26
Cell News 2/2015
action is required to load the Aurora-A-Cnn complex at the site
of microtubule nucleation in a centrosome
25
. Taken together, it
is important to study different PCM substrates and how they are
regulated by the interplay of mitotic kinases during centrosome
maturation.
Non-kinase-mediated PCM regulation of MTOC activity
Besides kinase-mediated regulation of PCM recruitment and cen-
trosome maturation, a recent finding suggested that cytoplasmic
tubulin dimers, which are known to function as building blocks
of microtubules, also play a role in regulating PCM structure and
function
26
. For clarity, in this text “tubulin dimer” refers to free
cytoplasmic tubulin dimers that are not part of microtubules.
Tubulin dimers are guanine nucleotide binding proteins and exist
in cells in either it's GTP- or GDP-bound form. Depending on the
nucleotide binding state of the tubulin dimer, it functions as a
molecular switch in regulating the dynamic instability of micro-
tubules. GTP-bound tubulin turns “on” the switch to enhance
microtubule growth, while GDP-bound tubulin turns “off” the
switch to promote microtubule disassembly.
To date, besides interacting with itself, tubulin dimers have been
shown to directly interact with only two centrosomal proteins
namely CPAP/Sas-4 and centrobin. The CPAP/Sas-4-tubulin in-
teraction has been studied in detail, showing that the interaction
is required for centriole elongation
26-28
. It is important to note
that CPAP does not bind centriolar microtubules via its tubulin
dimer-binding site. Thus, the CPAP/Sas-4-tubulin binding site is
specific for tubulin dimers but not for tubulins in their polyme-
rized form
29
. With these observations, it is clear that the CPAP/
Sas-4-tubulin interaction does not occur at the centriole, rather
takes place in the cytoplasm.
The regulation of PCM recruitment by tubulin is thought to be
unlikely due to its high-abundance and constant concentration
in a cell. However, the relative concentration between tubulin-
GTP and –GDP, which is known to oscillate during cell cycle, has
not been precisely quantified.
The significance of the cytoplasmic Sas-4-tubulin interaction
has been studied in great detail in the
Drosophila
model
26
. Bio-
chemical purification of Sas-4 complexes from
Drosophila
emb-
ryonic extracts revealed that while tubulin dimer-GDP favors the
formation of Sas-4 complexes with PCM proteins, tubulin dimer-
GTP limits the formation of Sas-4-mediated PCM complexes.
These findings point out that tubulin dimers critically regulate
the formation of Sas-4 mediated PCM complexes by functio-
ning as a molecular switch
26
. Indeed, genetic studies using flies
expressing a Sas-4 mutant that does not bind the tubulin dimer
confirmed the switching property of the tubulin dimer in regula-
ting the amount of PCM recruited during mitosis. In the absence
of tubulin dimer binding, centrosomes recruit increased amounts
of Cnn and thus exhibit enhanced microtubule nucleation
26
.
It is known that the conversion rate of tubulin-GDP into –GTP
is rapid in cells. Thus, at any given time, the tubulin dimer-GDP
complex only forms in a transient manner. If tubulin dimer-GDP
is a triggering factor for cytoplasmic PCM complex assembly, it
should be spatiotemporally regulated at the onset of G2 where
PCM starts to build on centrosomes before mitosis. In this scena-
rio, it would be crucial to identify the source of tubulin dimer-
GDP in cells that could function as a signaling cue to trigger
PCM complex assembly and recruitment. There are at least two
processes that could raise the availability of tubulin dimer-GDP,
including microtubule remodeling and cilium microtubule disas-
sembly, both of which occur parallel to the onset of G2. These
aspects await future experiments.
Potential linkers tethering the PCM in a centrosome
Earlier biochemical experiments revealed salt-stripped centroso-
mes to contain a salt-insensitive matrix of unknown compositi-
on, which could eventually complement microtubule nucleation.
This indicates that a component tightly associated with the cen-
triole wall could be a linker that assists in tethering the assem-
bled PCM around a centriole. Decades later, super resolution mi-
croscopy studies using a variety of antigens have unequivocally
resolved the mystery of complex PCM arrangement around cen-
trioles
11-14
. The molecular composition of the interphase between
PCM and centriole does indeed include S-CAP components. Thus,
it is likely that one of the components of the S-CAP complex
could function as a linker that mediates the tethering of PCM
in the functional assembly of a centrosome. Ideally, this should
be a protein component displaying an extended surface, able to
wrap around the centriole to seed an initial layer around it. Thus,
structural studies of S-CAP components at atomic resolution are
required to delineate this puzzle. Accordingly, three independent
studies have resolved crystal structures of the conserved TCP do-
main of Sas-4/CPAP from a variety of organisms revealing that
the conserved TCP domain could provide an extended surface-
like platform via its solvent-exposed single-layer of
β
-sheets
30
.
Functional studies in drosophila sperm cell confirmed this idea
as point mutations within the conserved TCP domain allowed
the centrioles to assemble but with depleted PCM
30
. Importantly,
the TCP mutant centrioles contained core centriolar structures,
as they were positive for the core centriolar proteins Bld-10 and
Ana2
30
. On the other hand, this does not seem to be the case
in
Drosophila
embryos as experiments where mutant TCPs were
expressed in wild type embryos revealed that Sas4-TCP mutants
failed to assemble centrioles
31
. However, in the absence of the
use of core centriolar markers, ultra structural analyses or ex-
periments with Sas-4 null embryos, these results remain insuf-
ficient to dissect the role of Sas-4’s TCP domain in centrosome
biogenesis in early embryos
31
.
Concluding remarks
Recent studies have enhanced our understanding of the comple-
xity of PCM assembly and the mechanisms that regulate their
structural changes during the cell cycle
11-14,23
. These structural
changes occurring at the PCM are required for the distinct func-
tions of centrosomes at interphase and mitosis. In our opini-
on, the major task in delineating the PCM puzzle is to combine
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