrafficking in cultured podocytes. These changes can be rescued by chemical inhibition or genetic silencing of SP1. The direct repression of AMPK, an energy sensor, replicates hyperglycemia-induced dynein expression by activating SP1. Mithramycin inhibition of SP1-directed dynein expression in streptozotocin-induced diabetic mice protected them from developing podocytopathy and prevented DN progression.
CONCLUSIONS
Our work implicates AMPK-SP1–regulated dynein expression as an early mechanism that translates energy disturbances in diabetes into podocyte dysfunction. Pharmaceutical restoration of dynein expression by targeting SP1 offers a new therapeutic strategy to prevent DN.
Topics: Animals; Humans; AMP-Activated Protein Kinases; Diabetic Nephropathies; Dyneins; Energy Metabolism; Sp1 Transcription Factor
PubMed: 38467599
DOI: 10.34067/KID.0000000000000392 -
EMBO Reports Apr 2024Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe...
Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.
Topics: Animals; Humans; Male; Mice; Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins; Dyneins; HSC70 Heat-Shock Proteins; Infertility, Male; Molecular Chaperones; Protein Folding; Semen; Sperm Head; Spermatogenesis; Spermatozoa; Teratozoospermia; Thiazoles
PubMed: 38454159
DOI: 10.1038/s44319-024-00112-x -
The EMBO Journal Apr 2024Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying...
Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.
Topics: Dyneins; Cryoelectron Microscopy; Biological Transport; Cilia; Flagella
PubMed: 38454149
DOI: 10.1038/s44318-024-00060-1