Mammalian PKA with PKI inhibitor bound

Chlamydomonas Flagella Research

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Flagella 101

Cilia and flagella are essentially the same things and are highly similar between all eukaryotes.  (Bacterial flagella are entirely different and probably shouldn't be called flagella at all.)  The inner mechanical works of a flagellum is called the axoneme and is based on microtubules.  Although there are exceptions, most axonemes have 9 doublet microtubules organized as a cylinder around 2 central pair microtubules.  This so-called 9 + 2 axoneme is diagrammed below.

Cross-section of Chlamydomonas axoneme

The force to create the bending of a flagellum is generated by the dynein arms.  Dyneins are motor proteins.  Flagella have at least 4 different kinds of dyneins arranged in a specific repeating pattern.  One end of each dynein is permanently bound to the A tubule of a microtubule doublet.  Using the energy of ATP hydrolysis, the other end of the dynein reaches out to the adjacent B tubule and walks along this doublet.  This dynein activity causes 2 microtubule doublets that are next to each other to slide relative to one another.  Because this microtubule sliding is restricted (probably by the nexin links), tension is created.  This tension causes the microtubules to bend.  In order to create the repeated bending as seen at the top of the page, dynein motors must be turned on and off.  Currently, how this is done is not known for any flagella.

Control of Flagellar Beating

It is not clear how the activity of dynein motors is controlled.  Many lines of evidence indicate that the regulatory mechanisms are quite complex.  The central pair microtubules and the radial spoke structures both play important roles in controlling flagellar beating.  In addition, there is a complex system of kinases and phosphatases that regulate dynein motors and thus flagellar motility.  Evidence also indicates that Ca2+ is an important regulator of beating in most flagella.  My lab has been involved in experiments investigating most of these aspects of flagellar control, but our central focus is on the kinase cAMP-dependent protein kinase (PKA).

What We Know About PKA in Chlamydomonas Flagella

In Chlamydomonas, PKA inhibits flagellar beating2, and our early work showed that active PKA slows the dynein motors down3.  Subsequent work by our collaborators (Win Sale, Emory University School of Medicine) indicates that one particular form of dynein called "inner arm I1" is the only type of dynein necessary for the PKA effect1.   As a kinase enzyme, the normal role of PKA is to phosphorylate proteins.  Putting this information together suggests the following model for the role of PKA in regulating flagellar motility.

Model of dynein regulation In this potential model, I1 dynein with a phosphate (P) attached (phosphorylated) is slower at creating microtubule sliding than dephosphorylated I1 dynein.  PKA phosphorylates I1 to slow  I1 down.  An opposing protein phosphatase (PP1 or PP2b) removes the phosphate to speed I1 dynein up.  The PKA and the phosphatases are themselves regulated by as yet unknown mechanisms.

What We Don't Know About PKA

Although all evidence points to a crucial role for PKA in controlling flagella in Chlamydomonas and other organisms, there is an awful lot that we do not know about flagellar PKA.  One other thing we are pretty sure of is that the PKA in Chlamydomonas flagella is a weird form of PKA.  Thus, the "rules" about the way PKA works in the cytoplasm of other cells may not apply in Chlamydomonas.  Here is a partial list of what we don't know:

  • The PKA protein in Chlamydomonas flagella has not been purified.  So, we do not know its exact identity.
  • We do not know which gene encodes the Chlamydomonas PKA.
  • We do not know what protein(s) PKA phosphorylates.  Evidence from our collaborators suggests that a different kinase (casein kinase I) actually directly phosphorylates I1 dynein5.  Thus, PKA may target some other protein, which in turn controls phosphorylation of I1 dynein.
  • We do not know how PKA is regulated.  Unlike typical cytoplasmic PKA, Chlamydomonas PKA does not seem to be controlled by the molecule cAMP.
  • We do not know where PKA is located in the axoneme.  Location is probably highly important for its function.
  • The role of PKA in a living cell is not known.  Thus far all of the work on understanding the role of PKA in flagellar motility has been performed on axonemes that were separated from cells.  So, a major question remains as to what PKA does during swimming.

What We Are Currently Doing to Find Out About PKA

1)  We are in the process of identifying and purifying the PKA protein from Chlamydomonas flagella.  We are using an affinity-based approach to this problem. 

Mammalian PKA with PKI bound One aspect in which Chlamydomonas PKA is similar to mammalian PKA (blue in diagram on left) is that both tightly bind the inhibitor peptide PKI (purple in diagram).  We are using the tight binding of PKA to PKI, to first identify and then to purify PKA.  One undergraduate is currently working on this project, but there is room for more researchers (MS or undergraduate).

2)  We are in the process of cloning a potential gene for Chlamydomonas PKA.  Undergraduates Colleen Trantow and Jason Wells successfully cloned most of the kinase domain of this gene.  However, there is some question as to whether this gene is actually a PKA or not.  We are in the process of cloning an additional part of the sequence which should greatly help our understanding of this gene.

3)  We are conducting experiments to try to determine the role of PKA in controlling flagellar motility.  One popular hypothesis is that PKA is required for Chlamydomonas to perform phototaxis.

Model of phototaxis PhototaxisChlamydomonas sense the direction of light by a single eyespot (red in the figure).  The position of the eyespot relative to the two flagella is always the same.  If a cell swimming toward the top of the screen (1) senses light coming in from the right, this causes an influx of Ca2+ into the flagella (2).  The 2 flagella respond differently to this increase in Ca2+; one flagellum becomes more active, and the other becomes less active (3).  This difference in activity causes the cell to turn toward the light (4).  Cells can be either positively phototactic (turn toward the light) or negatively phototactic (turn away from the light).

Former undergraduate researcher Mike Watson tested this hypothesis using lysed-cell models in which the flagella were still attached to the cell body but the membranes were removed.  Much to our surprise, he found that changing the activity of PKA or of phosphatases did not change the way that cells respond to Ca2+.  His results would suggest that PKA is not involved in phototaxis4.  However, there may be other explanations for his results.  For example, because these were dead, lysed cells, actual phototaxis could not be tested.  Perhaps in an intact cell PKA does play a role.  We are currently using other approaches to test the potential role of PKA in living cells.  These experiments use a variety of microscope-base motility assays.

Literature Cited

This was not intended to be an extensive review of PKA in flagella or flagellar motility.  Instead it is a simplistic introduction to what goes on in the Howard lab.  Numerous critically important works and concepts were left out for simplicity's sake.  My apologies to all the labs that were not cited here. 

  1. Habermacher G, Sale WS. 1997. Regulation of flagellar dynein by phosphorylation of a 138-kD inner arm dynein intermediate chain. J Cell Biol 136:167-76.
  2. Hasegawa E, Hayashi H, Asakura S, Kamiya R. 1987. Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP-dependent phosphorylation. Cell Motil Cytoskeleton 8:302-11.
  3. Howard DR, Habermacher G, Glass DB, Smith EF, Sale WS. 1994. Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase. J Cell Biol 127:1683-92.
  4. Watson, Jr. ME. & DR Howard. 1997.  Calcium-dependent regulation of axonemal beating in Chlamydomonas is apparently not modulated by phosphorylation. Molec. Biol. Cell 8:52a.
  5. Yang P, Sale WS. 2000. Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity. J Biol Chem 275:18905-12.

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Last Modified:  July 2005