scholarly journals IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

2009 ◽  
Vol 20 (13) ◽  
pp. 3055-3063 ◽  
Author(s):  
Raqual Bower ◽  
Kristyn VanderWaal ◽  
Eileen O'Toole ◽  
Laura Fox ◽  
Catherine Perrone ◽  
...  
Keyword(s):  
Double Mutant ◽  
Essential Role ◽  
Null Allele ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Mutant Strains ◽  
Dynein Arms ◽  
Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

scholarly journals The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

1998 ◽  
Vol 9 (12) ◽  
pp. 3351-3365 ◽  
Author(s):  
Catherine A. Perrone ◽  
Pinfen Yang ◽  
Eileen O’Toole ◽  
Winfield S. Sale ◽  
Mary E. Porter
Keyword(s):  
Insertional Mutagenesis ◽  
Gene Transformation ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Important Target ◽  
Dynein Intermediate Chain ◽  
Biochemical Analyses ◽  
Intermediate Chain

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm thatida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that theida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.

scholarly journals Analysis of the movement of Chlamydomonas flagella:" the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella.

1982 ◽  
Vol 92 (3) ◽  
pp. 722-732 ◽  
Author(s):  
C J Brokaw ◽  
D J Luck ◽  
B Huang
Keyword(s):  
Recovery Phase ◽  
Wild Type ◽  
Radial Spoke ◽  
Suppressor Mutations ◽  
Mutant Strains ◽  
Type Pattern ◽  
Dynein Arms ◽  
Beta Frequency ◽  
Amplitude Pattern ◽  
Asymmetric Bending

The mutation uni-1 gives rise to uniflagellate Chlamydomonas cells which rotate around a fixed point in the microscope field, so that the flagellar bending pattern can be photographed easily. This has allowed us to make a detailed analysis of the wild-type flagellar bending pattern and the bending patterns of flagella on several mutant strains. Cells containing uni-1, and recombinants of uni-1 with the suppressor mutations, suppf-1 and suppf-3, show the typical asymmetric bending pattern associated with forward swimming in Chlamydomonas, although suppf-1 flagella have about one-half the normal beta frequency, apparently as the result of defective function of the outer dynein arms. The pf-17 mutation has been shown to produce nonmotile flagella in which radial spoke heads and five characteristic axonemal polypeptides are missing. Recombinants containing pf-17 and either suppf-2 or suppf-3 have motile flagella, but still lack radial-spoke heads and the associated polypeptides. The flagellar bending pattern of these recombinants lacking radial-spoke heads is a nearly symmetric, large amplitude pattern which is quite unlike the wild-type pattern. However, the presence of an intact radial-spoke system is not required to convert active sliding into bending and is not required for bend initiation and bend propagation, since all of these processes are active in suppfpf-17 recombinants. The function of the radial-spoke system appears to be to convert the symmetric bending pattern displayed by these recombinants into the asymmetric bending pattern required for efficient swimming, by inhibiting the development of reverse bends during the recovery phase of the bending cycle.

scholarly journals Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein.

1991 ◽  
Vol 112 (3) ◽  
pp. 441-447 ◽  
Author(s):  
R Kamiya ◽  
E Kurimoto ◽  
E Muto
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cross Section ◽  
Double Mutant ◽  
The Other ◽  
Flagellar Motility ◽  
Wild Type ◽  
Single Group ◽  
Heavy Chains ◽  
Section Electron ◽  
Dynein Arms

Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.

scholarly journals ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

1993 ◽  
Vol 120 (3) ◽  
pp. 733-741 ◽  
Author(s):  
C J Horst ◽  
G B Witman
Keyword(s):  
Regulatory System ◽  
Structural Defects ◽  
Cell Models ◽  
Flagellar Motility ◽  
Wild Type ◽  
Structural Localization ◽  
Calcium Dependent ◽  
Mutant Strains ◽  
Radial Spokes ◽  
Dynein Arms

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

scholarly journals Vibrio choleraeadapts to sessile and motile lifestyles by cyclic di-GMP regulation of cell shape

2020 ◽  
Vol 117 (46) ◽  
pp. 29046-29054 ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Brian Y. Hsueh ◽  
Nguyen T. Q. Nhu ◽  
Joshua L. Franklin ◽  
Yann S. Dufour ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Guanosine Monophosphate ◽  
Human Pathogen ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Pathways ◽  
Matrix Components ◽  
Curved Rod

The cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. Alterations to the rod shape, such as the curved rod, occur through manipulating the process of cell wall synthesis. The human pathogenVibrio choleraetypically exists as a curved rod, but straight rods have been observed under certain conditions. While this appears to be a regulated process, the regulatory pathways controlling cell shape transitions inV. choleraeand the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape inV. choleraeis regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by posttranscriptionally repressing expression ofcrvA, a gene encoding an intermediate filament-like protein necessary for curvature formation inV. cholerae.This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated withV. cholerae’s induction of sessility. During microcolony formation, wild-typeV. choleraecells tended to exist as straight rods, while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straightV. choleraemutants have reduced swimming speed when using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic and biofilm lifestyles.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals A conserved CaM- and radial spoke–associated complex mediates regulation of flagellar dynein activity

2007 ◽  
Vol 179 (3) ◽  
pp. 515-526 ◽  
Author(s):  
Erin E. Dymek ◽  
Elizabeth F. Smith
Keyword(s):  
Mass Spectrometry ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Calcium Sensor ◽  
Wild Type ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Dynein Arms

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

scholarly journals Increased expression of Saccharomyces cerevisiae translation elongation factor 1 alpha bypasses the lethality of a TEF5 null allele encoding elongation factor 1 beta.

Genetics ◽  
1995 ◽  
Vol 141 (2) ◽  
pp. 481-489 ◽  
Author(s):  
T G Kinzy ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Elongation Factor ◽  
Wild Type ◽  
Translation Elongation Factor ◽  
Gene Encoding ◽  
Translation Elongation ◽  
Dominant Mutant ◽  
Cold Sensitive ◽  
Elongation Factor 1

Abstract Translation elongation factor 1beta (EF-1beta) catalyzes the exchange of bound GDP for GTP on EF-1alpha. The lethality of a null allele of the TEF5 gene encoding EF-1beta in Saccharomyces cerevisiae was suppressed by extra copies of the TEF2 gene encoding EF-1alpha. The strains with tef5::TRP1 suppressed by extra copies of TEF were slow growing, cold sensitive, hypersensitive to inhibitors of translation elongation and showed increased phenotypic suppression of +1 frameshift and UAG nonsense mutations. Nine dominant mutant alleles of TEF2 that cause increased suppression of frameshift mutations also suppressed the lethality of tef5::TRP1. Most of the strains in which tef5::TRP1 is suppressed by dominant mutant alleles of TEF2 grew more slowly and were more antibiotic sensitive than strains with tef5::TRP1 is suppressed by wild-type TEF2. Two alleles, TEF2-4 and TEF2-10, interact with tef5::TRP1 to produce strains that showed doubling times similar to tef5::TRP1 strains containing extra copies of wild-type TEF2. These strains were less cold sensitive, drug sensitive and correspondingly less efficient suppressor of +1 frameshift mutations. These phenotypes indicate that translation and cell growth are highly sensitive to changes in EF-1alpha and EF-1beta activity.

scholarly journals VIBRIO CHOLERAE ADAPTS TO SESSILE AND MOTILE LIFESTYLES BY CYCLIC DI-GMP REGULATION OF CELL SHAPE

2020 ◽  
Author(s):  
Nicolas L. Fernandez ◽  
Nguyen T. Q. Nhu ◽  
Brian Y. Hsueh ◽  
Joshua L. Franklin ◽  
Yann S. Dufour ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Morphology ◽  
Cell Shape ◽  
Guanosine Monophosphate ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Pathways ◽  
Matrix Components ◽  
Biofilm Matrix

AbstractThe cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. While V. cholerae grows into a curved shape under most conditions, straight rods have been observed. However, the signals and regulatory pathways controlling cell shape transitions in V. cholerae and the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape in V. cholerae is regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by repressing expression of crvA, a gene encoding an intermediate filament-like protein necessary for curvature formation in V. cholerae. This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated with V. cholerae’s induction of sessility. Wild-type V. cholerae cells adhering to a surface lose their characteristic curved shape to become as straight as cells lacking crvA while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straight V. cholerae mutants have reduced speed when swimming using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic or biofilm lifestyles.

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