scholarly journals Mutant strains of Chlamydomonas reinhardtii that move backwards only.

1984 ◽  
Vol 98 (6) ◽  
pp. 2026-2034 ◽  
Author(s):  
R A Segal ◽  
B Huang ◽  
Z Ramanis ◽  
D J Luck
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Protein Composition ◽  
Wave Form ◽  
Wild Type ◽  
Forward Movement ◽  
Type Composition ◽  
Mutant Strains ◽  
Motility Mutant ◽  
Dynein Arms ◽  
Mutant Cells

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.

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 Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella.

1982 ◽  
Vol 92 (1) ◽  
pp. 170-175 ◽  
Author(s):  
M R Kuchka ◽  
J W Jarvik
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cell Size ◽  
Size Control ◽  
Variable Number ◽  
Pool Size ◽  
Precursor Protein ◽  
Wild Type ◽  
Protein Pool ◽  
Mutant Cells ◽  
Pool Sizes

A mutant of Chlamydomonas reinhardtii with a variable number of flagella per cell has been used to investigate flagellar size control. The mutant and wild-type do not differ in cell size nor in flagellar length, yet the size of the intracellular pool of flagellar precursor protein can differ dramatically among individual mutant cells, with, for example, triflagellate cells having three times the pool of monoflagellate cells. Because cells of the same size, but with very different pool sizes, have flagella of identical length, it appears that the concentration of the unassembled flagellar precursor protein pool does not regulate flagellar length. The relation between cell size, pool size, and flagellar length has also been investigated for wild-type cells of different sizes and ploidies. Again, flagellar length appears to be maintained independent of pool size or concentration.

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 Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components.

1978 ◽  
Vol 76 (3) ◽  
pp. 729-747 ◽  
Author(s):  
G B Witman ◽  
J Plummer ◽  
G Sander
Keyword(s):  
Lattice Spacing ◽  
Sodium Dodecyl ◽  
Internal Standard ◽  
Protein Composition ◽  
Wild Type ◽  
Microscope Examination ◽  
Radial Spokes ◽  
Dynein Arms ◽  
High Molecular Weight Proteins ◽  
And Function

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice-spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described.

scholarly journals Aeromonas Flagella (Polar and Lateral) Are Enterocyte Adhesins That Contribute to Biofilm Formation on Surfaces

2004 ◽  
Vol 72 (4) ◽  
pp. 1939-1945 ◽  
Author(s):  
Sylvia M. Kirov ◽  
Marika Castrisios ◽  
Jonathan G. Shaw
Keyword(s):  
Cell Lines ◽  
Biofilm Formation ◽  
Glass Tube ◽  
Intestinal Cell ◽  
Wild Type ◽  
Wild Type Level ◽  
Lateral Flagellum ◽  
Mutant Strains ◽  
Motility Mutant ◽  
Characteristic Features

ABSTRACT Aeromonas spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only ∼60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by ∼60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of ∼50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp. (broth inoculum, with a 16- to 20-h incubation at 37°C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

Kinematics and Kinetics of Flagellar Locomotion in Chlamydomonas Reinhardtii

2011 ◽  
Author(s):  
P. V. Bayly ◽  
B. L. Lewis ◽  
E. C. Ranz ◽  
R. J. Okamoto ◽  
R. B. Pless ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Video Data ◽  
Wild Type ◽  
Viscous Forces ◽  
Sorting Algorithms ◽  
Theoretical Predictions ◽  
Dynein Arms ◽  
Kinetics Of ◽  
Kinematics And Kinetics ◽  
Surrounding Fluid

The forces exerted on the flagellum of the swimming alga Chlamydomonas reinhardtii by surrounding fluid are estimated from video data. “Wild-type” cells, as well as cells lacking inner dynein arms (ida3) and cells lacking outer dynein arms (oda2) were imaged (350 fps; 125 nm). Digital image registration and sorting algorithms provide high-resolution descriptions of the kinematics of the cell body and flagellum. The swimming cell is then modeled as an ellipsoid in Stokes flow, propelled by viscous forces that depend linearly on the velocity of the flagellum. The coefficients (CN and CT) that related normal and tangent forces on the flagellum to corresponding velocity components are estimated from equilibrium requirements. Their values are consistent among all three genotypes and similar to theoretical predictions.

An adenylyl cyclase that functions during late development of Dictyostelium

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5463-5471 ◽  
Author(s):  
F. Soderbom ◽  
C. Anjard ◽  
N. Iranfar ◽  
D. Fuller ◽  
W.F. Loomis
Keyword(s):  
Adenylyl Cyclase ◽  
Critical Role ◽  
Fruiting Bodies ◽  
Wild Type ◽  
Adenylyl Cyclase Activity ◽  
Extracellular Signals ◽  
Mutant Strains ◽  
Morphological Mutants ◽  
Low Levels ◽  
Mutant Cells

A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(−) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(−) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(−) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.

scholarly journals Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms.

1986 ◽  
Vol 103 (5) ◽  
pp. 1895-1902 ◽  
Author(s):  
T Okagaki ◽  
R Kamiya
Keyword(s):  
Functional Differentiation ◽  
The Other ◽  
Sliding Velocity ◽  
Wild Type ◽  
Michaelis Constant ◽  
The Poor ◽  
Special Procedure ◽  
Bacterial Protease ◽  
Mutant Strains ◽  
Dynein Arms

To clarify the functional differentiation between the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were assessed by measuring the sliding velocities of outer-doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild-type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which is difficult to achieve by ordinary methods. The flagella were first fragmented by sonication, demembranated by Nonidet P-40, and then perfused under a microscope with Mg-ATP and nagarse, a bacterial protease with broad substrate specificity. The sliding velocity varied with the Mg-ATP concentration in a Michaelis-Menten manner in the axonemes from the wild type and a motile mutant lacking the outer dynein arm (oda38). The maximal sliding velocity and apparent Michaelis constant for Mg-ATP were measured to be 13.2 +/- 1.0 micron/s and 158 +/- 36 microM for the wild type and 2.0 +/- 0.1 micron/s and 64 +/- 18 microM for oda38. These maximal sliding velocities were significantly smaller than those estimated in beating axonemes; the reason is not clear. The velocities in the presence or absence of 10(-5) M Ca2+ did not differ noticeably. The axonemes of nonmotile mutants lacking either outer arms (pf13A, pf22) or inner arms (pf23) were examined for their ability to undergo sliding disintegration in the presence of 0.1 mM Mg-ATP. Whereas pf13A axonemes underwent normal sliding disintegration, the other two species displayed it only very poorly. The poor ability of pf23 axonemes to undergo sliding disintegration raises the possibility that the outer dynein arm cannot function well in the absence of the inner arm.

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.

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