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PubMed1979

Dynein binds to and crossbridges cytoplasmic microtubules.

Dynein isolated from Chlamydomonas flagellar axonemes binds to microtubules assembled in vitro from 6S brain tubulin dimers. The dynein arms bind periodically along the length of the microtubules with a center-to-center spacing of 24 nm, equal to the periodicity of dynein arms on intact axonemes. The arms project from the in vitro assembled microtubules at an angle of approximately 55 degrees, thereby defining microtubule polarity. Dynein cosediments with microtubules through a sucrose gradient, as demonstrated by electron microscopy, gel electrophoresis, and ATPase analysis. In addition, dynein induces crossbridging between adjacent microtubules. Darkfield microscopy reveals that microtubules containing dynein are aggregated into large bundles; electron microscopy indicates that microtubules of the same polarity are crossbridged by a regular array of arms. Viewed by darkfield microscopy, addition of ATP to crossbridged microtubules causes their disaggregation; electron microscopy shows that the majority of these microtubules are no longer crossbridged. These observations are applicable to the determination of microtubule polarity and directionality of microtubule assembly in situ and suggest a role for dynein in cytoplasmic microtubule-based cellular movements.

Adenosine Triphosphatases↗
PubMed1977

Ciliary dynein from sea urchin embryos.

Axonemal dynein ATPase [EC 3.6.1.3] was extracted from cilia of sea urchin embryos for a study of its enzymatic properties. Sedimentation analysis on a sucrose density gradient revealed that ATPase activity was associated with the 12S particles. The partially purified 12S enzyme was characterized mainly with regard to the optimum pH, divalent cation and ionic strength requirments and substrate specificity. Comparative investigation of the data obtained indicates that the properties of the present dyneine ATPase resemble those of other dynein(-like) ATPase hitherto reported. In addition, the possible relationship among dyneins within a single species, in particular between the ciliary dynein and cytoplasmic dynein-like ATPase, is discussed.

Adenosine Triphosphatases↗
PubMed2026

[Genetic analysis of a fetus with Short-rib thoracic dysplasia 8 with or without polydactyly due to variants of DYNC2I1 gene].

OBJECTIVE: To investigate the clinical characteristics of a fetus with Short-rib thoracic dysplasia 8 with or without polydactyly (SRTD8) due to variants of DYNC2I1 gene. METHODS: A fetus identified to have short ribs, short long bones, and narrow thorax at 26+1 weeks of gestation at the Women and Children's Hospital of Ningbo University in September 2024 was selected as study subject. The fetus underwent termination of pregnancy at 35+5 weeks of gestation. Clinical data of the fetus were retrospectively collected. Whole exome sequencing (WES) was carried out on fetal tissue, and candidate variants were validated by Sanger sequencing. Difference between the wild type and variant DYNC2I1 proteins was analyzed using AlphaFold v3.0.1 and PyMOL v2.5.6 software. Pathogenicity of the variant was rated based on guidelines from the American College of Medical Genetics and Genomics (ACMG). Using keywords such as "DYNC2I1 gene", previous literature on patients due to biallelic DYNC2I1 gene variants were retrieved from the PubMed databases, CNKI, and Wanfang Data Knowledge Service Platform, and the genetic variant and clinical phenotypes of patients were analyzed. The literature retrieval time was set from the establishment of database to December 31, 2025. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2023-094). RESULTS: Prenatal ultrasound revealed that the fetus had short ribs, short long bones, and narrow thorax at 26+1 gestational weeks. WES and Sanger sequencing revealed that the fetus has harbored compound heterozygous variants of the DYNC2I1 gene, namely c.265_268 (p.Gln89GlyfsTer15) in exon 3 and c.1777C>T (p.Arg593Trp) in exon 14, which were inherited from his father and mother, respectively. Prediction of the DYNC2I1 protein structure suggested that the c.265_268del variant has formed a premature termination codon, which may significantly alter the protein's secondary structure. The c.1777C>T variant may disrupt the electrostatic interaction between Arg593 and Asp729. Based on the ACMG guidelines, the c.265_268del (p.Gln89GlyfsTer15) variant was predicted to be likely pathogenic (PM2_Supporting +PVS1), whilst the c.1777C>T(p.Arg593Trp) variant was rated as uncertain significance (PM2_Supporting+PM3+PP4). Literature search has identified five articles related to biallelic DYNC2I1 variants involving a total of 11 fetuses/patients. Together with the fetus from this study, typical phenotypes included short ribs (6 cases), narrow thorax (6 cases), short limb bones (6 cases), and hand polydactyly (6 cases), and foot polydactyly (5 cases), albeit with significant clinical heterogeneity. A total of 12 genetic variants were identified, among which c.44delC was the most common (16.7%, 4/24), followed by c.1777C>T, c.2246C>T, and c.2305G>A (each accounting for 12.5%). No mutational hotspot was identified. CONCLUSION: The c.265_268del (p.Gln89GlyfsTer15) and c.1777C>T (p.Arg593Trp) compound heterozygous variants of the DYNC2I1 gene probably underlay the pathogenesis of SRTD8 in this fetus. This study has enriched the mutational spectrum of the DYNC2I1 gene and facilitated etiological diagnosis and treatment of DYNC2I1-related diseases.

Humans↗
PubMed1979

The cytoskeleton and cell movement: general considerations.

The cytoskeletal proteins, actin, myosin, tubulin, dynein, and their associated proteins, are discussed selectively with regards to their biochemical and structural properties. Particular emphasis is placed on the comparison of non-muscle proteins to their muscle counterparts, and on the various mechanisms for regulating actin polymerization, actin-myosin interaction, and tubulin polymerization. This review as well as the bibliography accompanying it is selective. An attempt is made to stress the most recent findings and to emphasize those areas which appear to hold the greatest promise for future research.

Actins↗
PubMed1979

The mechanism of microtubule associated cytoplasmic transport. Isolation and preliminary characterisation of a microtubule transport system.

The nutritive tubes of telotrophic insect ovaries are cytoplasmic channels along which ribosomes are transported over distances of several mm from trophic cells to the developing oocytes. The presence within the nutritive tubes of a massive number of orientated microtubules renders them strongly birefringent in polarised light, a property which, together with their size, rendered them amenable to isolation by microdissection. Ultrastructurally the isolated tubes were indistinguishable from undissected controls. Polyacrylamide gels revealed a consistent pattern of some 30 bands of which tubulin was the most prominent. The tubes also contained a band which comigrated with the major high molecular weight microtubule associated protein (MAP) from mouse brain but no detectable actin, myosin or dynein. Microtubules in the isolated tubes were not depolymerised by treatments (cold, calcium and colchicine) which typically disrupt cytoplasmic microtubules. Following extraction of the membrane enclosing the tubes and the cytoplasmic matrix the microtubule cytoskeleton persisted, retaining its cylindrical organisation although no bridges between the microtubules were detected in the electron microscope. The possibility that the stability and spatial deployment of the nutritive tube microtubules is conferred by specific microtubule accessory proteins is discussed.

Animals↗
PubMed1978

Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis.

Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co-migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.

Chlamydomonas↗
PubMed1977

Binding of microtubules to pituitary secretory granules and secretory granule membranes.

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.

Adenosine↗
PubMed1976

Microtubules and associated microfilaments in the tentacles of the suctorian Heliophrya erhardi Matthes.

At the ultrastructural level length changes accompanying linear movements of resting (non-feeding) tentacles of the suctorian Heliophrya involve not only altered microtubule numbers, but also marked changes in the specific microtubule pattern of cross-sectioned tentacles. These changes in number and pattern indicate a sliding between axonemal microtubules. The visualization of microfilaments in the cytoplasm at the tentacle base and in the knob region could shed new light on the problem of whether microtubular sliding is an active or passive process. At the tentacle base, microfilaments are either arranged in a ring-shaped configuration around the axoneme, or they run parallel to the axonemal microtubules, whereas at the tentacle tip during the resting state, microfilaments are closely associated with the plasma membrane of the knob. They form a filamentous reticular layer, which is continuous at the anchorage site of axonemal microtubules with the dense epiplasmic layer of the tentacle shaft. Obiously, this filamentous layer is engaged in positioning the haptocysts at the plasma membrane and in holding the membrane itself under tension. The putative contractile nature of microfilaments and the epiplasmic layer is argued from ATP-sensitive glycerol models of tentacles and from the results of halothane treatment of native tentacles. Halothane treatment of resting tentacles also gave indications of the presence of differentially stable intermicrotubule-bridges. The role of micro-filaments and halothane-resistant dynein-like inter-row bridges in tentacle movement is discussed. As soon as the plasma membrane of the knob is 'sealed' with the prey pellicle during feeding, the microtubules of the sleeve region slide into the knob where they bend back and outwards. The microtubules now appear decorated and sometimes cross-connected by microfilaments which adhere closely to the plasma membrane- now acting as a peritrophic membrane-lining the prey cytoplasm against the microtubules of the inner tube. These microfilaments which show a close association with the microtubules of the active knob area, are thought to be engaged in microtubular bending and stretching during feeding. They may also be involved in the transport of the peritrophic membrane in distal tentacle regions. Microinematographically recorded oscillations in tentacle diameter in these regions are in agreement with the electron-microscopic findings of various states of collapsed tentacle axonemes. These observations, as well as the occurrence of helically twisted tentacles during feeding, suggest microfilament mediated sequential back and forth movements of sleeve microtubules in the knob region which generate a proximally migrating helical wave.

Adenosine Triphosphate↗