* Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of
California at San Diego, La Jolla, California 92093-0683; Program in Molecular Medicine, University of Massachusetts Medical
Center, Worcester, Massachusetts 01605; and § University of Utah, Department of Bioengineering, Salt Lake City, Utah 84112
The kinesin superfamily is a large group of proteins (kinesin-like proteins [KLPs]) that share sequence similarity with the microtubule (MT) motor kinesin. Several members of this superfamily have been implicated in various stages of mitosis and meiosis. Here we report our studies on KLP67A of Drosophila. DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain. To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction. We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types. In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern. Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled. These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.
In many cell types, mitochondria are localized in accordance with substrate availability or energy requiring
processes. In higher eukaryotes, this localization is
known to depend upon the microtubule (MT)1 cytoskeleton. The first convincing cytological demonstration of MT
dependence was the coimmunolocalization of mitochondria to MTs in cultured cells (Heggeness et al., 1978 Recent work indicates that mitochondrial translocation
may involve at least one member of the kinesin superfamily. Kinesin-like proteins (KLPs) all share a protein domain that has sequence homology to the kinesin heavy
chain (KHC) globular head or motor domain. The remainder of each KLP molecule is in most cases divergent and
suggested to be responsible for the attachment of specific cargoes within the cell (Vale and Goldstein, 1990 In this paper, we describe the characterization of KLP67A
and show that it is a plus end-directed motor. This KLP
was initially discovered in a PCR-based screen for new
members of the kinesin superfamily in Drosophila (Stewart et al., 1991 DNA Sequence Analysis
KLP67A was originally identified as a fragment in a PCR-based screen for
new members of the kinesin superfamily in Drosophila (Stewart et al.,
1991 Expression and Purification of KLP67A in E. coli
The vector pGEX-2T (Pharmacia LKB Biotechnology, Piscataway, NJ) was
used to express KLP67A as part of a glutathione S-transferase (GST) fusion protein (Guan and Dixon, 1991 E. coli 71/18 cells containing pGEX-KLP67A were grown at 22°C for
4-5 h, IPTG was added to a final concentration of 1 mM, and the culture
was incubated for an additional 1-2 h at 22°C. Cells were harvested by
centrifugation at 10,000 rpm, resuspended in sonication buffer (0.1 M sodium phosphate buffer, pH 7, 5 mM EDTA, 1 mM PMSF), recentrifuged,
and resuspended in 10 ml sonication buffer per 500 ml starting culture.
Cells were lysed by sonication followed by centrifugation to pellet insoluble material. The supernatant was incubated with glutathione-coupled
agarose beads (Sigma Chemical Co., St. Louis, MO) for 30 min on ice. The
agarose beads were washed three times with PEM 80 (80 mM K2Pipes/1 mM
EGTA/1 mM MgCl2, pH 7.2), 1 mM DTT, 0.1M NaCl and centrifuged
briefly in a microfuge. After the final wash, the agarose beads were resuspended in 0.1 M glutathione in PEM 80, pH 7.2, and incubated on ice for
20 min. The beads were pelleted in a microfuge and the supernatant was used immediately for motility assays.
Motility Assays
Soluble GST-KLP67A fusion protein was tested for motility activity with
MTs prepared from bovine brain tubulin as described previously (Stewart
et al., 1993 In Situ Hybridization to Embryos
In situ hybridization was carried out according to the method of Tautz and
Pfeifle (1989) In Situ Hybridization to Larval Tissue
Brains and imaginal discs were dissected from third instar larvae in 0.7%
NaCl, transferred to 4% paraformaldehyde in PBS, and fixed for a further
20 min. Pretreatment and hybridization was carried out according to the
method of Tautz and Pfeifle (1989) Antibody Production
Four rat antisera were raised against GST-KLP67A. GST-KLP67A was
purified by two different methods for immunization. Soluble GST-KLP67A
was prepared as for the motility assays described above. Approximately
10 µg of the fusion protein bound to 500 µl of glutathione agarose was
mixed with RIBI adjuvant (Immunochemical Research Inc., Hamilton,
MT) and injected subcutaneously into rat 1. The other three animals were injected with ~10 µg each of gel-purified GST-KLP67A suspended in RIBI
adjuvant. Rats were boosted on day 14 with an identical preparation. Test
bleeds 10 d later showed that three of the four rats (excluding rat 4) had
identical immunoreactivity patterns to Drosophila embryos as described
in the results section. 4 mo later, all four rats received another boost of the
same immunogen to maintain the anti-KLP67A antibody titer. After this
second boost, rat 4 antiserum exhibited an immunostaining pattern identical to those from the other three rats.
Gel-purified GST-KLP67A-70 (see below) was also used to immunize
two rabbits. After affinity purification, one of these rabbit antisera displayed a staining pattern identical to that of the four rat antisera described
below. The second antisera weakly stains spindle fibers. At this time we
do not know if this second staining pattern is due to a cross-reacting KLP.
Affinity Purification of Antisera
Each of the rat antisera were affinity purified using two different halves of
the protein. To make the construct encoding the carboxy half of KLP67A,
full-length GST-KLP67A (described above) was digested with BamHI and
NotI and repaired with the Klenow fragment of DNA polymerase and ligated. E. coli transformed with this subclone expresses an IPTG-inducible
70-kD protein (GST-KLP67A-70). To affinity purify antisera specific to
this part of the protein, ~100 µg of GST-KLP67A-70 were run on a 7.5%
polyacrylamide gel and transferred to Immobilon (Millipore Corp., Bedford, MA). The filter was stained with Ponceau S (Sigma Chemical Co.)
and the band containing GST-KLP67A-70 was excised and used for the
affinity purification (Olmsted, 1981 Immunolocalization
KLP67A was immunolocalized within methanol-fixed Drosophila embryos as described (Warn and Warn, 1986 To test for colocalization of KLP67A with mitochondria, embryos were
fixed with methanol minus EGTA or were used after 2 mo at Immunolocalization experiments using CHO cells were done using cells
grown on glass coverslips. To label mitochondria, MitoTracker Green
(Molecular Probes) was used according to the manufacturer's directions.
After labeling, the cells were washed with PBS and fixed in ice-cold methanol. Antibody labeling was as described (Doxsey et al., 1994 Western Blot Analysis
Drosophila lysate was prepared as described (Saxton et al., 1988 The complete amino acid sequence of KLP67A (Fig. 1 A)
was deduced from the DNA sequence of a KLP67A cDNA
isolated from a Drosophila embryonic cDNA library (Brown
and Kafatos, 1988
Many members of the kinesin superfamily can be
grouped into families on the basis of sequence similarity
within their motor domains (Goldstein, 1993 KLP67A Is a Plus End-directed MT Motor
The amino acid sequence of KLP67A predicts that this
protein is an MT motor. To verify this prediction, a GST-
KLP67A fusion protein was produced in E. coli, partially
purified by affinity chromatography, and used in an MT
motility assay. MT motility was observed in vitro after addition of GST-KLP67A fusion protein, MTs, and ATP to
the assay. The average speed of movement from three assays was .05 (±.02) µm/s, about 10 times slower than the speed measured for Drosophila kinesin (Kuznetsov et al.,
1989 Kinesin and related proteins exhibit unidirectional movement along the MT. MTs have an intrinsic polarity that is
generally defined by the relative rates of addition and loss
of tubulin subunits to their ends. This polarity is also related to MT orientation within the cell. The more stable
minus end of the MT is embedded in a centrosome or nucleation site, and the more rapidly growing plus end is in
the spindle midzone or at the cell periphery. A specific
motor protein therefore will reflect this polarity by causing
movement toward or away from the cell periphery or spindle pole. Since the directionality of a motor protein is critical for predictions of its cellular function, we determined
the direction of movement of KLP67A. To do this, a motility assay similar to that described above was carried out.
In this case, the minus ends of the MTs were labeled with
rhodamine-conjugated tubulin (Hyman, 1991
Expression Pattern of KLP67A mRNA
The expression pattern of a gene often provides information about functions of its encoded protein. We therefore
examined the pattern of KLP67A mRNA expression during embryogenesis by in situ hybridization (see Materials
and Methods). During the synchronous divisions of the
blastoderm embryo, KLP67A transcripts are uniformly distributed (Fig. 3 a). Their presence at high levels before the
onset of zygotic gene expression indicates that they are
maternally provided. Many maternal RNAs appear to be
degraded throughout the embryo upon cellularization, and
at this time KLP67A RNA is depleted (Fig. 3 b). After the
completion of cellularization, mitotic synchrony is lost, but
groups of cells referred to as mitotic domains continue to
divide in unison. These divisions take place in a precise
temporal and spatial pattern (Foe, 1989
To determine accurately the relationship between the
accumulation of KLP67A transcripts and the pattern of
mitosis, we performed in situ hybridization and stained
embryos with DAPI to visualize DNA and thus allow cells
that are undergoing mitosis to be distinguished from those
in interphase. The early mitotic domains 1 and 5 are located on the dorsal side of the embryo, anterior to the
cephalic furrow. Both are divided bilaterally, and, when
they are undergoing mitosis (Fig. 3 d, arrows), KLP67A
transcripts are present at levels higher than in the surrounding interphase cells (Fig. 3, d and e). Cells in domains 3 and 4, which lie at the anterior and posterior ends
of the embryo, respectively, also accumulate high levels
of KLP67A RNA at this time (Fig. 3, e and f, arrows). Similarly, in other cycle 14 domains, the accumulation of
KLP67A transcripts continues to follow closely the pattern of mitosis (Fig. 3, g, h, and i). In cycle 15, the mitotic
domains now consist of smaller groups of contiguous cells
that still accumulate KLP67A transcripts to high levels as
they divide (Fig. 3, j, k, and l, arrows). After the completion of the sixteenth mitotic cycle, divisions are confined to
the cells of the developing central nervous system (CNS)
where KLP67A transcripts are still present, whereas in
other nonproliferative regions of the embryo, the transcripts can no longer be detected (Fig. 3 c).
During larval development, mitosis is largely restricted
to the proliferative regions of the brain and imaginal tissue,
which have distinctive patterns of cell division (White and
Kankel, 1978 We find that the distribution of KLP67A transcripts reflects this pattern of cell division. Anterior to the furrow
are regions of cells, containing KLP67A transcripts, that
are likely to correspond to those undergoing asynchronous
mitoses. Posterior to the furrow, transcripts are restricted
to a narrow stripe of cells, including those undergoing synchronous mitoses (Fig. 4 a, arrows). Transcripts cannot be
detected in cells more posterior to the furrow, where mitosis has ceased. Similarly, in the optic lobes of the larval CNS, KLP67A transcripts can only be detected in cells
whose distribution is characteristic of those in the proliferative regions (White and Kankel, 1978
Immunolocalization of KLP67A
The observation that KLP67A mRNA is expressed in a
pattern indicative of a mitotic function prompted an examination of the distribution of KLP67A within the rapidly dividing nuclei of the precellular blastoderm. For these analyses, four rat antisera were raised against GST-KLP67A
as described in Materials and Methods. All four antisera
showed identical staining patterns on embryos before as
well as after affinity purification against two different parts of the protein (see Materials and Methods). Analysis
of the specificity of these antisera by Western blotting indicates that they give reactivity with a band of the appropriate size in embryo lysates. Rat 4 antisera, however, resulted in the strongest signal in Western blot experiments.
For example, rat 4 antisera affinity purified against the
KLP67A non-motor domain recognizes a predominant
protein species of ~90-kD from Drosophila embryo lysates (Fig. 5, lane 2). This size is very close to the molecular weight of 92 kD predicted from the KLP67A amino
acid sequence. This species is not recognized by preimmune sera from any of the four rats (data not shown). A
slightly smaller species is recognized in lysates prepared
from CHO cells (Fig. 5, lane 1).
Initially, immunofluorescence experiments with these
anti-KLP67A antisera were conducted with D. melanogaster infected with the endosymbiotic bacteria Wolbachia.
These bacteria are found in many laboratory stocks of
Drosophila grown in the absence of tetracycline (O'Neill
and Karr, 1990
An alternative embryo preparation allowed these particles to be released from the cytoskeleton, thereby becoming more visible. It was found that in embryos fixed in
methanol without EGTA, in embryos that have been stored
for a prolonged time at
It was also apparent that KLP67A staining is enriched at
the posterior pole of the blastoderm embryo (Fig. 9 B).
This observation is consistent with ultrastructural analyses
of the Drosophila oocyte and early embryo, which have
shown that a large number of mitochondria are concentrated at the posterior pole of the oocyte, where they are
attached or in close apposition to the polar granules until
about 30 min after fertilization (Mahowald, 1971
Some KLP67A staining is detectable in gastrula stage
embryos where the mRNA expression pattern is enriched
in mitotic domains. Surprisingly, however, at this stage
KLP67A staining is of apparently equivalent intensity in
interphase as in mitosis (data not shown). Whether this apparent inconsistency is a result of the level of the KLP67A
protein not following the mRNA amount, or whether it is
because immunofluorescence localization is not a reliable
indicator of actual protein amount in a cell is unclear at
present. Finally, staining for KLP67A late in embryogenesis is difficult to detect (e.g., the stage 15 embryo in Fig. 9,
A and B, upper right) relative to the high level of KLP67A
visible in early embryos (the stage 1 embryo in Fig. 9, A
and B, lower left can be used for comparison). Whether
this is due to an absence of KLP67A protein at later stages of embryogenesis or due to a detection problem is not
clear.
Finally, anti-KLP67A was also used to stain mammalian
cells. CHO cells labeled with anti-KLP67A and a vital dye
that is specifically taken up by mitochondria, Mitotracker,
show colocalization to mitochondria as shown in Fig. 10. This
staining pattern was observed with two different antisera,
one affinity purified against the carboxyl half of KLP67A
and the other affinity purified against the amino half. This
latter result is a strong verification that KLP67A is evolutionarily conserved and is associated with mitochondria in
both invertebrates and vertebrates.
MTs are used for translocating a number of different organelles within the cell. Several members of the kinesin superfamily are now known to be responsible for these
movements. Here we show that KLP67A is a plus end-
directed MT motor that is associated with mitochondria in
both Drosophila and mammalian cells. Although KLP67A
may persist after embryonic cells have ceased to divide (see below), our data suggest that it may be found primarily in proliferating tissue such as cultured cells and in
early Drosophila embryos. A survey of some third instar
larval tissue has shown that KLP67A is found in the imaginal discs and proliferation centers of the CNS but not in
some differentiated tissue such as neuropil and muscle
(Pereira, A., unpublished observations). It is therefore possible that a mitochondrial motor, similar to KIF1B of
mouse, is expressed in nonproliferating differentiated cell
types of Drosophila.
KLP67A is notable in that it does not readily fall into any
of the known KLP families that have been defined strictly
by sequence homology (Goldstein, 1993 The expression analysis of KLP67A indicates that while
KLP67A protein and mRNA levels appear to be equivalent
throughout the blastoderm embryo, the KLP67A mRNA
exhibits extraordinarily tight regulation later in embryogenesis. Specifically, KLP67A transcripts are present at
high levels in the syncytial embryo, and they appear to be
degraded at the time of cellularization before the onset of
zygotic transcription. After cellularization of the early Drosophila blastoderm embryo, the differential regulation of
the start of the 14th mitosis gives rise to what are called
mitotic domains (Foe, 1989 A requirement for KLP67A in mitotic cells is suggested
by the immunolocalization results. KLP67A is found associated with mitochondria near to or on the astral fibers
throughout mitosis in the early Drosophila embryo. A similar localization of mitochondria to the astral fibers in
mouse oocytes has been demonstrated, but the reason for
this localization is unknown (Van Blerkom, 1991). One
possibility is that the mitochondria are required for spindle function. The propinquity of the mitochondria to the
spindle poles may allow a locally high concentration of
ATP or calcium for anaphase or other movements. It is
also conceivable that the mitochondria have a structural
role as a support for the astral pulling force during spindle
assembly and anaphase B (Waters et al., 1993). Direct observation of the translocation of mitochondria along
MTs in extruded squid axoplasm has also been reported
(Brady et al., 1982
). At the ultrastructural level, mitochondria are also found surrounded by MTs in brain cells
(Raine et al., 1971
) and in the astral MTs of mouse oocytes (Van Blerkom, 1991). Finally, direct evidence for the requirement of MTs in mitochondrial localization has come
from genetic analyses in Schizosaccharomyces pombe
(Yaffe et al., 1996
).
). In the
case of mitochondrial movement, the mouse kinesin superfamily protein 1B (KIF1B) protein has recently been
found associated with mitochondria in neurons (Nangaku
et al., 1994
). Since KIF1B expression is enriched in neurons it may be, in part, specialized for the anterograde axonal transport system.
). Expression and immunolocalization studies
suggest that KLP67A may be a new type of mitochondrial
motor that is specialized for mitochondrial movement in
proliferative cells. Together with previous data on KIF1B, the results reported here suggest that eukaryotes may use
different mitochondrial motors adapted for specific MT
systems such as the axonal MT network, the interphase cytoskeleton, and the mitotic spindle.
Materials and Methods
). We used the original PCR fragment as a probe to screen a Drosophila 0-4-h embryonic cDNA library (Brown and Kafatos, 1988
). Several isolates from this library were found to each contain inserts of ~3 kb
in size. A 2.9-kb HindIII fragment was subcloned from one of these clones
into the Bluescript vector (Stratagene, La Jolla, CA) and a deletion series
was constructed using the Exo/mung kit (Stratagene) according to the
manufacturer's instructions. Deletion clones were sequenced on both
strands by the dideoxy chain termination method using a sequencing kit
(United States Biochem Corp., Cleveland, OH). The noncoding strand
was sequenced using a T7 primer. The coding strand was sequenced using a T3 primer as well as newly synthesized oligonucleotide primers. The resulting DNA sequence predicts a 2,442-bp open reading frame flanked by
a 138-bp 5
untranslated region and a 383-bp 3
untranslated region. The
original KLP67A cDNA clone within pNB40 was used to determine the
last 70 bp of sequence through the poly A tail.
). KLP67A was subcloned into this
vector in two steps. In the first step, a NcoI/HindIII fragment derived from
the KLP67A cDNA was cloned into NcoI/HindIII-digested pGEX-2T. This
subclone did not contain the first 350 bp of the KLP67A coding sequence.
In a second step, the 5
most 350-bp segment was ligated in the form of an
NcoI-digested PCR fragment. An NcoI site was created at the 5
end of
this PCR product through the use of a primer that encoded the initiator
methionine as part of the NcoI recognition sequence. The modified E. coli
KLP67A therefore begins with MET ALA rather than MET PRO. This
prediction was verified by DNA sequence analysis of pGEX-KLP67A. The
final construct was transformed into E. coli 71/18. Induction with isopropylthiogalactoside (IPTG) resulted in the production of a 122-kD GST fusion protein.
). A rabbit anti-GST antiserum was necessary to bind the fusion
protein to glass coverslips. The order in which the components of the assay
were combined was found to be critical. The GST-KLP67A fusion protein
(final concentration ~10 µg/ml) was added to a glass coverslip, followed
by the anti-GST antiserum (1:100 dilution), the MTs (final concentration
1-5 µg/ml), and ATP to a final concentration of 10 mM. MT movement
was observed by video-enhanced differential interference contrast microscopy. The direction of movement was determined using rhodamine-labeled MTs as described previously (Stewart et al., 1993
).
. After dechorionation by washing for 3 min in hypochlorite,
embryos were fixed in 4% paraformaldehyde in 120 mM NaCl, 10 mM
NaPO4 (PBS) with an equal volume of heptane. They were next devitalized by vigorous shaking in 1:1 heptane/methanol (Mitchison and Sedat,
1983
) and then pretreated for hybridization as described by Tautz and
Pfeifle (1989)
. After digestion with SalI, a digoxigenin-labeled anti-sense
RNA probe was transcribed from a KLP67A cDNA clone in the vector
pNB40 (Brown and Kafatos, 1988
). Before hybridization, probe RNA was
reduced in size by hydrolysis in 40 mM NaHCO3, 60 mM Na2CO3, pH 10, for 2 h at 60°C. The probe was precipitated and resuspended in 50 µl water; 5 µl was used for hybridization, which was carried out for 16 h at 47°C.
Hybridization solution and subsequent treatments were as described by
Tautz and Pfeifle (1989)
. Hybridization was detected by an alkaline phosphatase conjugated antidigoxigenin antibody (Boehringer Mannheim Corp.,
Indianapolis, IN) used at a dilution of 1:1,000. Embryos were washed in
PBS and then incubated for 15 min with 10 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) for visualization of DNA. Embryos were washed 3× 5 min in PBS and then mounted in 85% glycerol
and viewed using a microscope (model Axiophot; Carl Zeiss, Inc., Thornwood, NY). Embryos used to compare RNA levels between different
stages were drawn from the same collection and fixation batch.
except that the tissue was digested for
8 min with 50 µg/ml Proteinase K before hybridization. A probe was prepared by incorporation of digoxigenin-dUTP by random oligo-labeling (Feinberg and Vogelstein, 1984
) using a digoxigenin DNA labeling kit (Boehringer Mannheim Corp.). Tissue was dehydrated through an ethanol series
(30, 50, 70, 90, and 100%, 2 min each), mounted in Canada Balsam, and
viewed with a microscope (model Axiophot; Carl Zeiss, Inc.) using differential interference contrast optics.
). The amino half of the molecule was used in the same way for affinity purification. To make the construct encoding this half of the protein, the pGEX construct containing GST-KLP67A
was digested with NotI and HindIII, repaired as described above, and ligated. Antisera specific to the amino part of the protein were affinity purified as described above. Rat 1 antisera resulted in the most intense staining pattern. Antisera from the other rats exhibited an identical albeit
weaker staining pattern after affinity purification using either half of the
protein.
), with the following modifications. After fixation, the embryos were stored in 100% methanol at
20°C
from overnight to up to 1 mo with no change in tubulin staining quality, although mitochondria would gradually enlarge and detach from the MTs
and appear on the surface of the embryo. Washes after primary and secondary antibody incubations were overnight at 4°C with PBS, 0.1% Triton
X-100 (PBST). Primary antibody incubations were done using a 1:200 dilution of affinity-purified antisera together with one of the following: a 1:
10 dilution of mouse anti-tubulin monoclonal 3A5 (Piperno and Fuller,
1985
) , a 1:200 dilution of a rabbit anti-mitochondrial ATP synthase antisera (Pena and Garesse, 1993
), or a 1:200 dilution of an anticytochrome oxidase antibody (Molecular Probes, Eugene, OR). Dilutions were made
in PBST. The secondary antibodies used were fluorescein-conjugated goat
anti-mouse or anti-rabbit IgG and Cy3-conjugated goat anti-rat IgG (Cappel, Malvern, PA). Secondary antibodies were used at a dilution of 1:2,000
in PBST. Embryos were also fixed with formaldehyde as described (Theurkauf, 1992
).
20°C in
methanol. This resulted in a release of the mitochondria from the cytoskeleton and onto the surface of the embryo, which was necessary to visualize
the mitochondria with an anti-cytochrome oxidase antibody (Molecular
Probes, Eugene, OR). In embryos fixed as described above for MT preservation, the anti-cytochrome oxidase antibody staining pattern was very
difficult to visualize because of the high cytoplasmic background seen with
this antibody. It was still possible, however, to verify the colocalization of
KLP67A and cytochrome oxidase to mitochondria in the area surrounding the spindle poles.
). CHO cells
were observed and photographed using a microscope (model Axiophot;
Carl Zeiss, Inc.).
) and
loaded onto a 10% SDS-polyacrylamide gel. The protein was transferred
to Immobilon (Millipore Corp.), and the blot was then processed as described (Pereira et al., 1992
) except that a HRP-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used
to detect binding of the primary antibody. For the primary antibody incubation, rat 4 antisera affinity purified against GST-KLP67A-70 was used
at a 1:10 dilution. The HRP-conjugated anti-rat secondary antibody was
used at a dilution of 1:5,000. The HRP substrate was LumiGLO and was
used according to the manufacturer's instructions (Kirkegaard and Perry
Laboratories).
Results
). A fragment of the amino acid sequence of the KLP67A motor domain was previously published and was designated KLP3; it has now been renamed
according to its cytological location, 67A (Stewart et al.,
1991
). The KLP67A protein sequence includes 814 amino
acid residues. The putative initiator methionine is located
at nucleotide 138. Further analysis of the non-motor domain sequence using standard computer programs (Garnier et al., 1978
; Devereux et al., 1984
) reveals that the non-
motor domain, particularly between amino acid residues
340-660, has the potential to form an
-helix (Fig. 1 B).
Within this same stretch of amino acids, there is also a high
probability of
-helical coiled coil formation as predicted
by the method of Lupas et al. (1991)
as can be seen in Fig.
1 C. The secondary structure of the 200-amino acid carboxy-terminal region is unknown. Therefore, unlike the
KHC, KLP67A cannot be easily predicted to have a threedomain structure. This is also the case for several other
members of this superfamily such as nod (Zhang et al.,
1990
). Determination of the KLP67A secondary structure
therefore awaits biochemical analyses of the native protein.
Fig. 1.
KLP67A cDNA sequence. (A) The sequence of a KLP67A cDNA clone and its deduced amino acid sequence is shown. These
sequence data are available from EMBL/Genbank/DDBJ under accession number U89264. The amino acid sequence of a portion of the
presumptive motor domain was previously compared to 27 known members of the kinesin superfamily (Goldstein, 1993). (B) Structural
predictions from the UWGCG Peptide Structure program. (C)
-Helical coiled coil probability predicted from the algorithm of Lupas
et al. (1991)
.
[View Larger Version of this Image (72K GIF file)]
). For example, pairwise sequence comparisons of the predicted motor
domains among all members of the superfamily reveal several families that are more similar among themselves than
with other members of the superfamily (e.g., the bimC
family, the KHC family, and the KAR3 family). KLP67A
does not fall into any of these families, nor does it show
significantly more sequence similarity with the motor or
tail domain of any other kinesin superfamily member than
it does with the KHC. Direct comparisons to KIF1B (Nangaku et al., 1994
) also revealed no sequence similarity outside of the conserved motor domain.
) and KIF1B (Nangaku et al., 1994
) but similar to the
speed of Eg5, another kinesin-related protein (Sawin et al.,
1992
). Although a "no-insert" bacterial lysate control was
not done specifically during this series of motility experiments, previous work has demonstrated that E. coli lacks
an endogenous MT motility activity (data not shown).
; Stewart et al.,
1993
). Plus end-directed movement by KLP67A would cause
MT movement with the rhodamine seed leading, while minus end-directed movement would cause the rhodamine
seed to trail. In 12 assays, the addition of GST-KLP67A
clearly resulted in plus end-directed movement. For example, in Fig. 2 a the minus end of a rhodamine-seeded
MT is indicated by an arrow. Several minutes later the minus end has moved several microns beyond the point of
reference, as shown in Fig. 2 b.
Fig. 2.
Movement of polarity-marked microtubules in the plus
direction. The microtubule minus end is nearest the rhodamine seed
and is indicated by the arrow. (a) Microtubule position at t = 0. (b) Microtubule position at t = 6 min. The velocities are not as
fast as those in the non-fluorescence-based assays, presumably owing to photodamage. Bar, 5 µm.
[View Larger Version of this Image (119K GIF file)]
). We observed
that KLP67A transcripts assume a distribution that corresponds with this pattern.
Fig. 3.
The distribution of KLP67A transcripts in embryogenesis shown by in situ hybridization. (a) Syncytial embryo showing high levels of uniformly distributed KLP67A RNA. In the cellularizing embryo, the RNA is depleted (b). RNA is present in the embryonic central nervous system after mitosis has ceased in other tissues (c). In the cycle 14 embryo, KLP67A transcripts accumulate in mitotic
domains, indicated by arrows in d, e, and f, numbers refer to domains described by Foe (1989). Cells undergoing mitosis are distinguished by staining of DNA using DAPI, in f, i, and l. In later cycle 14 the distribution of KLP67A RNA continues to reflect the pattern
of mitotic domains (g). Comparison of h (KLP67A) and i (DNA staining) indicates that cells undergoing mitosis contain elevated levels of
KLP67A transcripts. In g, h, and i, adjacent regions of cells in interphase (I), and mitosis (M) are shown. In the cycle 15 embryo,
KLP67A RNA accumulation corresponds with the pattern of mitosis (j). Two groups of cells that contain high levels of KLP67A RNA
are indicated in j and k. DNA is visualized and the same groups of cells are indicated in l. All embryos are oriented anterior to left, dorsal up, except b, d, e, and f, where dorsal views are shown. Bars: (a-d, g, and j; shown in j) 100 µm; (e, f, h, i, k, and l; shown in l) 100 µm.
[View Larger Version of this Image (137K GIF file)]
). We therefore performed in situ hybridization (see Materials and Methods) to determine if KLP67A
transcripts were distributed correspondingly. In the eyeantenna disc, cells become incorporated into the ommatidia preclusters as a wave of differentiation, referred to as
the morphogenetic furrow, moves across the disc from
posterior to anterior. Anterior to the furrow, asynchronous mitoses provide cells for incorporation into the preclusters. Then, posterior to the furrow, specific cells of each
newly formed precluster undergo a final round of synchronous mitosis (Ready et al., 1976
; Wolff and Ready, 1991
).
; Truman and Bate,
1988
) (Fig. 4 b, arrows). Hence in embryogenesis and in
the tissues of the third instar larva that we examined, the
distribution of KLP67A transcripts closely follows the patterns of mitosis.
Fig. 4.
Distribution of KLP67A transcripts in tissues of the 3rd
instar larva, shown by in situ hybridization. (a) Transcripts are present in the anterior region of the eye disc where asynchronous mitoses are taking place (A). Transcripts are concentrated in a
stripe corresponding to cells undergoing synchronous mitoses (S), immediately posterior to the morphogenetic furrow (M). (b) In the larval brain, transcripts are concentrated in the proliferative regions of the optic lobes (arrows). In a and b, anterior is to the left. Bars: (a) 100 µm; (b) 200 µm.
[View Larger Version of this Image (59K GIF file)]
Fig. 5.
Western blot of Drosophila
embryo (lane 2) and CHO cell lysates
(lane 1) probed with rat 4 anti-KLP67A
rat antisera affinity purified against
GST-KLP67A-70.
[View Larger Version of this Image (30K GIF file)]
). This allowed the fortuitous discovery that
KLP67A is associated with bacteria found on the plus ends
of astral MT fibers (Pereira, A., and T. Karr, unpublished observation; the localization of KLP67A to Wolbachia will
be presented elsewhere). In embryos that are uninfected
by Wolbachia, KLP67A staining is still observed on particles in the region of the astral fibers, but these particles are
at least 10-fold smaller than Wolbachia. An example of this
localization within a blastoderm embryo double labeled
with anti-KLP67A and anti-tubulin antisera is shown in
Fig. 6. The antigen distribution relative to the spindle poles is most evident when a stepwise series of optical sections of the spindles is viewed simultaneously. Thus, four
0.8-µm optical sections through a sample of late anaphase
stage spindles are shown. Late anaphase B is shown specifically because at this stage the poles are more widely separated and the concentration of particles at the poles is
more evident than at other stages of mitosis. The small
particles stained by anti-KLP67A are predominantly seen in regions containing astral fibers radiating from each
spindle pole. To examine the possibility that these small
particles are mitochondria, embryos were also double labeled with affinity-purified anti-KLP67A antiserum and
an antiserum specific for the Drosophila mitochondrial
ATP synthase
subunit (Pena and Garesse, 1993
). A higher
magnification view (Fig. 7) shows that KLP67A staining is
primarily associated with particles that are also stained
with the ATP synthase antisera. However, KLP67A appears
to associate with a subset of mitochondria since not all of
the mitochondria that are stained by the ATP synthase antisera are also stained by anti-KLP67A. Similarly, localization of these particles to the region of the astral fibers is
also observed when embryos are double labeled with the
ATP synthase and tubulin antisera (data not shown).
Fig. 6.
A Z series of four consecutive optical sections through a Drosophila precellular blastoderm embryo double labeled with affinity-purified rat 4 KLP67A (top four panels) and antitubulin antibodies (bottom four panels). The leftmost section corresponds to the approximate middle of the spindle and the next three sections are progressing downward away from the surface of the embryo. Note that
anti-KLP67A stains circular areas of small particles concentrated at the spindle poles. One such area is indicated by the "p" in the first
pair of panels. Bar, 15 µm.
[View Larger Version of this Image (91K GIF file)]
Fig. 7.
Double labeling of a blastoderm embryo
similar to that shown in Fig. 6 with a Drosophila mitochondrial ATP synthase (A) and affinity-purified
rat 4 anti KLP67A (B) antiserum. Bar, 5 µm.
[View Larger Version of this Image (69K GIF file)]
20°C, or in embryos with poor astral MT preservation, anti-KLP67A stains mitochondrialike particles that are released from the cytoskeleton and
appear in aggregates on the surface of the egg (Fig. 8).
Since this aberration allows the particles to be visualized
more clearly, such a preparation was used to show that the
small particles detected by anti-KLP67A are indeed mitochondria. Thus, Fig. 8 also shows the surface of one such
embryo double labeled with anti-KLP67A (Fig. 8 C) and
the mitochondrial marker anti-cytochrome oxidase (Fig.
8 D). Although the cytochrome oxidase staining is not as intense and clear as that of the KLP67A staining, it can be
seen that the two colocalize completely to mitochondria
on the surface of this embryo.
Fig. 8.
Immunolocalization of KLP67A in the absence of astral fiber fixation. (A) Surface of a blastoderm embryo immunostained for KLP67A. (B) The corresponding DAPI-stained image
of anaphase chromosomes. Double labeling of the embryo preparation shown in Fig. 7 with anti-KLP67A (C) and anti-cytochrome
oxidase (D). Bar, 5 µm.
[View Larger Version of this Image (120K GIF file)]
). Indeed,
the high concentration of mitochondria in this region causes
intense DAPI staining in this area as well (Fig. 9 A);
KLP67A colocalizes with small DAPI-stained particles at the posterior pole of the embryo (data not shown). This
association suggests that KLP67A may be used for the
transport of mitochondria and possibly other polar granule components to the posterior pole during oogenesis.
Fig. 9.
Early and late Drosophila embryos double labeled with
DAPI (A) and affinity-purified rat 1 antisera (B) directed against
KLP67A. The arrowhead indicates the posterior polar end of the
stage 1 embryo. The second embryo is approximately at stage 15. Bar, 100 µm.
[View Larger Version of this Image (108K GIF file)]
Fig. 10.
Immunolocalization of KLP67A within CHO cells double labeled with Mitotracker (B and D). (A) Cells stained with
anti-KLP67A affinity purified against the motor domain portion
of KLP67A. (C) Cells stained with anti-KLP67A affinity purified
against the non-motor domain portion of KLP67A. The nucleus
is indicated by an "n" for registration purposes. Bar, 5 µm.
[View Larger Version of this Image (115K GIF file)]
Discussion
). Often, a functional homology among family members also exists. For
example, the bimC family consists of several different KLPs
that all separate spindle poles during spindle assembly (discussed in Heck et al., 1993
). Other families of KLPs also
appear to share similar cellular functions as well (Pereira
and Goldstein, 1994
). It is therefore curious that the Drosophila KLP67A associates with mitochondria in vivo, yet
it is not in the same family as the mouse mitochondrial
motor, KIF1B (Nangaku et al., 1994
). The sequence divergence of KLP67A and KIF1B may be due to several different factors. At the ultrastructural level, mitochondria
exhibit striking morphological differences depending on
the cell type examined. It is therefore possible that the receptors for motor binding may be different on differentiated versus embryonic mitochondria. The divergence may
also have resulted from the requirement for KIF1B to
move rapidly down the relatively long distance of the axon
or myofibril compared to the short distance of the astral
MTs traveled by KLP67A. Indeed, although velocity in
vitro may not be a reliable indicator of velocity in vivo, the
measured velocity of KLP67A driven movement in vitro
(~0.05 µm/s) is 10-fold slower than that reported for KIF1B
and for mitochondrial movement within the axon (Nangaku et al., 1994
).
). KLP67A mRNA is exclusively expressed in these mitotic domains and may therefore be regulated during the cell cycle. This pattern of transcript distribution is reminiscent of RNAs encoding proteins
known to be required for progression through mitosis in
Drosophila, such as cyclin A, cyclin B (Lehner and O'Farrell, 1990
; Dalby and Glover, 1993
), cdc2 (Jimenez et al., 1990
; Lehner and O'Farrell, 1990
), and polo (Llamazares
et al., 1991
). While the levels of these other RNA species
are elevated in regions of active mitosis, they do not appear to be as tightly coupled as KLP67A to the pattern of
mitotic domains in either the 14th or later cycles. In fact,
the pattern of KLP67A transcript distribution in these cycles is more similar to that of another kinesin-like protein,
KLP61F, which is required for correct separation of centrosomes in mitosis (Heck et al., 1993
). However, the levels of KLP61F RNA also appear less tightly coupled to the
pattern of mitotic domains than those of KLP67A. Of the
RNAs encoding proteins known to be required for correct
progression through mitosis in Drosophila, only the string
RNA (encoding a tyrosine phosphatase) (Edgar and O'Farrell, 1989
) appears to show coupling to the spatial and temporal pattern of mitosis that is as close as that of KLP67A
transcripts. Thus, the KLP67A expression pattern may be
a strong indication of function during mitosis. The patterns of transcript accumulation observed could be determined
by both cell cycle-regulated transcription and message stability. There is, however, an apparent contradiction between our failure to observe enrichment of KLP67A protein in these mitotic domains of mRNA expression. It is
possible that our immunofluorescent methods are simply
not reliable enough to preserve an actual difference in the
amount of KLP67A between interphase and mitotic cells, i.e., there may be differences in the amounts of KLP67A
extracted from these two cell types during fixation, or the
antibody staining may not reflect the amounts of protein
depending on organization of the protein in the cells. Alternatively, there may be near equivalent amounts of
KLP67A in mitotic and interphase cells at this stage of development; this apparent equivalence could result from
persistence of the KLP67A protein from earlier stages when the mRNA is more abundant. In this case, the mitotic cycling of KLP67A mRNA may be used to ensure that there
are sufficient amounts of KLP67A for the dividing cell. In
this regard, at least two other genes have been reported to
have a pattern of cycling mRNA during the cell cycle but
constant protein expression similar to that of KLP67A.
The yeast SPC110/NUF1 transcript is cell cycle-regulated, yet the corresponding protein levels are not (Kilmartin et al., 1993
). Also in yeast, the CDC9 (DNA ligase) transcript is
tightly regulated, while the stability of the protein allows it
to persist through the cell cycle (Johnson et al., 1986
). In
each of these cases, cell cycle control may ensure that the
gene product will be present in sufficient quantities during
mitosis, whereas protein stability may result in its persistence through interphase.
). Alternatively, the spindle may be required to segregate mitochondria during division. In the case of the large acellular
Drosophila embryo, a spindle attachment could ensure
that the mitochondria will be localized to the dividing cells
rather than to the yolk region or areas that are less metabolically active. The genetic analysis of KLP67A should
allow us to distinguish between these various possibilities.
Received for publication 10 June 1996 and in revised form 12 November 1996.
A.J. Pereira would like to express her gratitude to William S. Gelbart of Harvard University (Cambridge, MA) in whose lab she did a large part of the work reported. She would like to thank Nicholas Lim (Harvard University) for technical assistance, Aruna Purohit (University of Massachusetts, Worcester), Jason Dictenberg (University of Massachusetts, Worcester), Tim Karr (University of Chicago), and Paul Dobner (University of Massachusetts, Worcester) for scientific discussion and advice, and also Kristin White (Brandeis University, Boston, MA) for the use of her confocal microscope and Fuji printer. A.J. Pereira would also like to thank Rafael Garesse (Universidad Autonoma de Madrid, Spain) for an aliquot of anti-Drosophila mitochondrial ATP synthase antisera.This work was supported by National Institutes of Health grant GM35252 to L.S.B. Goldstein. B. Dalby was supported by a European Molecular Biology Organization fellowship. S.J. Doxsey is supported through the American Heart Association. L.S.B. Goldstein is an investigator of the Howard Hughes Medical Institute.
CNS, central nervous system; DAPI, 4,6-diamidino-2-phenylindole; GST, glutathione S-transferase; KIF1B, kinesin superfamily protein 1B; KHC, kinesin heavy chain; KLP, kinesinlike protein; IPTG, isopropylthiogalactoside; MT, microtubule.