scholarly journals Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

2005 ◽  
Vol 16 (12) ◽  
pp. 5661-5674 ◽  
Author(s):  
Linda M. DiBella ◽  
Oksana Gorbatyuk ◽  
Miho Sakato ◽  
Ken-ichi Wakabayashi ◽  
Ramila S. Patel-King ◽  
...  
Keyword(s):  
Motor Function ◽  
Beat Frequency ◽  
Immunoblot Analysis ◽  
Null Mutant ◽  
Dynein Motor ◽  
Flagellar Beat ◽  
A Subunit ◽  
Type Motor ◽  
A Minor ◽  
Intermediate Chains

Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.

scholarly journals Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

2008 ◽  
Vol 19 (9) ◽  
pp. 3724-3734 ◽  
Author(s):  
Christopher A. Tanner ◽  
Panteleimon Rompolas ◽  
Ramila S. Patel-King ◽  
Oksana Gorbatyuk ◽  
Ken-ichi Wakabayashi ◽  
...  
Keyword(s):  
Motor Function ◽  
Light Chain ◽  
Beat Frequency ◽  
Chain Complex ◽  
Genomic Analysis ◽  
Dynein Light Chain ◽  
Enzyme Systems ◽  
Dynein Motor ◽  
Flagellar Beat ◽  
Dynein Arms

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ∼2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3′ end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.

scholarly journals Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

2011 ◽  
Vol 22 (15) ◽  
pp. 2690-2701 ◽  
Author(s):  
Melissa D. Stuchell-Brereton ◽  
Amanda Siglin ◽  
Jun Li ◽  
Jeffrey K. Moore ◽  
Shubbir Ahmed ◽  
...  
Keyword(s):  
Light Chain ◽  
Direct Evidence ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Intermediate Chains ◽  
Activator Complex

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

Development of a Portable-type Motor Function Evaluation System using Compact RGB-D Sensor

2021 ◽  
Vol 2021 (0) ◽  
pp. 2P1-D05
Author(s):  
Keisuke MOCHIDA ◽  
Naohiko HANAJIMA ◽  
Makoto OHATA ◽  
Tatsunori MIMASA ◽  
Youhei MURAOKA ◽  
...  
Keyword(s):  
Motor Function ◽  
Evaluation System ◽  
Function Evaluation ◽  
Type Motor

Temperature effects on anaphase chromosome movement in the spermatocytes of two species of crane flies (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius)

1979 ◽  
Vol 39 (1) ◽  
pp. 29-52
Author(s):  
C.J. Schaap ◽  
A. Forer
Keyword(s):  
Sex Chromosomes ◽  
Temperature Effects ◽  
Sex Chromosome ◽  
Beat Frequency ◽  
General Pattern ◽  
Force Production ◽  
Temperature Shift ◽  
Ciliary Beat Frequency ◽  
Chromosome Movement ◽  
Flagellar Beat

Using phase-contrast cinemicrography on living crane fly (Nephrotoma suturalis Loew and Nephrotoma ferruginea Fabricius) spermatocytes, we have studied the effects of a range of temperatures (6–30 degrees C) on the anaphase I chromosome-to-pole movements of both autosomes and sex chromosomes. In contrast to previous work we have been able to study chromosome-to-pole velocities of autosomes without concurrent pole-to-pole elongation. In these cells we found that the higher the temperature, the faster was the autosomal chromosomes movement. From reviewing the literature we find that the general pattern of the effects of temperature on chromosome movement is similar whether or not pole-to-pole elongation occurs simultaneously with the chromosome-to-pole movement. Changes in cellular viscosities calculated from measurements of particulate Brownian movement do not seem to be able to account for the observed velocity differences due to temperature. Temperature effects on muscle contraction speed, flagellar beat frequency, ciliary beat frequency, granule flow in nerves, and chromosome movement have been compared, as have the activation energies for the rate-limiting steps in these motile systems: no distinction between possible mechanisms of force production is possible using these comparisons. The data show that even the different autosomes within single spermatocytes usually move at different speeds. These velocity differences cannot simply be related to chromosome size as the autosomes are visually indistinguishable. The sex chromosomes start their anaphase poleward movement after that of the autosomes, and move more slowly (by a factor of about 4), but their velocities appear to be affected by temperature in the same fashion as those of the autosomes. The interval between the onset of autosome anaphase and sex chromosome anaphase is also affected by temperature: the higher the temperature, the shorter the interval between the 2 stages. We have observed abnormalities in sex chromosome segregation, which may be due to temperature, but have not determined what the exact temperature shift conditions are that cause these abnormalities.

scholarly journals The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

1997 ◽  
Vol 8 (4) ◽  
pp. 607-620 ◽  
Author(s):  
S H Myster ◽  
J A Knott ◽  
E O'Toole ◽  
M E Porter
Keyword(s):  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Northern Blot Analysis ◽  
Immunoblot Analysis ◽  
Transcription Unit ◽  
Peptide Sequence ◽  
Dynein Heavy Chain ◽  
The Individual ◽  
Intermediate Chains ◽  
Specific Peptide

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

scholarly journals Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

1995 ◽  
Vol 198 (3) ◽  
pp. 645-653 ◽  
Author(s):  
C Shingyoji ◽  
K Yoshimura ◽  
D Eshel ◽  
K Takahashi ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
Beat Frequency ◽  
Distal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Flagellar Beat ◽  
Tripneustes Gratilla ◽  
Sea Urchin Sperm

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly and function of Chlamydomonas flagellar mastigonemes as probed with a monoclonal antibody

1996 ◽  
Vol 109 (1) ◽  
pp. 57-62 ◽  
Author(s):  
S. Nakamura ◽  
G. Tanaka ◽  
T. Maeda ◽  
R. Kamiya ◽  
T. Matsunaga ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Beat Frequency ◽  
Distal Portion ◽  
Distal Region ◽  
Ring Structure ◽  
Live Cells ◽  
Swimming Velocity ◽  
Flagellar Beat ◽  
Original Length ◽  
And Function

Mastigonemes are hair-like projections on the flagella of various kinds of lower eukaryotes. We obtained a monoclonal antibody (mAb-MAST1) to mastigonemes of Chlamydomonas reinhardtii, and found that it reacts with a single flagellar glycoprotein of about 230 kDa. Interestingly, immunofluorescence microscopy demonstrated that mAb-MAST1 recognizes not only the flagellar mastigonemes but also a ring composed of 10 or more particles located in the anterior end of the cell body close to the flagellar bases. The ring structure may be the pool of the mastigoneme protein. When the flagella are amputated, they regenerate to their original length in 90–120 minutes. We found that mastigonemes appear on the new flagellar surface as early as 15 minutes after deflagellation, and that new mastigonemes are mostly assembled onto the distal region of the flagellar surface. Mastigonemes thus appear to be inserted into the membrane only in the distal region of the flagellum. Alternatively, mastigonemes may be inserted at the base and transported very rapidly to the distal portion where they are trapped. When live cells are treated with mAb-MAST1, mastigonemes disappear from the flagellar surface. In these mAb-MAST1 treated cells, the swimming velocity decreases to 70–80% of the normal value, although the flagellar beat frequency increases to approximately 110% of the control. These findings demonstrate vectorial transport of mastigonemes to their assembly sites, and show that mastigonemes function to increase flagellar propulsive force by increasing the effective surface of the flagellum.

Heat shock protein 80 ofNeurospora crassa: Sequence analysis of the gene and expression during the asexual phase

2000 ◽  
Vol 46 (11) ◽  
pp. 981-991 ◽  
Author(s):  
T L Girvitz ◽  
P M Ouimet ◽  
M Kapoor
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Immunoblot Analysis ◽  
Sequence Motifs ◽  
Gene Encoding ◽  
Putative Promoter Region ◽  
Calculated Molecular Mass ◽  
Transcription Start Sites ◽  
Oligomeric Complex ◽  
A Minor

Heat shock protein 80 (Hsp80) of Neurospora crassa, a member of the stress-90 protein family, is a cytosolic molecular chaperone that interacts directly with Hsp70 to form a hetero-oligomeric complex. The complete nucleotide sequence of the gene encoding this protein, along with the 5'- and 3'-flanking DNA, is reported. The coding sequence is interrupted by two introns, 61 and 30 nucleotides, respectively, in length. The deduced amino acid sequence corresponds to a 695-residue polypeptide with a calculated molecular mass of 78 894 Da and an average pI of 4.94. Primer extension experiments demonstrated two transcription start sites, a major and a minor one. No sequence motifs resembling the standard eukaryotic heat shock elements were evident in the putative promoter region. Immunoblot analysis showed Hsp80 protein to be present in the mature, dormant conidia, while the hsp80 transcripts were not detected. Both the transcripts and the protein were present in the germinating conidia in the absence of externally applied stress.Key words: Hsp90, filamentous fungi, sequence, conidia, germination.

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 Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome

2013 ◽  
Vol 24 (20) ◽  
pp. 3205-3214 ◽  
Author(s):  
Olga N. Zhapparova ◽  
Artem I. Fokin ◽  
Nadezhda E. Vorobyeva ◽  
Sofia A. Bryantseva ◽  
Elena S. Nadezhdina
Keyword(s):  
Phosphorylation Site ◽  
Cell Polarization ◽  
Microtubule Organization ◽  
Microtubule Binding ◽  
Major Function ◽  
Cytoskeleton Organization ◽  
Dynein Motor ◽  
Microtubule Binding Domain ◽  
A Minor ◽  
Radial Organization

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end–directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150Gluedsubunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150Gluedand does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150Gluedto the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150Gluedexpressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150Gluedsuggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.

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