scholarly journals Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

2012 ◽  
Vol 23 (18) ◽  
pp. 3554-3565 ◽  
Author(s):  
Panteleimon Rompolas ◽  
Ramila S. Patel-King ◽  
Stephen M. King
Keyword(s):  
Heavy Chain ◽  
Beat Frequency ◽  
Tight Binding ◽  
Cytoplasmic Dynein ◽  
High Load ◽  
Flagellar Motility ◽  
Wild Type ◽  
Regulatory Factor ◽  
Flagellar Beat ◽  
Load Conditions

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.

scholarly journals The sup-pf-2 mutations of Chlamydomonas alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

1996 ◽  
Vol 135 (6) ◽  
pp. 1853-1865 ◽  
Author(s):  
G Rupp ◽  
E O'Toole ◽  
L C Gardner ◽  
B F Mitchell ◽  
M E Porter
Keyword(s):  
Linkage Analysis ◽  
Heavy Chain ◽  
Biochemical Analysis ◽  
Beat Frequency ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Flagellar Beat ◽  
Regulatory Mutations ◽  
Components Analysis ◽  
Dynein Arms

The sup-pf-2 mutation is a member of a group of dynein regulatory mutations that are capable of restoring motility to paralyzed central pair or radial spoke defective strains. Previous work has shown that the flagellar beat frequency is reduced in sup-pf-2, but little else was known about the sup-pf-2 phenotype (Huang, B., Z. Ramanis, and D.J.L. Luck. 1982. Cell. 28:115-125; Brokaw, C.J., and D.J.L. Luck. 1985. Cell Motil. 5:195-208). We have reexamined sup-pf-2 using improved biochemical and structural techniques and by the analysis of additional sup-pf-2 alleles. We have found that the sup-pf-2 mutations are associated with defects in the outer dynein arms. Biochemical analysis of sup-pf-2-1 axonemes indicates that both axonemal ATPase activity and outer arm polypeptides are reduced by 40-50% when compared with wild type. By thin-section EM, these defects correlate with an approximately 45% loss of outer dynein arm structures. Interestingly, this loss is biased toward a subset of outer doublets, resulting in a radial asymmetry that may reflect some aspect of outer arm assembly. The defects in outer arm assembly do not appear to result from defects in either the outer doublet microtubules or the outer arm docking structures, but rather appear to result from defects in outer dynein arm components. Analysis of new sup-pf-2 mutations indicates that the severity of the outer arm assembly defects varies with different alleles. Complementation tests and linkage analysis reveal that the sup-pf-2 mutations are alleles of the PF28/ODA2 locus, which is thought to encode the gamma-dynein heavy chain subunit of the outer arm. The sup-pf-2 mutations therefore appear to alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

scholarly journals A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain.

1991 ◽  
Vol 113 (3) ◽  
pp. 615-622 ◽  
Author(s):  
H Sakakibara ◽  
D R Mitchell ◽  
R Kamiya
Keyword(s):  
Cross Section ◽  
Heavy Chain ◽  
Fragment Length Polymorphism ◽  
Beat Frequency ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Heavy Chains ◽  
Flagellar Beat ◽  
Section Electron ◽  
In The Wild

A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha heavy chain of outer arm dynein but retaining the beta and gamma heavy chains was isolated. Restriction fragment length polymorphism analysis with an alpha heavy chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha heavy chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha heavy chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha heavy chain, and that the outer arm missing the alpha heavy chain retains partial function.

scholarly journals Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures

1999 ◽  
Vol 144 (2) ◽  
pp. 293-304 ◽  
Author(s):  
David R. Mitchell ◽  
Winfield S. Sale
Keyword(s):  
Beat Frequency ◽  
Live Cells ◽  
Flagellar Motility ◽  
Wild Type ◽  
Electron Microscopic ◽  
Central Pair ◽  
Radial Spoke ◽  
Flagellar Beat ◽  
Sds Page

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

scholarly journals IC138 Is a WD-Repeat Dynein Intermediate Chain Required for Light Chain Assembly and Regulation of Flagellar Bending

2004 ◽  
Vol 15 (12) ◽  
pp. 5431-5442 ◽  
Author(s):  
Triscia W. Hendrickson ◽  
Catherine A. Perrone ◽  
Paul Griffin ◽  
Kristin Wuichet ◽  
Joshua Mueller ◽  
...  
Keyword(s):  
Light Chain ◽  
Beat Frequency ◽  
Wild Type ◽  
Central Pair ◽  
Flagellar Beat ◽  
C Terminus ◽  
Dynein Intermediate Chain ◽  
Radial Spokes ◽  
Calculated Mass ◽  
Wd Repeat

Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform.

scholarly journals Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms.

1992 ◽  
Vol 118 (5) ◽  
pp. 1163-1176 ◽  
Author(s):  
M E Porter ◽  
J Power ◽  
S K Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Synergistic Effects ◽  
Beat Frequency ◽  
Suppressor Mutation ◽  
Swimming Velocity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Flagellar Beat ◽  
Extragenic Suppressors

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.

scholarly journals Functional Recombination of Outer Dynein Arms with Outer Arm-Missing Flagellar Axonemes of a Chlamydomonas Mutant

1989 ◽  
Vol 92 (1) ◽  
pp. 77-83 ◽  
Author(s):  
HITOSHI SAKAKIBARA ◽  
RITSU KAMIYA
Keyword(s):  
Cell Model ◽  
Beat Frequency ◽  
High Salt ◽  
Type Model ◽  
Cell Models ◽  
Wild Type ◽  
Flagellar Beat ◽  
Recombination System ◽  
Dynein Arms

A flagellar mutant of Chlamydomonas, oda, lacks the entire outer dynein arm but can swim at a speed of one third to half of that of the wild type. We found that the addition of a high-salt extract of wild-type axonemes to demembranated oda cell models restored up to 83% of the outer arms normally present on the outer-doublet microtubules of wild-type axonemes. Furthermore, when reactivated in the presence of ATP after being mixed with the extract, the oda cell models gained a higher level of motility, close to that of the wild type. The increase in flagellar beat frequency parallelled the increase in the number of restored outer dynein arms. These observations indicate that the axoneme of the oda mutant retains the binding sites for the outer dynein arms, and that the outer arms solubilized with high salt are functionally active. This in vitro recombination system with the oda mutant should be useful as an assay system for various preparations of outer-arm dynein. Evidence is presented that the two axonemes on an oda cell model beat at the same frequency, whereas those on a wild-type model beat at different frequencies. The two oda axonemes beat at the same frequency even after the higher level of motility has been restored by addition of crude dynein extract. We propose that a heterogeneity in the outer dynein arms is responsible for the frequency imbalance between the two flagella of wild-type Chlamydomonas.

The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility

1996 ◽  
Vol 109 (6) ◽  
pp. 1545-1553 ◽  
Author(s):  
C. Gagnon ◽  
D. White ◽  
J. Cosson ◽  
P. Huitorel ◽  
B. Edde ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Cortical Microtubule ◽  
Flagellar Motility ◽  
Microtubule Network ◽  
Alpha Tubulin ◽  
Oxyrrhis Marina ◽  
Terminal Domain ◽  
Flagellar Beat ◽  
Sea Urchin Sperm

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

scholarly journals Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

scholarly journals Mutations in Hydin impair ciliary motility in mice

2008 ◽  
Vol 180 (3) ◽  
pp. 633-643 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Philippe Delmotte ◽  
Michael L. Robinson ◽  
Michael J. Sanderson ◽  
George B. Witman
Keyword(s):  
Fluid Transport ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Flagellar Motility ◽  
Wild Type ◽  
Central Pair ◽  
Ciliary Motility ◽  
Pair Defect ◽  
Radial Spokes ◽  
The Brain

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.

scholarly journals Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes during Mitosis in Drosophila

1999 ◽  
Vol 146 (3) ◽  
pp. 597-608 ◽  
Author(s):  
John T. Robinson ◽  
Edward J. Wojcik ◽  
Mark A. Sanders ◽  
Maura McGrail ◽  
Thomas S. Hays
Keyword(s):  
Heavy Chain ◽  
Spatial Organization ◽  
Critical Role ◽  
Genetic System ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
And Migration

Cytoplasmic dynein is a multisubunit minus-end–directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immu-nofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

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