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

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.

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Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas.

The Journal of Cell Biology ◽

10.1083/jcb.118.5.1145 ◽

1992 ◽

Vol 118 (5) ◽

pp. 1145-1162 ◽

Cited By ~ 123

Author(s):

D N Mastronarde ◽

E T O'Toole ◽

K L McDonald ◽

J R McIntosh ◽

M E Porter

Keyword(s):

Cross Sections ◽

Morphological Data ◽

Wild Type ◽

Cross Sectional ◽

Radial Spoke ◽

Single Region ◽

Radial Spokes ◽

Longitudinal View ◽

Dynein Arms ◽

Additional Loss

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.

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The CSC is required for complete radial spoke assembly and wild-type ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0271 ◽

2011 ◽

Vol 22 (14) ◽

pp. 2520-2531 ◽

Cited By ~ 52

Author(s):

Erin E. Dymek ◽

Thomas Heuser ◽

Daniela Nicastro ◽

Elizabeth F. Smith

Keyword(s):

Calcium Binding ◽

Critical Role ◽

Artificial Microrna ◽

Structural Studies ◽

Wild Type ◽

Radial Spoke ◽

Radial Spokes ◽

Dynein Arms ◽

Cilia And Flagella

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.

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Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components.

The Journal of Cell Biology ◽

10.1083/jcb.88.1.73 ◽

1981 ◽

Vol 88 (1) ◽

pp. 73-79 ◽

Cited By ~ 123

Author(s):

G Piperno ◽

B Huang ◽

Z Ramanis ◽

D J Luck

Keyword(s):

Cellular Proteins ◽

Two Dimensional ◽

Wild Type ◽

Molecular Weight Range ◽

Radial Spoke ◽

Radial Spokes ◽

32P Incorporation ◽

Electrophoretic Techniques ◽

Dynein Arms

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e09-04-0277 ◽

2009 ◽

Vol 20 (13) ◽

pp. 3055-3063 ◽

Cited By ~ 43

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.

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Secondary Messenger Systems in PTSD

10.1093/med/9780190215422.003.0014 ◽

2016 ◽

Author(s):

Ulrike Schmidt

Keyword(s):

Second Messengers ◽

Cyclic Adenosine Monophosphate ◽

Second Messenger ◽

Adenosine Monophosphate ◽

Cyclic Guanosine Monophosphate ◽

Brain Regions ◽

Guanosine Monophosphate ◽

Intracellular Camp ◽

Post Traumatic Stress ◽

Secondary Messenger

Second messengers such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate, and diacylglycerol (DAG) are a prerequisite for the signal transduction of extracellular receptors. The latter are central for cellular function and thus are implicated in the pathobiology of a variety of disorders, such as schizophrenia, bipolar disorder, major depression, and post-traumatic stress disorder (PTSD). This chapter focuses on the involvement of second messenger molecules and their regulators as direct targets in human and animal PTSD and aims to stimulate the underdeveloped research in this field. The synthesis of literature reveals that second messengers clearly play a central role in PTSD-associated brain regions and processes. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), an important regulator of intracellular cAMP levels, as well as protein kinase c, the major target of DAG, belong to the hitherto most promising PTSD candidate molecules directly involved in second messenger signaling.

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

The Journal of Cell Biology ◽

10.1083/jcb.92.3.722 ◽

1982 ◽

Vol 92 (3) ◽

pp. 722-732 ◽

Cited By ~ 128

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.

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Adenylate-cyclase Activity in Obsessive-compulsive Patients

European Psychiatry ◽

10.1016/j.eurpsy.2017.01.1060 ◽

2017 ◽

Vol 41 (S1) ◽

pp. S641-S642

Author(s):

D. Marazziti ◽

S. Baroni ◽

F. Mucci ◽

L. Palego ◽

A. Piccinni

Keyword(s):

Adenylate Cyclase ◽

Second Messengers ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Obsessive Compulsive ◽

Biological Assays ◽

Compulsive Disorder ◽

Healthy Control ◽

Competing Interest

IntroductionA possible role of second messengers, such as cyclic adenosine monophosphate (cAMP) signalling, in the development of obsessive-compulsive disorder (OCD) has been recently postulated.AimsThe aim of the present study was to explore and to compare the adenylate cyclase (AC) activity in both basal conditions and after the stimulation by isoprenaline (ISO) in platelets of OCD patients and healthy control subjects. The AC activity was measured both in the absence and in the presence of α- and β- adrenoreceptor antagonists.Materials and methodsForty patients were included in the study and compared with healthy volunteers. Biological assays were carried out with a method developed by us.ResultsThe basal AC activity was similar in both groups. The addition of 10 μM ISO enhanced significantly (P < .05) platelet basal AC in both groups. A stimulatory response following ISO in all subjects even without α-antagonists was also observed.DiscussionNo difference in the basal AC activity in platelet membranes of healthy subjects and OCD patients was found. Our findings showed that there is an inhibitory component of ISO effect on platelet AC, due to the agonist interaction with α2 receptors, at its higher concentrations (>1 μM), as well as a condition of supersensitive β-receptors. Our study suggests the presence of cathecolamine system disturbances in OCD.Disclosure of interestThe authors have not supplied their declaration of competing interest.

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

The Journal of Cell Biology ◽

10.1083/jcb.76.3.729 ◽

1978 ◽

Vol 76 (3) ◽

pp. 729-747 ◽

Cited By ~ 256

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.

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Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1311 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1311-1325 ◽

Cited By ~ 123

Author(s):

L C Gardner ◽

E O'Toole ◽

C A Perrone ◽

T Giddings ◽

M E Porter

Keyword(s):

Liquid Chromatography ◽

Cross Sections ◽

Close Association ◽

Longitudinal Section ◽

Radial Spoke ◽

Sds Page ◽

Radial Spokes ◽

Regulatory Complex ◽

Dynein Arms ◽

The Relationship

Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is though to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown (Huang et al., 1982; Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) to analyze the relationship between the DRC and the dynein arms. Our results suggest that some of the DRC components are located at the base of the second radial spoke in close association with the inner dynein arms. (a) Averages of axoneme cross-sections indicate that inner arm structures are significantly reduced in three DRC mutants (pf3 < pf2 < sup-pf-3 < wt). (b) These defects are more pronounced in distal/medial regions of the axoneme than in proximal regions. (c) Analysis of flagellar extracts by fast protein liquid chromatography and SDS-PAGE indicates that a specific dynein I2 isoform is missing in pf3 and reduced in pf2 and sup-pf-3. Comparison with ida4 and pf3ida4 extracts reveals that this isoform differs from those missing in ida4. (d) When viewed in longitudinal section, all three DRC mutants lack a crescent-shaped density above the second radial spoke, and pf3 axonemes lack additional structures adjacent to the crescent. We propose that the crescent corresponds in part to the location of the DRC, and that this structure is also directly associated with a subset of the inner dynein arms. This position is appropriate for a complex that is thought to mediate signals between the radial spokes and the dynein arms.

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