ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 441–446 doi:10.1242/jcs.160077
SHORT REPORT
The septation initiation network controls the assembly of nodes
containing Cdr2p for cytokinesis in fission yeast
Kai-Ming Pu1, Matthew Akamatsu1,2 and Thomas D. Pollard1,2,3,*
In the fission yeast Schizosaccharomyces pombe, cortical protein
structures called interphase nodes help to prepare the cell for
cytokinesis by positioning precursors of the cytokinetic contractile
ring, and the septation initiation network (SIN) regulates the onset of
cytokinesis and septum formation. Previous work has noted that
one type of interphase node disappears during mitosis providing
SIN activity is high. Here, we used time-lapse fluorescence
microscopy to provide evidence that SIN activity is necessary and
sufficient to disperse the type 1 node proteins Cdr2p and Mid1p into
the cytoplasm, so these nodes assemble only during interphase
through early mitosis when SIN activity is low. Activating the SIN in
interphase cells dispersed Cdr2p and anillin Mid1p from type 1
nodes a few min after the SIN kinase Cdc7p–GFP accumulated at
spindle pole bodies. If the SIN was then turned off in interphase
cells, Cdr2p and Mid1p reappeared in nodes in parallel with the
decline in SIN activity. Hyperactivating SIN during mitosis dispersed
type 1 nodes earlier than normal, and prolonged SIN activation
prevented nodes from reforming at the end of mitosis.
KEY WORDS: Cytokinesis, Fission yeast, Interphase nodes,
Septation initiation network, Confocal microscopy
INTRODUCTION
During cell division, fungal and animal cells use a contractile ring
composed of actin filaments and myosin to cleave in two. The
contractile ring in the fission yeast Schizosaccharomyces pombe
forms from discrete, protein structures called nodes (Martin and
Berthelot-Grosjean, 2009; Moseley et al., 2009; Paoletti and
Chang, 2000; Wu et al., 2006). Two types of nodes appear at
different times and places during interphase and combine to form
the cytokinetic nodes that assemble into the contractile ring
(Akamatsu et al., 2014). Type 1 nodes, which are marked by the
kinases Cdr1p and Cdr2p, appear in early G2 in a broad band
around each daughter nucleus (Morrell et al., 2004; Moseley
et al., 2009; Paoletti and Chang, 2000; Wu et al., 2006). Cdr1p
and Cdr2p negatively regulate the kinase Wee1p, which inhibits
the cyclin-dependent kinase Cdk1p as part of the mechanism
controlling the entry into mitosis (Moseley et al., 2009; Wu and
1
Departments of Molecular Cellular and Developmental Biology, Yale University,
PO Box 208103, New Haven, CT 06520-8103 USA. 2Departments of Molecular
Biophysics and Biochemistry, Yale University, PO Box 208103, New Haven, CT
06520-8103 USA. 3Department of Cell Biology, Yale University, PO Box 208103,
New Haven, CT 06520-8103 USA.
*Author for correspondence ([email protected])
Received 17 July 2014; Accepted 4 December 2014
Russell, 1993). The anillin Mid1p moves from the nucleus and
joins type 1 nodes during G2 (Morrell et al., 2004; Moseley et al.,
2009; Paoletti and Chang, 2000; Wu et al., 2006). Type 2 nodes,
composed of contractile ring proteins Blt1p, Klp8p, Nod1p and
Gef2p (Jourdain et al., 2013; Moseley et al., 2009; Zhu et al.,
2013), emerge from the contractile ring as it disassembles at the
end of cytokinesis. During interphase, type 2 nodes diffuse in the
cortex from the previous division site until they bind stationary
type 1 nodes around the cell equator to form cytokinetic nodes
(Akamatsu et al., 2014). Early in mitosis, these nodes accumulate
additional contractile ring proteins (Moseley et al., 2009; Saha
and Pollard, 2012). During anaphase, the type 2 node proteins,
Mid1p and other proteins condense into the contractile ring
(Vavylonis et al., 2008), while type 1 nodes move away from the
equator as their proteins disperse into the cytoplasm for the
duration of mitosis, a time called the eclipse period (Akamatsu
et al., 2014).
A signaling cascade called the septation initiation network
(SIN) originates from spindle pole bodies (SPBs) and regulates
the onset of cytokinesis and septation (Johnson et al., 2012;
Sparks et al., 1999). A GTPase-activating protein (GAP)
composed of Byr4p and Cdc16p negatively regulates the
GTPase Spg1p at the top of the SIN. As a cell enters mitosis,
cell cycle kinases Cdk1p and Plo1p phosphorylate Byr4p,
inhibiting the GAP activity and activating the SIN (Rachfall
et al., 2014). Activating Spg1p on one SPB during mitosis turns
on a cascade of three kinases – Cdc7p, Sid1p and Sid2p
(Fankhauser and Simanis, 1994; Guertin et al., 2002; Johnson
et al., 2012; Krapp and Simanis, 2008; Sohrmann et al., 1998).
Low Cdk1p activity during anaphase allows Sid1p to accumulate
on the SPB (Guertin et al., 2000) and to activate Sid2p, which
moves with its binding partner Mob1p to the contractile ring to
initiate constriction (Hou et al., 2004; Sparks et al., 1999).
We recently found that Cdr2p does not disperse from type 1
nodes during mitosis in sid2-250 mutant cells, suggesting that the
SIN controls the assembly of type 1 nodes (Akamatsu et al.,
2014). Here, we use a conditional mutation to turn the SIN on and
off to establish that the SIN is both sufficient and necessary to
disperse type 1 nodes.
RESULTS AND DISCUSSION
SIN signaling correlates with type 1 node dispersal
We used the presence of the SIN kinase Cdc7p–GFP on spindle
pole bodies (SPBs) to track SIN activity across the cell cycle
(Garcı́a-Cortés and McCollum, 2009; Sohrmann et al., 1998). We
define the separation of the SPBs as cell cycle time zero (Wu
et al., 2003). Cdc7p–GFP was dispersed in the cytoplasm during
interphase, but concentrated at the single or both duplicated SPBs
early in mitosis at cell cycle time +261.5 min (6s.d.; Fig. 1A,C),
marking activation of the SIN. Active Cdc7p–GFP remained on
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Journal of Cell Science
ABSTRACT
SHORT REPORT
Journal of Cell Science (2015) 128, 441–446 doi:10.1242/jcs.160077
Fig. 1. Localization of type 1 nodes and SIN proteins during mitosis. (A,B) Images are time series of confocal fluorescence micrographs with time in min
from SPB separation. Color images and the bottom row in A are merged maximum intensity projections; all others are reverse contrast sum projections. Scale
bars 2 mm. Images of mitotic cells expressing (top and middle rows) Cdr2p–mEGFP, Cdc7p–GFP and Sad1p–mCherry or (bottom row) Sid2p–mEGFP. The
middle row is a reverse contrast sum projection of the GFP channel of the top row. (A) Early in mitosis Cdc7p–GFP and Sid2p–mEGFP appeared at the SPBs
while type 1 nodes (marked by Cdr2p–mEGFP) moved away from the equator and dispersed into the cytoplasm by time 2665 min (mean6s.d.; n521).
(B) A separating cell showing nodes reforming around the lower SPB without Cdc7p–GFP before the more active upper SPB with Cdc7p–GFP. (C) Outcome
plots of the time course of the fraction of cells with: white circles, Cdr2p–mEGFP concentrated in type 1 nodes around the equator (n528); black squares, cells
with Cdc7p–GFP present on at least 1 SPB (n510); and black triangles, Sid2p–mEGFP present in the contractile ring (n56). (D) Time course of the
reappearance of Cdr2p–mEGFP in type 1 nodes around the equators of pairs of daughter cells late in mitosis with time zero set as when Cdc7p–GFP
disappeared from the active SPB. White circles, daughter cell with the inactive SPB; black circles, daughter cell with the active SPB (n520). The cartoon shows
the organization of the cells. w/o, without; w/, with.
442
cells, first around the inactive mother SPB lacking Cdc7p–GFP
and then around the active daughter SPB 362 min later as
Cdc7p–GFP disappeared from that SPB (Fig. 1D).
Ectopic activation of the SIN reversibly disperses type
1 nodes
We tested for a cause and effect relationship between SIN activity
and the dispersal of type 1 nodes by turning the SIN on and off
with a temperature sensitive cdc16-116 mutation of the SIN
inhibitor GAP Cdc16p (Fig. 2A,B; supplementary material Fig.
S2A). Starting 14 min after shifting interphase cdc16-116 cells to
the restrictive temperature of 35 ˚C, SIN turned on asynchronously
in the population of cells as indicated by accumulation of Cdc7p–
GFP on the single interphase SPBs (Fig. 2C,E; supplementary
material Movie 1). The fraction of interphase cells with type 1
nodes marked with Cdr2p–mEGFP declined in direct proportion
to the fraction of cells with Cdc7p–GFP on their SPB (Fig. 2G).
Activating SIN during interphase also dispersed the anillin Mid1p
from type 1 nodes (supplementary material Fig. S2). After
100 min at the restrictive temperature, ,80% of interphase cells
accumulated Cdc7p–GFP at the SPB and dispersed their type 1
nodes (Fig. 2E,G). In contrast to wild-type mitotic cells, these
cells retained no Cdr2p–mEGFP around the middle of the cell
Journal of Cell Science
both SPBs throughout anaphase but disappeared from the old SPB
approximately at the end of anaphase B when the mitotic spindle
reached its maximum length (Fig. 1A) (Garcı́a-Cortés and
McCollum, 2009). The SIN kinase Sid2p–mEGFP was visible
at the SPB during interphase and persisted on both SPBs through
mitosis. At the end of anaphase, Sid2p–mEGFP partially
relocalized to the contractile ring at time +3064 min where it
persisted until the daughter cells separated (Fig. 1A,C) (Hou
et al., 2004; Hou et al., 2000; McCormick et al., 2013; Salimova
et al., 2000; Sparks et al., 1999).
During the cell cycle the presence of type 1 nodes marked by
the kinase Cdr2p was inversely correlated with SIN activity
(Akamatsu et al., 2014), so nodes were present around the equator
when SIN was inactive from early G2 phase until early mitosis
(Fig. 1C). During anaphase, type 1 nodes separated from the
forming contractile ring and then dispersed into the cytoplasm
,20 min after Cdc7p appeared on SPBs (Fig. 1A), leaving a
small fraction (,15%) of Cdr2p–mEGFP at the division site
(Fig. 1A) (Akamatsu et al., 2014; Morrell et al., 2004; Saha and
Pollard, 2012). Cdr2p nodes reappeared in the cortex around the
nuclei of the daughter cells at time +6764 min, coincidently with
disappearance of Cdc7p–GFP from the SPBs at +6865 min
(Fig. 1B,C). Cdr2p nodes reappeared asymmetrically in 75% of
Journal of Cell Science (2015) 128, 441–446 doi:10.1242/jcs.160077
Fig. 2. Activating SIN during interphase in temperature sensitive cdc16-116 cells reversibly disperses Cdr2p-mEGFP from type 1 nodes. (A–D) Wildtype and cdc16-116 cells expressing Cdr2p–mEGFP, Cdc7p–GFP and Sad1p–mCherry are shown. Scale bars: 2 mm. (A,B) Reversed contrast and merged
color images of (top) wild-type (WT) and (bottom) cdc16-116 cells. (A) Cells were incubated at 37˚C for 1 h and imaged at 35˚C. Cdc7p–GFP accumulated at the
SPB of most interphase cdc16-116 cells, indicating that the SIN was activated. Cdr2p–mEGFP was dispersed into the cytoplasm of cdc16-116 cells, but
not in wild-type cells (n576). The arrow shows one cdc16-116 cell that did not accumulate Cdc7p–GFP at SPB and retained Cdr2p–mEGFP at type 1 nodes.
(B) Cells were incubated at 37˚C for 1 h and then returned to 23˚C for 1 h. Cdr2p–mEGFP reappeared in nodes of cdc16-116 cells without Cdc7p–GFP at the
SPB. The arrowhead shows one cdc16-116 cell that retained Cdc7p–GFP at SPB and did not accumulate Cdr2p–mEGFP in type 1 nodes. (C,D) Time
series of merged maximum intensity projections of confocal fluorescence micrographs of (top) wild-type and (bottom) cdc16-116 interphase cells with the time in
min after temperature shifts. (C) Shifting the temperature from 23 to 35˚C activated the SIN in the cdc16-116 cell but not the wild-type cell, resulting in
accumulation of Cdc7p–GFP at the SPB (arrow) and the disappearance of Cdr2p–mEGFP from nodes (n.30 cells). (D) Shifting the temperature from 37 to 23˚C
inactivated the SIN in the cdc16-116 cell, resulting in loss of Cdc7p–GFP from the SPB (arrowhead) and the reappearance of Cdr2p–mEGFP in nodes (n.60
cells). Interphase wild-type cells retained their nodes throughout. (E,F) Time courses of the fractions of interphase cdc16-116 cells with, white circles,
Cdr2p–mEGFP in nodes and, black squares, Cdc7p–GFP at the SPB after shifting temperatures at time zero. (E) Temperature shift from 23 to 35˚C (n553).
(F) Temperature shift from 37 to 23˚C (n576). (G) Plots of times after temperature shifts when Cdc7p–GFP appeared or disappeared at SPBs and when
Cdr2p–mEGFP appeared or disappeared in nodes of the same cells. White circles, time after temperature shift from 23 to 35˚C when Cdc7p–GFP accumulated
at the SPB and Cdr2p-mEGFP disappeared from nodes of individual cells (n556 cells). Black circles, time after temperature shift from 37 to 23˚C when Cdc7p–
GFP disappeared from the SPB and Cdr2p–mEGFP reappeared in nodes of individual cells (n576 cells).
when their nodes dispersed (Fig. 2C). On a cell-by-cell basis,
Cdc7p–GFP appeared at the SPB (Fig. 2C) 563 min (6s.d.)
before Cdr2p–mEGFP dispersed from nodes (Fig. 2E,G).
Similarly, in early G2 cells still connected by a septum the
SPB in one daughter cell accumulated Cdc7p–GFP 765 min
before the second SPB followed by the disappearance of Cdr2p–
mEGFP from type 1 nodes (supplementary material Fig. S1A).
Returning interphase cdc16-116 cells from 35˚C to the permissive
temperature of 23˚C turned off the SIN and allowed type 1 nodes to
reform with Cdr2p and Mid1p (Fig. 2B,D; supplementary material
Fig. S2B; Movie 2). In a population of cells, the time that Cdc7p–
GFP disappeared from SPBs was highly correlated with the time
Cdr2p–mEGFP reappeared in type 1 nodes, with a delay of only
465 min (Fig. 2F,G).
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SHORT REPORT
During interphase, wild-type cells had broad bands of type 1
nodes around their equators and no Cdc7p at their SPBs at 23 ˚C
(Fig. 2A,D). When shifted to 35 ˚C, wild-type cells did not
accumulate Cdc7p–GFP at the SPBs or disperse Cdr2p–mEGFP
from nodes (Fig. 2A,C).
Node reformation at the end of mitosis depends on
inactivation of the SIN
We used cdc16-116 cells expressing Cdr2p–mEGFP and Sad1p–
mCherry to investigate how SIN activity influences Cdr2p
during mitosis. These cells entered mitosis normally at all
temperatures, allowing for observation of the effect of the SIN
activity on mitotic entry. At the restrictive temperature, where
the SIN is hyperactive during mitosis, nodes dispersed at time
062 min (6s.d.) simultaneously with SPB separation in cells
entering mitosis, far earlier than in wild-type cells in which this
occurred at time +1664 min (Fig. 3A) (Vavylonis et al., 2008).
We do not know when Cdc7p associated with SPBs in these
experiments, because genetic interactions between tagged
Cdc7p–GFP and cdc16-116 during mitosis precluded including
this Cdc7p marker. The cdc16-116 cells expressing Cdc7p–
GFP, Cdr2p–mEGFP and Sad1p–mCherry entered mitosis
normally at the permissive temperature of 23 ˚C, but at the
restrictive temperature no SPBs separated during 100 min of
imaging. This mitotic defect impaired growth of the tripletagged strain.
We took advantage of an effect of cdc16-116 to show that the
SIN must be inactivated for type 1 nodes to reform at the end of
mitosis. If cdc16-116 cells entered mitosis prior to shifting the
Journal of Cell Science (2015) 128, 441–446 doi:10.1242/jcs.160077
temperature from 23 ˚C to 35 ˚C, Cdc7p–GFP never disappeared
from the old SPB (Sohrmann et al., 1998) and the type 1 nodes
did not reform (Fig. 3B; supplementary material Movie 3). This
result was duplicated in the cdc16-116 strain without Cdc7p–
GFP, confirming that continued SIN activation inhibited node
reformation and that this was not a result of the genetic
interaction between cdc16-116 and Cdc7p–GFP (supplementary
material Fig. S1B).
The reversibility of SIN activation and node dispersal during
interphase provides evidence that the cell cycle and node
assembly are regulated independently. SIN activity alone
disperses nodes in both mitosis and interphase.
Output pathways
The terminal SIN kinase, Sid2p, is part of the pathway that
regulates nodes, given that nodes do not disperse during mitosis
in cells containing the conditional allele sid2-250 (Akamatsu
et al., 2014). One possible output pathway is from Sid2p to the
NIMA kinase Fin1p (Grallert et al., 2012) and then Pom1p
kinase. Pom1p phosphorylates Cdr2p, inhibiting its kinase
activity (Bhatia et al., 2014; Deng et al., 2014; Martin and
Berthelot-Grosjean, 2009; Moseley et al., 2009) and restricting
it to the medial cortex during interphase (Rincon et al., 2014).
This pathway might also regulate the assembly of Cdr2p in type
1 nodes, but fin1D cells dispersed Cdr2p from nodes ,4 min
earlier (not later) and nodes reappeared ,8 min earlier than
in wild-type cells (Fig. 3C). This behavior is consistent with
Fin1p influencing the time of mitotic entry rather than the
dispersal of type 1 nodes. Thus, a parallel pathway from
Fig. 3. Behavior of type 1 nodes when SIN is hyperactivated during mitosis and in fin1D cells. (A,B) Effect on type 1 nodes of prolonged activation of the
SIN during and after mitosis. Images are time series of maximum intensity projections of confocal fluorescence micrographs with time in min on two scales:
(upper line) after shifting the temperature from 23 to 35˚C; and (lower line) relative to SPB separation at time zero. Scale bars: 2 mm. (A) Mitotic cells expressing
Cdr2p–mEGFP and Sad1p–mCherry. (Top) Wild-type (WT) cell dispersed nodes 1664 min (mean6s.d.; n515) after SPB separation, whereas (bottom) cdc16116 cell dispersed nodes when SPBs separated. (B) Cells at the end of the eclipse period expressing Cdr2p–mEGFP, Cdc7p–GFP and Sad1p–mCherry. (Top)
Wild-type and (bottom) cdc16-116 cells. In cdc16-116 cells with a hyperactivated SIN, Cdc7p–GFP distributed symmetrically between the two SPBs and no
nodes reformed for the duration of imaging (100 min). (C) Time course of the localization of Cdr2p–mEGFP to type 1 nodes in, black circles, wild-type and, white
circles, fin1D cells at 23˚C. Cdr2p–mEGFP disperses from nodes at time +2262 min and reforms into nodes at time +6064 min (mean6s.d.; n510).
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SHORT REPORT
SHORT REPORT
Timing of Cdr2p dispersal relative to SIN activation
The presence of Cdr2p–mEGFP in type 1 nodes was inversely
correlated with SIN activity, but Cdc7p always appeared at SPBs
before Cdr2p dispersed. The 20 min lag in wild-type cells
entering mitosis (Fig. 1C,D) shows that the pathway through the
SIN to Cdr2p must have one or more slow steps. The lag is
shorter when the SIN is hyperactivated at 37 ˚C in cdc16-116 cells
(Fig. 2G), so these delays are overwhelmed.
Several steps along the SIN cascade from Cdc7p to Sid1p and
Sid2p might slow the signal before it reaches the level required to
disperse Cdr2p. First, the slow accumulation of ,400 Cdc7p
molecules on SPBs over 40 min (Wu and Pollard, 2005) is a good
candidate to be the rate-limiting factor in the pathway, and the
timing in wild-type cells suggests that a threshold of ,200
molecules of Cdc7p–mYFP on the active SPB is required to
sustain enough SIN activity to disperse type 1 nodes. Second,
high Cdk1p activity early in mitosis inhibits Sid1p, so that it
localizes to the active SPB only after Cdk1p activity drops in
anaphase (Guertin et al., 2000). The low Cdk1p activity during
interphase allows the SIN to respond quickly to ectopic activation
(Guertin et al., 2000), explaining the rapid dispersal of type 1
nodes during interphase (Fig. 2C,E,G). Strong SIN activation
before mitosis might overwhelm the modulation of Sid1p during
mitosis (Fig. 3A). Finally, Sid2p activation might also be rate
limiting; SPBs have only ,50 molecules of Sid2p in late
interphase and then accumulate ,200–300 Sid2p molecules
during mitosis (McCormick et al., 2013).
We conclude that SIN activity is both necessary and sufficient
to disperse type 1 nodes based on our previous observation that
SIN-deficient cells do not disperse Cdr2p from nodes in mitosis
and our present finding that hyperactivation of the SIN forces
rapid dispersal of type 1 nodes in interphase and mitotic cells.
MATERIALS AND METHODS
Strain construction
We constructed strains of S. pombe with genetically encoded fluorescent
fusions proteins and temperature sensitive alleles by genetic crosses
(supplementary material Table S1). We deleted fin1+ with custom
primers using a PCR-based gene-targeting method (Bähler et al., 1998)
and the pFA6a-kanMX6 plasmid JW85. Fluorescently tagged strains
were obtained from laboratory stocks or by crossing laboratory stock
strains. We tested strains depending on fluorescent fusion proteins for
functionality by colony growth at 25 ˚, 30 ˚ and 37 ˚C and measuring the
timing of cell cycle events (SPB separation, contractile ring constriction,
septation and cell separation). Cdc16-116 cells expressing Cdr2p–
mEGFP, Sad1p–mCherry and Cdc7p–GFP did not enter mitosis
(n.50), but cells with of the same genotype but without a tag on
Cdc7p entered mitosis normally.
Microscopy and image analysis
Cells were grown for 36 h in liquid YE5S at 25 ˚C prior to imaging on
gelatin pads at 23 ˚C. For imaging at elevated temperatures, cells were
incubated at 37 ˚C for 1 h in suspension prior to imaging on agar pads
with an objective heater to maintain the temperature at 35 ˚C. We used an
Olympus IX-71 microscope (Olympus America, Center Valley, PA) with
either a 1006 1.4 NA objective or a 606 1.4 NA objective with a 1.66
optical magnifier, argon-ion lasers (Melles Griot), a spinning disk
confocal head (Yokogawa CSU- X1) and an electron multiplying CCD
camera (Andor iXon 897) using Andor iQ2 acquisition software.
Time lapse images were a collected as a z-series of 20 confocal slices
at 360-nm intervals encompassing the entire cell every 3 min at 23 ˚C or
every 2 min at 35 ˚C. Images were visualized as maximum intensity or
sum projections using ImageJ (National Institutes of Health, Bethesda,
MD). We defined SPB separation as time zero of the cellular clock (Wu
et al., 2003). We defined type 1 nodes as punctate Cdr2p–mEGFP or
Mid1p–mEGFP structures near the medial cortex in two or more
sequential images. We judged Cdc7p–GFP to be present in a SPB if it
colocalized with Sad1p–mCherry in at least two sequential images.
Outcome plots
Outcome plots in Fig. 1C,D, Fig. 2E,F and Fig. 3C were constructed
by plotting the cumulative proportion of cells with the specified
characteristics against time. Cell populations were drawn from at least
two fields of view, but for each given plot are limited to a subset of cells,
e.g. those in interphase (Fig. 2E,F) or those that undergo mitosis
(Fig. 1C). The plot in Fig. 2G was constructed by plotting (white circles)
the timing of node disappearance versus the timing of Cdc7p–GFP
appearance on the SPB, or (black circles) the timing of node appearance
versus the timing of Cdc7p–GFP disappearance on a cell-by-cell basis.
Acknowledgements
The authors thank the Raymond and Beverly Sackler Institute for support, Rajesh
Arasada for helpful discussions, Julien Berro for sharing ImageJ plugins and
Chad McCormick for sharing strains. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the National
Institutes of Health.
Competing interests
The authors declare no competing or financial interests.
Author contributions
K.-M.P., M.A. and T.D.P. designed the experiments; K.-M.P. and M.A. performed
experiments; K.-M.P. and M.A. analyzed data; K.-M.P., M.A. and T.D.P. wrote the
paper.
Funding
This work was supported by National Institute of General Medical Sciences of the
National Institutes of Health [grant number R01GM026132]. K.-M.P. was funded
by the Yale College Dean’s Research Fellowship in the Sciences. Deposited in
PMC for release after 12 months.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.160077/-/DC1
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