CILIA
Flagellar microtubule doublet
assembly in vitro reveals a regulatory
role of tubulin C-terminal tails
M. Schmidt-Cernohorska^1 *, I. Zhernov2,3, E. Steib^1 , M. Le Guennec^1 , R. Achek^4 ,
S. Borgers^1 , D. Demurtas^5 , L. Mouawad^4 , Z. Lansky^2 , V. Hamel^1 †, P. Guichard^1 †
Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of
a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport
and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism
governing their formation is unknown. We used a cell-free assay to demonstrate a crucial
inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly.
Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of
a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament
inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with
binding protein partners. The dynamics of B-microtubule nucleation and its distinctive
isotropic elongation was elucidated by using live imaging. Thus, inherent interaction
properties of tubulin provide a structural basis driving flagellar MTD assembly.
T
he cilium is an organelle crucial for mo-
tility, as well as for sensing environmen-
tal cues such as signaling molecules, light,
and mechanical stimuli ( 1 ). The core
structure of the cilium is characterized
by nine microtubule doublets (MTDs) ( 2 ). In
Chlamydomonas, MTDs form a double-track
railway for intraflagellar transport trains ( 3 ),
which carry ciliary building blocks along mi-
crotubules during the assembly and disassembly
of the cilium ( 4 ). These MTDs display distinctive
structural features and are formed by a com-
plete A-microtubule composed of 13 protofila-
ments and an incomplete B-microtubule of 10
protofilaments, which starts from the outer
junction (OJ) between protofilaments A10 and
A11 of the A-microtubule (Fig. 1A and fig. S1A)
( 5 – 8 ). Cryo–electron microscopy (cryo-EM) anal-
ysis of theTetrahymenaciliary MTD reveals
that this OJ involves noncanonical, surface-to-
side tubulin-tubulin contacts ( 9 ). It also reveals
an inner sheath composed of microtubule inner
proteins (MIPs) inside the MTD.
MTD assembly occurs at the centriolar level, with
the B-microtubule nucleating and elongating bi-
directionally onto the surface of the A-microtubule,
as assessed by cryo–electron tomography (cryo-ET)
in human centrosomes ( 10 ). However, the molec-
ular mechanism enabling B-microtubule nucle-
ation at the surface of the A-microtubule is
unclear. C-terminal tails of tubulin may play
arole( 11 ) because their limited proteolytic
digestion by subtilisin induces the nucleation of
hooked microtubules and protofilament bundles
(fig. S1, B and C).We hypothesized that B-microtubule assembly
could be mediated solely through tubulin-tubulin
interactions. We first set out to address whether
tubulin devoid of the C termini alone could as-
semble MTDs. We developed an in vitro assay to
mimic the sequential assembly of MTDs by A-
microtubule formation followed by B-microtubule
nucleation. First, stable microtubules ( 12 )were
assembled (Fig. 1B) and subsequently incubated
with subtilisin-treated tubulin without C-terminal
tails (Tub_S) ( 11 , 13 ) (materials and methods in
the supplementary materials). This did not result
in MTD formation (Fig. 1C). We next assessed
whether the removal of C-terminal tails of the
A-microtubule would promote MTD formation.
Microtubules treated with subtilisin (MT_S)
( 13 , 14 ) (fig. S1D) looked identical to untreated
microtubules (Fig. 1, B and D). When we added
free tubulin to subtilisin-treated microtubules,
72% of these microtubules formed assemblies
that resembled MTDs (Fig. 1, E and F), reaching
a median length of 0.66 ± 0.5mm after 15 min
(fig. S1E). By contrast, only ~7.5% of MTD-like
structures were observed among microtubules
treated with subtilisin alone. This possibly reflects
some depolymerization of the tubulin lacking
C-terminal tails; this tubulin would reattach at
the surface of the A-microtubule and nucleate
efficiently because of having a lower critical
concentration than untreated tubulin ( 15 ) (Fig.
1F). Thus, C-terminal tails of the A-microtubule
negatively regulate a noncanonical, surface-to-
side tubulin interaction, allowing microtubule
branching.RESEARCH
Schmidt-Cernohorskaet al.,Science 363 , 285–288 (2019) 18 January 2019 1of4
(^1) Department of Cell Biology, Sciences III, University of
Geneva, Geneva, Switzerland.^2 Institute of Biotechnology of
the Czech Academy of Sciences, BIOCEV, Vestec, Czech
Republic.^3 Faculty of Mathematics and Physics, Charles
University in Prague, Prague, Czech Republic.^4 Institut Curie,
PSL Research University, CNRS UMR 9187–INSERM U1196,
Paris-Saclay University, F-91405 Orsay, France.
(^5) Interdisciplinary Centre for Electron Microscopy, Swiss
Federal Institute of Technology (EPFL), Lausanne,
Switzerland.
*Present address: Laboratory of Adaptive Immunity, Institute of
Molecular Genetics, Academy of Sciences of the Czech Republic,
Prague, Czech Republic.
†Corresponding author. Email: [email protected] (V.H.);
[email protected] (P.G.)
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Fig. 1. MTD assembly in vitro.(A) Schematic of a procentriole with A- and B-microtubules (green)
and the procentriole cartwheel structure (gray). B-microtubule branching occurs at the OJ,
highlighting the protofilaments A10, A11, and B1. The dashed box corresponds to the closer view to
the right. Black arrows, seam of the A-microtubule and inner junction. (BtoE) Representative
cryo-EM images of microtubules (MT) (B), microtubules supplemented with tubulin pretreated with
subtilisin (MT+Tub_S) (C), subtilisin-treated microtubules (MT_S) (D), and MT_S incubated with
tubulin (MTD) (E) with their corresponding schematics. Arrowhead, MTD. Tub, tubulin. Scale bars,
25 nm. (F) Percentage of MTD formation (three independent experiments): 0% for MT (n= 825
microtubules), 3 ± 2% for MT+Tub_S (n= 729), 8 ± 6% for MT_S (n= 1515), and 72 ± 8% for MTDs
(n= 2341). Errors bars represent SD.
on January 17, 2019^
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