Download Supplementary Legends - Word file

Supplementary Movie 1
This movie shows a C. elegans zygote at metaphase-to-anaphase
transition. Note the displacement of the spindle toward the
posterior (right) and the disappearance of the metaphase plate at
anaphase onset. (QuickTime; 1.5 MB)
Supplementary Movie 2
This movie shows asymmetric spindle severing: the anterior
centrosome (left) is chopped off. The position and time of laser
ablation is marked with a white circle. (QuickTime; 1.3 MB)
Supplementary Movie 3
This movie shows cytokinesis in an unsevered control C. elegans
zygote. (QuickTime; 2.5 MB)
Supplementary Movie 4
This movie shows cytokinesis after anterior ASS: Note the
formation of two distinct furrows after ASS. The severed region is
highlighted with a grey bar. The first furrow does not complete,
pauses, and regresses. (QuickTime; 6 MB)
Supplementary Movie 5
This movie shows cytokinesis after posterior ASS: Note the furrow
correction observed after ASS. The cut region is highlighted with a
grey bar. The focus is rapidly changed during the correction
process to reveal the position of the nuclei and the complexity of
the cytokinesis furrow. (QuickTime; 3.6 MB)
Supplementary Movie 6
This movie shows asymmetric spindle severing with subsequent
disintegration of the chopped-off aster. The time and place of laser
ablation is marked with a white circle. (QuickTime; 2.8 MB)
Supplementary Movie 7
This movie shows cytokinesis after ASS with disintegration of the
chopped-off aster. The posterior centrosome (highlighted with a
white circle) is chopped off and disintegrated. The frames during
which the aster was disintegrated were removed (see movie 6 for
details of the disintegration assay). Note that the first furrow sets
up further away from the remaining aster compared with wildtype
or conventional ASS. (QuickTime; 3.9 MB)
Supplementary Movie 8
This movie shows cytokinesis in a zen-4(RNAi) depleted one-cell
embryo. Note the spindle snapping at anaphase. The furrow
regresses and cytokinesis fails. (QuickTime; 2.2 MB)
Supplementary Movie 9
This movie shows cytokinesis after ASS in a zen-4(RNAi)
depleted embryo. The severed region is highlighted with a grey
bar. The posterior aster is chopped off. Note that the ingression of
the first furrow is reduced compared with wildtype (movie 4).
(QuickTime; 1.8 MB)
Supplementary Movie 10
This movie shows cytokinesis in a klp-7(RNAi) depleted zygote.
Note the spindle snapping at anaphase. (QuickTime; 2.3 MB)
Supplementary Movie 11
This movie shows cytokinesis after posterior ASS in a klp7(RNAi) depleted zygote. The severed region is highlighted with a
grey bar. (QuickTime; 2.5 MB)
Supplementary Movie 12
This movie shows cytokinesis in a mel-11(it26) mutant zygote.
(QuickTime; 2.9 MB)
Supplementary Movie 13
This movie shows cytokinesis after posterior ASS in a mel-11(it26)
mutant zygote. The severed region is highlighted with a grey bar.
Note that the aster-dependent furrow completes before the
midzone-dependent furrow appears, leading to the formation of
three cells. The furrow that separates the anterior aster from the
anterior nucleus was not stable and regressed (not part of the
movie).
(QuickTime; 3.3 MB)
Supplementary Movie 14
This movie shows cytokinesis in an NMY2::GFP strain observed
by spinning disk microscopy.
(QuickTime; 1.3 MB)
Supplementary Movie 15
This movie shows cytokinesis after posterior ASS plus aster
ablation in an NMY2::GFP strain observed by spinning disk
microscopy. Note that after completion of the furrows, cortical
NMY2 patches continue to flow into the midzone-positioned
furrow and into the part of the aster-positioned furrow that
separates the two nuclei. The part of the aster-positioned furrow
that separates the posterior aster and the posterior nucleus did not
show such flow, was not stable, and regressed.
(QuickTime; 2.8 MB)
Supplementary Table 1
Genes with known or potential involvement in the first cytokinesis
of the C. elegans zygote, and their roles in redundant cleavage
plane specification as analysed by asymmetric spindle severing
(ASS): Shown are gene names, loci, knockout method and
phenotypes after ASS.
Gene knockout was performed using established protocols, either
by using feeding clones obtained form the MRC Geneservice1
(labeled Ahringer) or injection of double-stranded RNA obtained
from Cenix Bioscience2. For zen-4 we repeated RNAi with a
different feeding clone (a gift from M. Glotzer)3. If genetic mutants
were used, the strain and allele is specified.
Generally, analysis after ASS was impossible if microtubule-based
pulling forces were strongly reduced, if spindle assembly or
positioning was severely affected, or if the cortex of the embryo
displayed vigorous movements. In some cases we could obtain a
result by performing ASS plus aster ablation. If both assays failed
to give a result they are marked with a dash. We excluded genes
from our analysis that failed in cytokinesis because of a missing
furrow. If several genes acted in the same pathway, we did not test
all components of this pathway but only some examples. Genes
that were not tested are labeled “not tested”. The list excludes
genes that are required for formation of the mitotic apparatus or
that cause osmotic defects. All genes classes associated with
cytokinesis and their role in cleavage plane determination as
analysed by ASS are listed below:
Embryonic polarity defect. The first cell division of C. elegans is
asymmetric4. We hence probed polarity genes with ASS to see if
polarity influences cytokinesis furrow positioning. None of the
genes tested lead to a cytokinetic furrow specification defect after
ASS. In order to verify that polarity is not involved in the control
of cleavage plane specification, we performed ASS in a
symmetrically cleaving cell (AB)4. We observed two furrows in
AB (data not shown). These results establish that polarity has no
effect on redundant cleavage plane specification.
Reduced contractility. Several genetic defects lead to a reduced
contractility of the cortex, manifested by an absence of early
ruffling before the polarization of the cortex, lack of a
pseudocleavage furrow, or slow cytokinesis. We wondered
whether reduced contractility is a subtle manifestation of a
cleavage plane determination defect. Tested genes were let-502(no
pseudocleavage furrow, slow cytokinesis)5, C34C12.3 (S/TPhosphatase-like, no pseudocleavage furrow)2, C47G2.5
(phosphatase regulatory subunit, no pseudocleavage furrow)2,
K06A4.3 (gelsolin-like protein, reduced pseudocleavage furrow,
our unpublished data), nop-1(reduced early ruffling, no
pseudocleavage furrow)6, cdc-42(reduced pseudocleavage
furrow)7,8, unc-59/unc619 (septin homologues double knockout,
reduced early ruffling and reduced pseudocleavage furrow, slow
cytokinesis, our unpublished data), Y49E10.19 (putative Anillin
homolog, reduced early ruffling)2, arx-1(slow cytokinesis, our
unpublished data), ZC204.11 (kelch-like protein, reduced ruffling,
reduced pseudocleavage furrow)2. All genes in this phenotypic
class subjected to ASS analysis still displayed redundant cleavage
plane specification and correction of the furrow were normal. We
hence could not find any relation between reduced contractility and
cleavage plane specification.
Increased contractility – fast cytokinesis. In contrast to the first
phenotypic class, the mel-11 mutation leads to increased
contractility and fast ingression. In mel-11 embryos cytokinesis
only takes half the time compared with wildtype embryos5. In mel11 embryos subjected to ASS the first furrow completes before the
second is formed, ultimately leading to the generation of three
cells.
Hypercontractility – ectopic furrowing. Several mutations lead
to the generation of hypercontractile and ectopic furrows during
cytokinesis. Examples are csn-210, rfl-111, ubc-12, cul-111,
K09H11.32, Y75B7AL.42. The effect of these genes onto the two
furrows, however, is impossible to study after ASS, because of the
vigorous movements of the embryo cortex during the attempts at
cytokinesis.
Sister-chromatid separation defect – regression of the furrow.
Several gene knockouts lead to a defect in spindle midzone
formation and sister chromatid separation. These mutant embryos
specify and ingress a furrow but do not complete cytokinesis. Most
of the genes with this phenotype belong to the air-2 (Aurora B)
pathway (icp-1, bir-1, csc-1)12,13. We tested the role of air-2 in
cleavage plane determination subjecting the mutant or20714 to
ASS: A single furrow is specified by the asters, ingresses weakly,
and finally regresses. The nonseparated chromatin has no effect on
furrow positioning. The apparent lack of the second furrow can be
explained by the absence of a functional spindle midzone.
Spindle midzone defect – cytokinesis furrow regresses. ZEN-4
and CYK-4 constitute the centralspindlin complex which is
controlled by CDC-1415-17. Depletion of these proteins leads to a
spindle midzone defect, and absence of spindle microtubules, and a
failure in cytokinesis. ASS in central spindle mutants reveals the
same phenotype as for the sister-chromatid separation defect class.
The spindle midzone does not seem to provide a signal. This defect
is combined with a reduced contractility of the first furrow
(maximum ingression of the first furrow is 81  3 % embryo width
in wildtype and 42  4 % embryo width in zen-4(RNAi). We
observed comparable results with the zen-4 mutant or15314),
explaining the failure in cytokinesis. We verified the absence of
midzone microtubules under our RNAi conditions by using tubulin
fluorescence (data not shown).
Spindle midzone defect – cytokinesis generally completes.
Defects in spindle midzone formation are often accompanied by a
failure in cytokinesis (reviewed in 18). Mutants in klp-7 and spd-1,
however, display spindle midzone defects manifested by a
snapping or breaking of the mitotic spindle, and an absence of
midzone microtubules. Though, cytokinesis generally
completes19,20(and this work). After ASS in spd-1 and klp-7
mutants a single furrow is specified by the asters and completes
irrespective of the position of the “spindle midzone” between the
two nuclei. The lack of the second furrow can be explained by the
absence of a functional spindle midzone, while the completion of
the first furrow suggests that the spindle midzone is dispensable for
the completion of cytokinesis. However, there seem to be proteins
necessary for completion of cytokinesis like ZEN-4 and CYK-4
that localize in the vicinity of the spindle midzone21,22.
Cytokinesis completes but the furrow is unstable. Several
mutants display apparently normal cytokinesis. After the furrow
appears to have completed, the furrow rips apart and regresses.
Examples for this phenotype are K04D7.123 and Y18D10A.172.
Genes associated in this phenotypic class seem to be important for
abscission or sealing of the membrane, a process taking place far
after the cleavage plane is determined. We did not test genes
required in this process.
No cytokinetic furrow. Several gene knockouts (unc-60a24, mlc425, cyk-126, nmy-227, syn-428, act-22, act-52, let-212, pfn-126, rho12) lead to the complete absence of a cytokinesis furrowing. These
genes are either required for furrow ingression, in general, or are
involved in specification of both furrows. We did not subject these
genes to our assay.
1.
2.
3.
4.
5.
6.
7.
Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans
genome using RNAi. Nature 421, 231-7 (2003).
Sonnichsen, B. et al. Full-genome RNAi profiling of early embryogenesis in
Caenorhabditis elegans. Nature 434, 462-9 (2005).
Dechant, R. & Glotzer, M. Centrosome separation and central spindle assembly
act in redundant pathways that regulate microtubule density and trigger cleavage
furrow formation. Dev Cell 4, 333-44 (2003).
Goldstein, B., Hird, S. N. & White, J. G. Cell polarity in early C. elegans
development. Dev Suppl, 279-87 (1993).
Piekny, A. J. & Mains, P. E. Rho-binding kinase (LET-502) and myosin
phosphatase (MEL-11) regulate cytokinesis in the early Caenorhabditis elegans
embryo. J Cell Sci 115, 2271-82 (2002).
Rose, L. S., Lamb, M. L., Hird, S. N. & Kemphues, K. J. Pseudocleavage is
dispensable for polarity and development in C. elegans embryos. Dev Biol 168,
479-89 (1995).
Gotta, M., Abraham, M. C. & Ahringer, J. CDC-42 controls early cell polarity
and spindle orientation in C. elegans. Curr Biol 11, 482-8 (2001).
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Kay, A. J. & Hunter, C. P. CDC-42 regulates PAR protein localization and
function to control cellular and embryonic polarity in C. elegans. Curr Biol 11,
474-81 (2001).
Nguyen, T. Q., Sawa, H., Okano, H. & White, J. G. The C. elegans septin genes,
unc-59 and unc-61, are required for normal postembryonic cytokineses and
morphogenesis but have no essential function in embryogenesis. J Cell Sci 113 Pt
21, 3825-37 (2000).
Pintard, L. et al. Neddylation and deneddylation of CUL-3 is required to target
MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans.
Curr Biol 13, 911-21 (2003).
Kurz, T. et al. Cytoskeletal regulation by the Nedd8 ubiquitin-like protein
modification pathway. Science 295, 1294-8 (2002).
Schumacher, J. M., Golden, A. & Donovan, P. J. AIR-2: An Aurora/Ipl1-related
protein kinase associated with chromosomes and midbody microtubules is
required for polar body extrusion and cytokinesis in Caenorhabditis elegans
embryos. J Cell Biol 143, 1635-46 (1998).
Romano, A. et al. CSC-1: a subunit of the Aurora B kinase complex that binds to
the survivin-like protein BIR-1 and the incenp-like protein ICP-1. J Cell Biol 161,
229-36 (2003).
Severson, A. F., Hamill, D. R., Carter, J. C., Schumacher, J. & Bowerman, B. The
aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at
metaphase and is required for cytokinesis. Curr Biol 10, 1162-71 (2000).
Mishima, M., Kaitna, S. & Glotzer, M. Central spindle assembly and cytokinesis
require a kinesin-like protein/RhoGAP complex with microtubule bundling
activity. Dev Cell 2, 41-54 (2002).
Gruneberg, U., Glotzer, M., Gartner, A. & Nigg, E. A. The CeCDC-14
phosphatase is required for cytokinesis in the Caenorhabditis elegans embryo. J
Cell Biol 158, 901-14 (2002).
Mishima, M., Pavicic, V., Gruneberg, U., Nigg, E. A. & Glotzer, M. Cell cycle
regulation of central spindle assembly. Nature 430, 908-13 (2004).
Straight, A. F. & Field, C. M. Microtubules, membranes and cytokinesis. Curr
Biol 10, R760-70 (2000).
Grill, S. W., Gonczy, P., Stelzer, E. H. & Hyman, A. A. Polarity controls forces
governing asymmetric spindle positioning in the Caenorhabditis elegans embryo.
Nature 409, 630-3 (2001).
Verbrugghe, K. J. & White, J. G. SPD-1 Is Required for the Formation of the
Spindle Midzone but Is Not Essential for the Completion of Cytokinesis in C.
elegans Embryos. Curr Biol 14, 1755-60 (2004).
Raich, W. B., Moran, A. N., Rothman, J. H. & Hardin, J. Cytokinesis and
midzone microtubule organization in Caenorhabditis elegans require the kinesinlike protein ZEN-4. Mol Biol Cell 9, 2037-49 (1998).
Jantsch-Plunger, V. et al. CYK-4: A Rho family gtpase activating protein (GAP)
required for central spindle formation and cytokinesis. J Cell Biol 149, 1391-404
(2000).
23.
24.
25.
26.
27.
28.
Skop, A. R., Liu, H., Yates, J., 3rd, Meyer, B. J. & Heald, R. Dissection of the
mammalian midbody proteome reveals conserved cytokinesis mechanisms.
Science 305, 61-6 (2004).
Ono, K., Parast, M., Alberico, C., Benian, G. M. & Ono, S. Specific requirement
for two ADF/cofilin isoforms in distinct actin-dependent processes in
Caenorhabditis elegans. J Cell Sci 116, 2073-85 (2003).
Shelton, C. A., Carter, J. C., Ellis, G. C. & Bowerman, B. The nonmuscle myosin
regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior
polarity, and body morphology during Caenorhabditis elegans embryogenesis. J
Cell Biol 146, 439-51 (1999).
Severson, A. F., Baillie, D. L. & Bowerman, B. A Formin Homology protein and
a profilin are required for cytokinesis and Arp2/3-independent assembly of
cortical microfilaments in C. elegans. Curr Biol 12, 2066-75 (2002).
Guo, S. & Kemphues, K. J. A non-muscle myosin required for embryonic polarity
in Caenorhabditis elegans. Nature 382, 455-8 (1996).
Jantsch-Plunger, V. & Glotzer, M. Depletion of syntaxins in the early
Caenorhabditis elegans embryo reveals a role for membrane fusion events in
cytokinesis. Curr Biol 9, 738-45 (1999).