A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

Altschul
S. F.
,
Madden
T. L.
,
Schäffer
A. A.
,
Zhang
J.
,
Zhang
Z.
,
Miller
W.
,
Lipman
D. J.
(
1997
).
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucl. Acids Res
25
,
3389
3402
Anderson
R. G. W.
(
1974
).
Isolation of ciliated or unciliated basal bodies from the rabbit oviduct.
J. Cell Biol
60
,
393
404
Auclair
W.
,
Siegel
B. W.
(
1966
).
Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins.
Science
154
,
913
915
Beck
K.
,
Brodsky
B.
(
1998
).
Supercoiled protein motifs: the collagen triple-helix and alpha-helical coiled-coil.
J. Struct. Biol
122
,
17
29
Debuchy
R.
,
Purton
S.
,
Rochaix
J. D.
(
1989
).
The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus.
EMBO J
8
,
2803
2809
Eyal
Y.
,
Neumann
H.
,
Or
E.
,
Frydman
A.
(
1999
).
Inverse single-strand RACE: An adaptor-independent method of 5RACE.
BioTechniques
27
,
656
658
Fernandez
E.
,
Schnell
R.
,
Ranum
L. P. W.
,
Hussey
S. C.
,
Silflow
C. D.
,
Lefebvre
P. A.
(
1989
).
Isolation and characterization of the nitrate reductase structural gene of Chlamydomonasreinhardtii.
Proc. Nat. Acad. Sci. USA
86
,
6449
6453
Ferris
P. J.
,
Goodenough
U. W.
(
1994
).
The mating-type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences.
Cell
76
,
1135
1145
Finst
R. J.
,
Kim
P. J.
,
Quarmby
L. M.
(
1998
).
Genetics of the deflagellation pathway in Chlamydomonas.
Genetics
149
,
927
936
Gitelman
S. E.
,
Witman
G. B.
(
1980
).
Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.
J. Cell Biol
87
,
764
70
Hartman
J. J.
,
Mahr
J.
,
McNally
K.
,
Okawa
K.
,
Iwamatsu
A.
,
Thomas
S.
,
Cheesman
S.
,
Heuser
J.
,
Vale
R. D.
,
McNally
F. J.
(
1998
).
Katanin, a microtubule-severing protein, is a novel AAA ATPase that target to the centrosome using a WD40-containing subunit.
Cell
93
,
277
287
Hastie
A. T.
,
Dicker
D. T.
,
Hingley
S. T.
,
Kueppers
F.
,
Higgins
M. L.
,
Weinbaum
G.
(
1986
).
Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms.
Cell Motil. Cytoskel
6
,
25
34
Jarvik
J. W.
,
Suhan
J. P.
(
1991
).
The role of the flagellar transition region: Inferences from the analysis of a Chlamydomonas mutant with defective transition region structures.
J. Cell Sci
99
,
731
740
Heimburg
T.
,
Schunemann
J.
,
Weber
K.
,
Geisler
N.
(
1999
).
FTIR-spectroscopy of multistranded coiled coil proteins.
Biochemistry
38
,
12727
12734
King
S. M.
,
Otter
T.
,
Witman
G. B.
(
1986
).
Purification and characterization of Chlamydomonas flagellar dyneins.
Meth. Enzymol
134
,
291
305
Kozminski
K. G.
,
Diener
D. R.
,
Rosenbaum
J. L.
(
1993
).
High level expression of nonacetylatable-tubulin in Chlamydomonas reinhardtii.
Cell Motil. Cytoskel
25
,
158
170
LeDizet
M.
,
Piperno
G.
(
1995
).
ida4-1, ida4-2 and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms.
Mol. Biol. Cell
6
,
713
723
Lefebvre
P. A.
,
Rosenbaum
J. L.
(
1986
).
Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration.
Annu. Rev. Cell Biol
2
,
517
546
Lewin
R. A.
(
1953
).
Studies on the flagella of algae. II. Formation of flagellaby Chlamydomonas in light and darkness.
Ann. NY Acad. Sci
56
,
1091
1093
Lohret
T. A.
,
McNally
F. J.
,
Quarmby
L. M.
(
1998
).
A role for katanin-mediated axonemal severing during Chlamydomonas deflagellation.
Mol. Biol. Cell
9
,
421
437
Lohret
T. A.
,
Zhao
L.
,
Quarmby
L. M.
(
1999
).
Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation.
Cell Motil. Cytoskel
43
,
221
231
Lupas
A.
,
Van Dyke
M.
,
Stock
J.
(
1991
).
Predicting coiled coils from protein sequences.
Science
252
,
1162
1164
McNally
F. J.
,
Vale
R. D.
(
1993
).
Identification of katanin, an ATPase that severs and disassembles stable microtubules.
Cell
75
,
419
429
Mohammed
B. J.
,
Mitchell
T. J.
,
Andrew
P. W.
,
Hirst
R. A.
,
O'Callaghan
C.
(
1999
).
The effect of the pneumococcal toxin, pheumolysin on brain ependymal cilia.
Microb. Pathog
27
,
303
309
Pazour
G. J.
,
Sineshchekov
O. A.
,
Witman
G. B.
(
1995
).
Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.
J. Cell Biol
131
,
427
440
Quarmby
L. M.
,
Hartzell
H. C.
(
1994
).
Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii.
J. Cell Biol
124
,
807
815
Quarmby
L. M.
(
1996
).
Ca2+influx activated by low pH in Chlamydomonas.
J. Gen. Physiol
108
,
351
361
Quarmby
L. M.
,
Lohret
T. A.
(
1999
).
Microtubule severing.
Cell Motil. Cytoskel
43
,
1
9
Rhoads
A. R.
,
Friedberg
F.
(
1997
).
Sequence motifs for calmodulin recognition.
FASEBJ
11
,
331
340
Romero
M. F.
,
Kanai
Y.
,
Gunshin
H.
,
Hediger
M. A.
(
1998
).
Expression cloning using Xenopus laevis oocytes.
Meth. Enzymol
296
,
17
52
Sager
R.
,
Granick
S.
(
1953
).
Nutritional studies with Chlamydomonas reinhardtii.
Ann. NY Acad. Sci
56
,
831
838
Sanders
M. A.
,
Salisbury
J. L.
(
1989
).
Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.
J. Cell Biol
108
,
1751
1760
Sanders
M. A.
,
Salisbury
J. L.
(
1994
).
Centrin plays an essential role in microtubule severing during flagellar excision in Chlamydomonas reinhardtii.
J. Cell Biol
108
,
795
806
Shoeman
R. L.
,
Traub
P.
(
1993
).
Assembly of intermediate filaments.
BioEssays
15
,
605
611
Smith
E. F.
,
Sale
W. S.
(
1991
).
Microtubule binding and translocation by inner dynein arm subtype I1.
Cell Motil. Cytoskel
18
,
258
268
Stephens
R. E.
(
1975
).
The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets.
J. Cell Biol
64
,
408
420
Taillon
B. E.
,
Adler
S. A.
,
Suhan
J. P.
,
Jarvik
J. W.
(
1992
).
Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.
J. Cell Biol
119
,
1613
1624
Vale
R.
(
1991
).
Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts.
Cell
64
,
827
839
Watson
M. R.
,
Hopkins
J. M.
(
1962
).
Isolated cilia from Tetrahymena pyriformis.
Exp. Cell Res
28
,
280
295
Wilkerson
C. G.
,
King
S. M.
,
Witman
G. B.
(
1994
).
Molecular analysis of theheavy chain of Chlamydomonas flagellar outer-arm dynein.
J. Cell Sci
107
,
497
506
Witman
G. B.
(
1986
).
Isolation of Chlamydomonas flagella and flagellar axomenes.
Meth. Enzymol
134
,
280
290
This content is only available via PDF.