Supplementary Figure Legends
Figure S1
a, Illustration describing the structural organization and MT polarity patterns within the
metaphase spindle. In animal cells, the spindle forms from two radial arrays of MTs
nucleated by duplicated centrosomes1. These arrays interact with chromosomes and one
another to assemble a bipolar structure in which MTs are oriented with their dynamic
plus-ends facing the spindle equator and their minus-ends focused near the centrosomes
at the spindle poles. The primary interface between spindle MTs and chromosomes is a
multiprotein chromosomal structure termed the kinetochore, which is specialized to
capture and stabilize MTs at their plus-ends (MTs that bind kinetochores are termed
kMTs3).
b, Overview of the mitotic stages. In general terms, this occurs as follows: 1) following
nuclear envelope breakdown at prometaphase, chromosomes capture spindle MTs and
congress to the spindle equator, or metaphase plate, 2) the equatorial position of
chromosomes is maintained through metaphase, and 3) at the onset of anaphase, sister
chromatids disjoin and translocate toward opposite spindle poles in a dramatic movement
termed anaphase A. Spindle elongation occurs during anaphase B. Lastly,
chromosome decondensation and daughter nuclear envelopes form during telophase.
c, There are currently two predominant models to explain poleward chromosome
movement, both of which invoke the active depolymerization of kMT ends. In the first,
termed “Pacman”, kinetochores directly induce the depolymerization of kMT plus-ends,
allowing chromosomes to move poleward by ‘chewing up’ MT tracks4,5. d, In the second
model, termed “poleward flux”, kinetochores serve primarily as anchors to kMTs and are
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‘reeled-in’ to the poles via minus-end kMT depolymerization. This model stems from in
vivo studies showing the continuous poleward movement of labeled tubulin subunits
through kMTs6 (termed “poleward MT flux”).
Figure S2
a, Linear maps of the three Drosophila KinI kinesin polypeptides illustrate the Nterminal (NT) sequences selected to make GST fusion proteins that were used to raise
antisera. KLP10A; CG1453 mapped onto genomic P1 clone AC007521 and encodes a
cDNA (EST GM26945) with an ORF of 2,409 bp, predicting a 88.4 kDa polypeptide.
KLP59C; CG3219 mapped onto genomic P1 clone AC006245 and encodes a cDNA
(EST LP01010) with an ORF of 1,878 bp, predicting a 70 kDa polypeptide. KLP59D;
CG12192 mapped onto genomic P1 clone AC006245, approximately 14 kb upstream and
in an opposite orientation from KLP59C, and encodes a cDNA (EST AT18571) with an
ORF of 2,187 bp, predicting a 81.9 kDa polypeptide. ESTs were purchased from
ResGen. Sequence data are available from GenBank/EMBL/BDGP under indicated
accession numbers. The KinI-NT sequences encode the functionally important
intracellular targeting domain11 (see Supplementary Fig. S10). In addition, 10A-NT and
59C-NT recombinant proteins were microinjected into early Drosophila embryos and act
as dominant/negative proteins (Supplementary Fig. S9).
b, Dendrogram generated by multiple sequence alignment with hierarchical clustering
indicating the relationship between the Drosophila KinI proteins and KinI family
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members from other systems. Vertebrate KinI family members with demonstrated or
putative mitotic functions are indicated in bold text.
c, Coomassie-stained SDS-7.5% PAGE of the three recombinant glutathione-Sepharose
purified GST-tagged N-terminal (NT)-KinI kinesin polypeptides and purified GST that
was used as a negative control in our in vivo studies. Expression plasmids for GST and
MBP fusions with NT domain of each KinI kinesin were made by directionally
subcloning fragments of KLP10A nucleotides 1-685, KLP59C nucleotides 1-414, and
KLP59D nucleotides 1-615 into both pGEX-6p2 (Amersham Pharmacia) and pMAL-c2x
(New England Biolabs).
Figure S3
KinI kinesin MT-depolymerization assays analyzed visually (a) and by sedimentation
(b). Rhodamine-labeled MTs (2.5M) were stabilized using the non-hydrolyzable GTP
analog, GMPCPP, and incubated for 30 minutes at room temperature with MgATP
(1.5mM) in buffer alone or containing the catalytic domain of KLP10A (500nM) or
KLP59C (500nM) or full-length XKCM1 (50nM). -tubulin in the supernatants (S) and
pellets (P) was analyzed by Western blotting for -tubulin. c, KLP10A and KLP59C
depolymerize stabilized MTs in an ATP-dependent manner. Visual assay of
depolymerization of GMPCPP-stabilized rhodamine-labeled MTs (2.5M) by the
minimal catalytic domains of recombinant KLP10A or KLP59C (500nM). MT
depolymerization requires MgATP (1.5mM) (middle panels), is inhibited with MgAMPPNP (5mM) (lower panels) and is not observed in buffer alone (upper panels).
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Figure S4
To monitor the RNAi-induced reduction of KinI expression, polyclonal antibodies were
generated against recombinant non-conserved regions of each KinI protein (see
Supplementary Fig. S2c).
a and b, Immunoblots of Drosophila S2 cell (a) and embryonic (b) lysates using three
different anti-KLP10A antibodies (numbers above lanes identify sera; numbers to left are
kDa of standards). All react specifically with a single 111 kDa polypeptide.
c, Immunoblot of S2 cell lysate with an anti-KLP59D antibody identifies a single 107
kDa polypeptide.
d-f, Two different anti-KLP59C antibodies react with a single 70 kDa polypeptide in S2
cell lysates (d) and a fractionated embryonic extract (e). The embryonic extract was
fractionated on a 5-20% sucrose gradient and the peak fraction for the 70 kDa band (the
only band detected in any fraction) is shown in e. KLP59C is also detected on isolated
mitotic chromosomes (f). Coomassie-stained SDS-7.5%PAGE of isolated mitotic S2 cell
chromosomes (IMC) and immunoblots of IMC samples using the two anti-KLP59C
antibodies.
g, Quantitative immunoblots of four kinesins and -tubulin (loading control) in S2 cell
cultures treated with KLP10A, KLP59C, KLP59D or KLP61F dsRNA for 1-7 days. All
lanes loaded with equal total protein. Compared with day 1, each kinesin was markedly
reduced after 7 days of RNAi-treatment (KLP10A, 98% reduction; KLP59C, 95%;
KLP59D, 94%; KLP61F, 97%). RNAi-induced phenotypes are very similar after days 4
or 7 even though, in some cases, protein is still detected after day 4. Templates for in
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vitro transcription (Megascript T7 kit; Ambion) were generated by using primers that
began with the T7 promoter 5'-TAATACGACTCACTATAGGG-3' and continue with
gene specific sequences: primers 5'-GATCAAGAACTTGCGGTCC-3' and 5'GGCTACGTTTTCTTTGTTGTGCC-3' to PCR amplify the last 994 bp of KLP61F
coding sequence, primers 5'-TAAATTGTAAGCGTTAATATTTTG-3' and 5'AATTCGATATCAAGCTTATCGAT-3' to PCR amplify a non-coding 900 bp fragment
from pBluescript SK, primers 5'-ATGGATAAGTTGTCGATCG-3' and 5'ACCAGGTTCACATGCTTGCG-3' to PCR amplify the first 689 bp of KLP59C coding
sequence, primers 5'-ATGATTACGGTGGGGCA-3' and 5'-GACATCGATCT
CCTTGCG-3' to PCR amplify the first 894 bp of KLP10A coding sequence and primers
5'-GGATCGCATCAAAATTGG-3' and 5'-CGTAGACCAGCGCATTG-3' to PCR
amplify the first 901 bp of KLP59D coding sequence. Quantitation of RNAi-induced
reduction of protein levels was performed using video densitometry (MetaMorph,
Universal Imaging) of scanned ECL Western blots.
Figure S5
Mitotic indices determined from 8 different KLP-RNAi-treated S2 cell cultures after 1, 4
and 7 days of dsRNA treatment. Along with RNAi-treatments for each Drosophila KinI
individually and in pairs (labeled), RNAi for KLP61F, a Drosophila kinesin essential for
maintaining mitotic spindle shape28, was used as a positive control. With the exception
of KLP59C RNAi, changes in the mitotic index at day 4 and 7 RNAi treatments were
found to be statistically significant (for all treatments p<0.035). Values are averages (+
S.E.M.) of mitotic indices of experimental and non-coding dsRNA-treated control
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cultures. The number of independent experiments performed per treatment is shown in
each bar (an average of 560 day 1 cells, 659 day 4 cells and 665 day 7 cells were
analyzed per experiment). Cells were analyzed by immunofluorescence for tubulin and
chromosome staining. For one-to-one comparisons of the mitotic index (control versus
experimental on days 4 and 7), statistical significance was determined using 2-tailed 2sample t-tests for independent samples after first testing variance equality using an F test.
For multiple comparisons with the group, a one-way ANOVA test with a Tukey’s
multiple comparison post-test was used.
Figure S6
KLP10A and KLP59C differ spatially in their localization on mitotic centromeres.
Immunolocalization of KLP10A (top panels) and KLP59C (middle panels) in mitotic S2
cells and isolated chromosomes. KLP10A is found on mitotic centromeres and partially
overlaps with the inner kinetochore protein Cid. However, KLP59C does not localize to
kinetochores and is restricted to centromeres throughout mitosis.
(bottom panels) KLP10A localizes to centrosomes and centromeres in a MT-independent
manner and, conversely, to spindle poles in a MT-dependent manner. KLP10A
immunolocalizes to spindle poles, centrosomes and centromeres in mitotic S2 cells (colchicine row). After a 16 hour treatment with the MT-depolymerizing drug colchicine
(+ colchicine rows), KLP10A properly localized to centromeres and partially colocalized with the centrosomal protein tubulin (arrows). In addition, KLP10A was
found as discrete aggregates within the cytoplasm (arrowheads).
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Figure S7
Analyses of mitotic phenotypes are limited to defined regions within microinjected
Drosophila syncytial blastoderm-stage embryos.
Low magnification confocal micrographs of two different live embryos microinjected
with anti-KLP10A (#656; upper panel) or anti-KLP59C (#698; lower panel) antibody. A
GFP-tubulin (labeling MTs) expressing embryo is shown in the upper panel. The embryo
in the lower panel expresses GFP-histone (labeling chromosomes) and contains
rhodamine-tubulin (not shown). Both embryos displayed a gradient of mitotic
phenotypes emanating from the microinjection site (arrows) where the severity of the
antibody-induced phenotype is highest. Analyses of mitotic phenotypes were restricted
to a region (approximately 1/3rd of the embryo) originating from the site of injection
(yellow tracings). The boundaries of this region were clearly defined by a striking
discontinuity of the inhibitor-induced phenotype (upper panel inset). In addition, the
classification of the anaphase chromosome segregation defects observed in KLP10A and
KLP59C inhibited embryos is shown in the lower right panel (N-normal anaphase; Type
I-lagging chromosomes; Type II-bilobed chromosomal mass; Type III-stretched
chromosomal mass; asterisks mark the position of the spindle poles).
Figure S8
Microinjection of anti-KLP10A or anti-KLP59C Fab fragments into Drosophila embryos
phenocopies mitotic defects observed in whole anti-KinI antibody containing embryos.
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a, Coomassie-stained SDS-10% PAGE shows the efficiency of papain digestion of two
different affinity-purified anti-KinI antibodies (anti-KLP10A #656 and anti-KLP59C
#696). Lane 1 shows the heavy chain (HC) of the precleaved IgG. Lane 2 shows the
products of the papain treatment: ~25kDa Fab fragments and > 25kDa Fc fragments (note
the disappearance of the HC and the removal of the Fc fragments from the anti-KLP10A
Fab preparation). ~1g of protein loaded per lane.
b, Time-series of still images from a 4-D data set showing mitosis in an anti-KLP10A
Fab fragment injected, GFP-tubulin expressing embryo (labeling MTs). Time in seconds
at bottom left. * indicate the opposite poles of a spindle in each image. At a final
concentration of 0.05mg/ml, anti-KLP10A Fab fragments produced results
indistinguishable from the intact antibody (compare with Fig. 2a).
c, Still images from a 4-D time series showing mitosis in an anti-KLP59C Fab fragment
injected embryo containing rhodamine-labeled tubulin (red) and GFP-histone to label
chromosomes (green). Time in seconds at bottom left. At a final concentration of 0.08
mg/ml, anti-KLP59C Fab fragments produced results indistinguishable from the intact
antibody (compare with Fig. 3a).
Figure S9
Microinjection of purified GST-tagged 10A-NT or 59C-NT polypeptides into Drosophila
embryos phenocopies mitotic defects observed in anti-KinI antibody-containing
embryos. Still images from a time series showing metaphase and telophase in GST-10ANT (left panels) and GST-59C-NT (right panels; both used at 6 M final concentration)
protein-injected embryos containing either GFP-tubulin (left panels) or rhodamine-
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labeled tubulin (red) and GFP-histones to label chromosomes (green) (right panels).
Time in seconds at upper left of each panel. * follows the position of centrosomes of an
individual bipolar spindle in each embryo. The purity of the recombinant polypeptides is
shown in Supplementary Figure S2c. The recombinant GST-tagged constructs encode
the N-terminal (NT) intracellular targeting domain of each KinI kinesin11 (see
Supplementary Figure S2a, S10). (Homologous constructs used in CHO cells and
Xenopus extracts localized to mitotic centromeres and acted as dominant/negative
inhibitors of KinI kinesin function11,13 by preventing the endogenous KinI from
assembling onto centromeres13). Injection of these constructs results in phenotypes that
are similar to those resulting from anti-KinI antibody injection (compare with Fig. 2a,
3a).
Figure S10
Microinjection of anti-KinI antibodies results in the mis-localization of spindleassociated KinI targeting domains.
a, Still images showing a mitotic domain from three different living GFP-tubulin (green)
expressing embryos microinjected with a low concentration (1.8M) of rhodaminelabeled KLP10A-N-terminal targeting domain (10A-NT) protein (red) and either buffer
(upper panels), Cy5-labeled anti-KLP10A antibody (#656; blue) (middle panels) or Cy5labeled anti-KLP59C antibody (#698; blue) (lower panels). In each case, the domains
shown are in metaphase and * mark the positions of selected centrosomes on bipolar
spindles. Fluorescent 10A-NT protein localizes to spindle poles, centrosomes and
centromeres in buffer and anti-KLP59C antibody injected embryos, but is displaced from
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the spindle within two minutes of introducing anti-KLP10A antibody. The mis-localized
10A-NT protein forms cytoplasmic aggregates with the Cy5-labeled anti-KLP10A
antibody (arrows). Note that high concentrations of 10A-NT protein phenocopy the
injection of anti-KLP10A antibodies (Supplementary Fig. S9).
b, Still images showing a mitotic domain from three different living GFP-histone (green)
expressing embryos microinjected with a low concentration (1.4M) of rhodaminelabeled KLP59C-N-terminal targeting domain (59C-NT) protein (red) and either buffer
(upper panels), Cy5-labeled anti-KLP59C antibody (#698; blue) (middle panels) or Cy5labeled anti-KLP10A antibody (#656; blue) (lower panels). Again, each domain is in
metaphase and * mark the positions of selected centrosomes on bipolar spindles.
Fluorescent 59C-NT protein localizes to centromeres in buffer and anti-KLP10A
antibody injected embryos, but is displaced from chromosomes within two minutes of
introducing anti-KLP59C antibody. The mis-localized 59C-NT protein forms
cytoplasmic aggregates with the Cy5-labeled anti-KLP59C antibody (arrows). Note that
the injection of Cy5-labeled anti-KLP10A antibody still forms cytoplasmic aggregates
(arrows) that presumably contain endogenous KLP10A but does not mis-localize the
59C-NT protein. Also note that high concentrations of 59C-NT protein phenocopy the
injection of anti-KLP59C antibodies (Supplementary Fig. S9).
Figure S11
Microinjection of anti-KLP10A or anti-KLP59C antibodies into Drosophila embryos
does not mislocalize centrosome, kinetochore or centromere proteins. Still images from
4-D data sets showing metaphase in living control (top panels), anti-KLP10A (middle
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panels) and anti-KLP59C (bottom panels) antibody injected embryos containing
rhodamine-labeled tubulin (red) and either GFP-Rod (an outer kinetochore protein), GFPDTACC (a component of the centrosome), GFP-MeiS332 (a centromere protein required
for sister chromatid cohesion) or GFP-Polo kinase (a centrosome and outer kinetochore
protein)(green). Anti-KLP10A antibody #656 (final concentrations of 0.22-0.26mg/ml)
and anti-KLP59C antibody #698 (final concentrations of 0.28-0.36mg/ml) were used in
these experiments. In each condition, the GFP-tagged proteins displayed an appropriate
localization to their mitotic structures.
Figure S12
Distribution histogram of tubulin speckle movements.
Rates of tubulin speckle-to-pole movement in buffer control- (55 speckles from 37
spindles in 3 different embryos), anti-KLP10A antibody- (72 speckles from 61 spindles
in 4 different embryos) and anti-KLP59C antibody- (50 speckles from 42 spindles in 3
different embryos) injected embryos.
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