the extended conformation (12)], and the sheath
length of 5 of 7 structures matches the length of
a fired MAC (table S2).
We averaged 20 and 25 subtomograms of
MACs in the “extended” (Fig. 4P, movie S6) and
“contracted without tube” (Fig. 4Q, movie S7)
states, respectively. Averages of subtomograms
show the different sheath diameters, helical surface ridges on contracted sheath (ridges indicated
in Fig. 4K), baseplate symmetry and tail fibers in
longitudinal (Fig. 4, J and O) and cross-sectional
views (Fig. 4, F to I and K to N). In both the
extended and contracted forms twelve fibers
emerge from the baseplate, cross paths, and separate to meet at the ring-shaped vertices of the
hexagonal net surrounding individual MACs
(Fig. 4R, movie S8). We speculate that six of
the tail fibers originate from a single MAC, with
the remaining six fibers stemming from neighboring MACs to connect the array (Fig. 4, P to
S, orange). This six-tail fiber per MAC model
(Fig. 4S) is supported by the fact that the two
arms of a phage tail fiber have a length ratio of
1:1 (23) and that the length is similar to the tail
fiber connections in MACs (fig. S10). The model
also predicts the presence of an as-yet-unidentified
protein that forms the hexagonal net. A set of
six tail pins (Fig. 4P, red) face outwards. Because the tail pins are the most distal structure
in the arrays, they are likely the first structure to
engage and sense MAC targets.
We have shown that an ordered array of con-
tractile phage tail–like structures produced by
an environmentally occurring bacterium induces
metamorphosis of a marine invertebrate larva.
This discovery begins to explain how marine
biofilms can trigger metamorphosis of benthic
animals. Our data suggest that MAC arrays are
synthesized intracellularly by P. luteoviolacea,
released by cell lysis, and expand extracellularly
into an ordered multi-MAC array. How these ar-
rays engage with larvae of H. elegans is an open
question. In the arrays imaged, all contracted
MACs were clustered together, which suggests
that their linkages might support cooperative
firing. Array formation might also multiply the
total payload delivered per interaction or favor
specific engagement sites and/or geometries with
MAC targets. The evolutionary pressure to produce
MACs is probably strong, given that MAC pro-
duction leads to the lysis and death of a sub-
population of cells. Whether this represents an
instance of altruistic behavior that facilitates group
selection, or a neutral lytic event with a set fre-
quency remains to be determined. Although MAC
production is beneficial for tubeworm larvae by
inducing metamorphosis, it is currently unclear
how larval settlement and metamorphosis might
benefit the bacterium. It is equally possible that
MACs evolved for a completely different pur-
pose. Note that P. luteoviolacea has been found
to induce the metamorphosis of coral and sea-
urchin larvae (24, 25). Other bacterial species
also induce metamorphosis of H. elegans lar-
vae (8, 26, 27), and mac-like gene clusters have
been identified in the genomes of other marine
bacteria (28). Future research into how MACs
interact with larvae might yield new insights
into the mechanisms underpinning marine ani-
mal development and ecology, with potentially
important practical applications for aquaculture
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Acknowledgments: We thank B. Pernet for help with locating
and identifying tubeworms and for giving us the algal strain
used in this work; A. McDowall for help with EM; Y. Huang, who
created the StrR-strain (7); A. Asahina and S. Wilbur for laboratory
assistance; J. Levine for help with time-lapse microscopy; J. Ricci
for help with phylogenetic analyses; and members of the Newman
group for discussions and comments on the manuscript. The
Howard Hughes Medical Institute, Z. Yu, and J. de la Cruz are
acknowledged for providing access to the FEI Titan Krios at Janelia
Farm and support in data collection. N.J.S. was supported by a
California Institute of Technology (Caltech) Division of Biology
Postdoctoral Fellowship. This collaboration was supported
by the Caltech Center for Environmental Microbiology Interactions,
the Howard Hughes Medical Institute (D.K.N. and G.J.J.), Office of
Naval Research grants N00014-08-1-0413 and N00014-05-1-0579
(M.G.H.), NIH grant GM094800B (G.J.J.), and a gift from the
Gordon and Betty Moore Foundation (Caltech). D.K.N. and G.J.J. are
Investigators of the Howard Hughes Medical Institute. Strains
obtained from the American Type Culture Collection listed in table S2
(ATCC 33492, ATCC 14393, ATCC 15057). DNA sequences encoding
for mac, T6SS, and bacteriocin-2 genes are deposited under GenBank
accession numbers KF724687, KF724688, and KF724689,
respectively. Subtomogram averages were deposited in the Electron
Microscopy Data Bank (accession numbers EMD-2543, EMD-2544, and
EMD-2545). Author contributions: All authors designed research. N.J.S.,
M.P. and G.L. W. performed research. All authors wrote the paper.
Materials and Methods
Figs. S1 to S10
Tables S1 to S4
Movies S1 to S8
3 October 2013; accepted 23 December 2013
Published online 9 January 2014;
Reversal of Female Infertility by Chk2
Ablation Reveals the Oocyte DNA
Damage Checkpoint Pathway
Ewelina Bolcun-Filas,1 Vera D. Rinaldi,1 Michelle E. White,1 John C. Schimenti1*
Genetic errors in meiosis can lead to birth defects and spontaneous abortions. Checkpoint
mechanisms of hitherto unknown nature eliminate oocytes with unrepaired DNA damage, causing
recombination-defective mutant mice to be sterile. Here, we report that checkpoint kinase 2
(Chk2 or Chek2), is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA
double-strand breaks (DSBs). Female infertility caused by a meiotic recombination mutation or
irradiation was reversed by mutation of Chk2. Both meiotically programmed and induced DSBs
trigger CHK2-dependent activation of TRP53 (p53) and TRP63 (p63), effecting oocyte elimination.
These data establish CHK2 as essential for DNA damage surveillance in female meiosis and indicate
that the oocyte DSB damage response primarily involves a pathway hierarchy in which ataxia
telangiectasia and Rad3-related (ATR) signals to CHK2, which then activates p53 and p63.
Fertility, offspring health, and species suc- cess depend on production of gametes with intact genomes. Particularly crucial is the proper synapsis and segregation of homologous chromosomes at the first meiotic division, pro- cesses requiring homologous recombination (HR),