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Digestion of the Radial Spoke in Chlamydomonas Flagella and the
Homology of Radial Spoke Proteins in Tetrahymena thermophila
Zagum
Bhatti
University
of Wisconsin-Milwaukee
Milwaukee,
WI
Mentor:
Dr. Pinfen Yang
The
radial spoke is an axonemal structure, required for the control of dynein-driven
flagellar motility. It is proposed that the radial spoke acts in signal
transduction to convey messages from the central pair to the surrounding
microtubules. Furthermore, flagellar beating changes suggest the radial
spoke is composed of an array of protein regulators. Chlamydomonas motility
mutants provide an opportunity to study the radial spoke as subunits of
an aggregate complex. Sucrose gradient results of mutant pf24, defective
in translation of radial spoke protein 2 (RSP2), reveals sedimentation
of proteins into at least three distinct radial spoke particles. To use
an alternate method to separate the spoke into subunit particles, a procedure
was sought for the limited digestion of the Chlamydomonas radial spoke,
with the intent to further resolve the positions of these individual proteins.
Previous findings indicate that the radial spoke in sea urchin axonemes
is digested by trypsin at a position in the spoke stalk. Using trypsin
to digest the Chlamydomonas radial spoke, an ideal digestion time and concentration
of trypsin were determined for proteolysis at the position of RSP3, an
AKAP. With this information, sucrose gradient analysis will be used for
the identification of the components of the resulting radial spoke particles.
Determining
the homology of radial spoke proteins in other species can provide clues
to the conservation of the mechanism behind the radial spoke in both flagella
and cilia, as most cilia also possess the fundamental “9+2” axonemal structure.
Prokaryotic Tetrahymena thermophila provides a relatively resourceful opportunity
to isolate ciliar radial spoke, and in contrast to Chlamydomonas, it has
proven to be an ideal organism for gene knockout procedures. Our findings
show that four radial spoke proteins in Chlamydomonas flagella show a degree
of homology in Tetrahymena cilia: RSP2, RSP3, RSP11, and RSP16. |
