Biology Reference
In-Depth Information
vi) No proof is available of an intrinsic asymmetry around the preexisting cen-
triole before the onset of centriole duplication
These observations suggest that a yet unidentified mechanism inherent to the
centrosome assures that only one new centriole is formed near a preexisting
centriole (Sluder and Khodjakov
2010
).
Analysis of centriole duplication is challenged by a combination of factors.
Centrosomes are essential for development in animals. Centrosomes and centrioles
are in low-abundance, found only in one or two copies per cell, thus challenging
biochemical approaches. Furthermore, new centriole intermediates are few, small,
short-lived, and form too close to the preexisting centriole to be observed as a
distinct entity, making it extremely challenging to study centriole intermediates by
traditional light microscopy (the internal structure of the centriole is beyond the
resolution of standard light microscopy).
Despite these challenges, many proteins involved in centriole duplication have
been identified over the past 15 years. The recent identification of many proteins
involved in centriole duplication opens new ways to overcome these barriers. One
commonly used way is to overexpress the centriolar protein, usually in immor-
talized cells (in vitro), and observe the consequences using microscopy
(Dzhindzhev et al.
2010
; Gopalakrishnan et al.
2010
; Tang et al.
2009
). While
sometimes informative, interpreting overexpression data is problematic due to the
fact that proteins are studied at non-physiological levels. Also, immortalized cells
often have abnormal centrioles, suggesting they already carry mutations that
prevent normal centriole duplication. Therefore, to balance these limitations, it is
important to develop approaches to study centriole duplication in vivo, when
proteins are expressed at physiological levels.
For a number of reasons, Drosophila is ideal for developing such a balanced
approach for studying centriole duplication and centrosome biogenesis:
First: mutants defective in centrosome biogenesis are available (see Table
1.1
).
In flies, even null mutations in essential centrosomal proteins are not embryonic
lethal and the fly can often develop to maturity. This is due to maternal contri-
bution which allows the embryo to form centrosomes when they are critical for
development, namely during early embryonic development. Later during pupal
development when the adult fly is forming, maternal contribution becomes
depleted but centrosomes are no longer essential for development. This allows
extensive characterization of defective centrosome biogenesis in the testes and
sensory neurons of pupae (Basto et al.
2006
; Blachon et al.
2009
; Blachon et al.
2008
; Mottier-Pavie and Megraw
2009
).
Second: techniques are available to introduce newly-engineered proteins with
modified capabilities into a null mutant background, allowing their specific
function to be studied with expression at near physiological levels and in the
absence of the wild-type protein (Blachon et al.
2008
; Gopalakrishnan et al.
2011
).
This is especially useful in the study of centrosomes, as many centrosomal proteins
form multiprotein complexes and may have more than one function. The ability to
engineer a mutant that is deficient in one or limited interactions is very insightful
