How to assemble an organelle: elongation and length
control of eukaryotic flagella.
CHLAMYDOMONAS FLAGELLA
Fig. 2. (A) Thin section electron micrograph through an isolated, demembranated flagellar axoneme (bar � 100 n
gram of major structural components of the axoneme and mutations that affect their assembly. The view is looking distally
the cell. Inner row dyneins have been divided into an outer lobe (green) and an inner lobe (yellow) whose densities are
the ida1 and ida4 mutants, respectively.
Supervisor: Dr. Marco Polin (m.polin@warwick.ac.uk)*
plex differs between cis and trans flagella in a manner
important for phototaxis (see below). Functionally,
most mutations that block outer row dynein assembly
reduce beat frequency by about 60% but have little effect on waveform, whereas loss of the inner row dyneins changes waveform rather than frequency
(Brokaw and Kamiya 1987).
Inner row dyneins have been fractionated into
seven complexes (a–g) that likely correspond to seven
unique enzymatic units (Kagami and Kamiya 1992).
Eight different catalytic heavy chains are associated
with these complexes, two in complex f (also referred
to as I1 dynein) and one in each of the other six inner
row dyneins. Mutations at ida1, ida2, ida3, and ida7
block assembly of the two-headed I1 dynein and deplete a single tri-lobed density from each 96 nm re-
peat (Fig. 3). Three of these I1-disrupting
now been cloned and found to encode the
chains and a 140 kDa intermediate chain
gous to outer row dynein intermediate cha
(Porter et al. 1996, Myster et al. 1997, Per
1998). All six of the “single-headed” inne
neins are associated with monomeric actin
gene product (Kato et al. 1993, Kato-Mino
1997). Curiously, disruption of the actin g
lethal and only prevents assembly of four o
dyneins (complexes a, c, d, and e). The
two complexes (b and g) recruit an actin-re
tein (not a component of wild-type flagella
ternative light chain (Kato-Minoura et al.
though these actin-associated inner row dyn
been designated as I2 and I3 dyneins, based
their presumed distribution within each 96
(Piperno et al. 1990, Piperno and Ramanis 1
tations that individually remove each dynein
yet been identified and some confusion e
cerning their axonemal locations. Bioc
however, they can be divided into two classe
their association with common subunits.
these actin-associated dyneins (complexes a
have a common light chain (p28), the prod
ida4 locus (LeDizet and Piperno 1995a, b
function mutations at this locus prevent a
these three dyneins and deplete three den
each 96 nm repeat (Fig. 3; Mastronarde et
The motility defects associated with ida4 are
those of other inner row dynein mutation
cells swim slowly as a result of waveform
(Kamiya et al. 1991). The remaining three
dyneins (containing heavy chains b, e, an
have a common light chain, centrin, the p
the vfl2 locus (Piperno et al. 1990). Althou
is a calcium binding protein of the EF-ha
(Salisbury 1989, 1995), significance of calc
ing to inner row dynein function is not k
only available Chlamydomonas centrin mu
missense mutations (Taillon et al. 1992) th
affect assembly of these dyneins. The ida6
Living organisms possess a striking abiltransversal
a)
b)
ity to generate and maintain complex spatial
arrangements, from organelles of predictable
shape and size to complex macroscopic patterns generated during embryonic development.
longitudinal
Spatial organisation is critical to cellular and
organismal functioning, but the mechanisms
behind its development and whether general
governing laws exist, are still largely unknown.
Solving this mystery is a major scientific challenge for the next century.
The project will take a close look at the
process of flagellar assembly in the unicellular Figure 1: (a) Individual C. reinhardtii cell
biflagellate green alga Chlamydomonas rein- held by a micropipette (scale bar 10 µm). (b)
hardtii, the preferred model organism for bi- Transversal and longitudinal schematics of the
ological studies of the eukaryotic flagellum. axoneme. (D.R. Mitchell J. Phycol. (2000))
Flagella and cilia, collectively known as undulipodia, are micrometric hair-like organelles responsible for a wide variety of tasks (e.g.
motility, transport, sensing). Their sophisticated internal structure, the axoneme, is highly
conserved across eukaryotic species. This allows general aspects of flagellar physiology, valid
e.g. in humans, to be studied directly in simple organisms like C. reinhardtii. Pioneering studies in this alga have revealed that growth and maintenance of eukaryotic flagella depend on
an evolutionarily conserved transport mechanism known as Intra Flagellar Transport (IFT),
which constantly ferries axonemal proteins bidirectionally between cell body and flagellar tip.
While the structure of individual IFT trains is by now well established, the link between IFT
transport and the actual dynamics of flagellar growth remains largely unknown.
You will study in detail the dynamics of flagellar elongation and beating during regrowth,
and their link with the arrival of individual IFT trains. Flagellar length and beating force are
intimately linked as both structural proteins (tubulins) and molecular motors (responsible for
flagellar motion) have to be transported by the same IFT particles. These studies will then
contribute to our understanding of how IFT load is partitioned between structural and nonstructural proteins (possibly dependent on flagellar length). Your research will provide new
insights into how flagellar proteins are incorporated into the growing axoneme and how they
manage to work cooperatively and generate spontaneous oscillations.
The project will include both experiments and modelling/simulation, so it is essential to
keep an open mind and be willing to challenge yourself in new and diverse areas of research.
You will be warmly encouraged to think independently and develop your own ideas, learning
important skills for a competitive professional profile.
Technical requirements: basic programming knowledge (e.g. C++, Matlab) is fundamental; basic scientific computing beneficial but not necessary; previous laboratory experience
beneficial but not necessary (but you need to have an interest in learning experimental techniques).
Fig. 3. Diagram of dyneins and related structures seen
along the A tubule of each outer doublet in thin section electron micrographs. Outer arm dyneins repeat with a 24 nm period, other structures with a 96 nm period. Colors for dynein
isoforms correspond to those in Fig. 2. The dynein regulatory
complex (DRC, blue) is situated between inner and outer row
dyneins and close to spoke S2. Unknown structures (black) may
include inner row dynein isoforms b and g, whose locations
have not been identified. Adapted from Porter, 1996.
*The theoretical/simulation component of this project will be done in close collaboration with
Dr. Hermes Gadelha (http://www.damtp.cam.ac.uk/user/habg2/ )