640
Chlamydomonas
tubules have rigid flagellae, while mutants
lacking functional radial spokes are usu-
ally paralyzed. Radial spokes participate
in the regulation of dynein arm activity
and, thus, in flagellar bending. The radial
spoke consists of a thin stalk attached to
the A tubule of the outer doublet and of
a head that projects close to the central
pair of microtubules. A set of 17 axone-
ma
lpo
lypept
idesappearstobeassoc
iated
with radial spokes because radial spoke-
less mutants lack all these polypeptides.
Several of the corresponding genes have
been isolated and characterized.
A common feature observed in many
paralyzed mutants is the loss of a set
of polypeptides corresponding to an en-
tire complex, presumably because loss of
one subunit leads to increased turnover of
the other subunits or because the miss-
ing subunit is important for anchoring
the complex. In some cases, it has been
possible to identify the speciFc polypep-
tide that is affected by the mutation by
performing a dikaryon rescue experiment.
During wild-type crosses, biflagellate ga-
metes of plus and minus mating-type pro-
duce quadriflagellate temporary dikaryons.
It was originally shown that flagellar motil-
ity is restored in pairwise crosses between
paralyzed mutants and the wild-type, sug-
gesting that the polypeptides contributed
by both parents can be assembled into
functional flagellar complexes after mat-
ing. This approach has been extended by
labeling the proteins of the mutant and
by mating the labeled mutant cells with
unlabeled wild-type cells. Restoration of
flagellar function is allowed to occur in the
presence of a protein synthesis inhibitor to
block new protein synthesis. Under these
conditions, the polypeptide that is deF-
cient in the mutant appears only in the
unlabeled form, since it is derived from the
wild-type parent, whereas the other normal
proteins from the mutant are labeled.
Dikaryon rescue has also been used
to study the polarity of assembly of the
flagellar microtubules. In these studies,
transformants that expressed an epitope-
tagged
α
-tubulin were mated to recipient
cells that had half-length flagellae. Upon
fusion, the shorter pair of flagellae grew to
full length using a common pool of precur-
sors that included epitope-tagged tubulin.
Immunodetection methods revealed that
the flagellar microtubules elongate by ad-
dition of new subunits at their distal end.
Similar results have been obtained with
the assembly of radial spoke proteins onto
radial spokeless full-length flagella, sug-
gesting that both tubulin and radial spoke
proteins are transported to the distal end
of the flagellum prior to their assembly
into flagellar structures.
Because flagellar structure has been
conserved throughout evolution, results
obtained with
Chlamydomonas
are relevant
for understanding human diseases. These
include primary ciliary dyskinesis that af-
fects the motility of cilia, polycystic kidney
disease that involves, in some cases, a de-
fect in the assembly of the primary cilia,
and retinitis pigmentosa that causes retinal
degeneration through a defect in transport
of proteins through the connecting cil-
ium of the photoreceptor cells and thereby
leads to blindness. Several of the
Chlamy-
domonas
flagellar proteins are remarkably
similar to the human proteins associated
with some of these diseases.
Other mutations affecting the size of the
flagella have also been examined. Some of
these mutants have short flagella but other-
wise normal axonemal structures. Others,
called
stumpy mutants
,h
a
v
ev
e
r
ysh
o
r
t
,
nonfunctional flagella. Mutants lacking
flagella, called
bald mutants
, are deFcient in
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