The complete amino acid sequence of KLP67A (Fig. A
) was deduced from the DNA sequence of a KLP67A
cDNA isolated from a Drosophila
embryonic cDNA library (Brown and Kafatos, 1988
). 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 α-helix (Fig. B
). Within this same stretch of amino acids, there is also a high probability of α-helical coiled coil formation as predicted by the method of Lupas et al. (1991)
as can be seen in Fig. 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.
Figure 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 (more ...)
Many members of the kinesin superfamily can be grouped into families on the basis of sequence similarity within their motor domains (Goldstein, 1993
). 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.
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
) 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).
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
; 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. 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. b.
Figure 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 (more ...)
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. 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. 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
). We observed that KLP67A transcripts assume a distribution that corresponds with this pattern.
Figure 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 (more ...)
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. d, arrows), KLP67A transcripts are present at levels higher than in the surrounding interphase cells (Fig. , 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. , e and f, arrows). Similarly, in other cycle 14 domains, the accumulation of KLP67A transcripts continues to follow closely the pattern of mitosis (Fig. , 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. , 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. 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 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
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. 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
; Truman and Bate, 1988
) (Fig. b
). 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.
Figure 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 (more ...)
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. , 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. , lane 1).
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.
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
). 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. . 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 β subunit (Pena and Garesse, 1993
). A higher magnification view (Fig. ) 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).
Figure 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 (more ...)
Double labeling of a blastoderm embryo similar to that shown in Fig. with a Drosophila mitochondrial ATP synthase (A) and affinity-purified rat 4 anti KLP67A (B) antiserum. Bar, 5 μm.
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 −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. ). 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. also shows the surface of one such embryo double labeled with anti-KLP67A (Fig. C) and the mitochondrial marker anti–cytochrome oxidase (Fig. 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.
Figure 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. (more ...)
It was also apparent that KLP67A staining is enriched at the posterior pole of the blastoderm embryo (Fig. 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
). Indeed, the high concentration of mitochondria in this region causes intense DAPI staining in this area as well (Fig. 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.
Figure 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. (more ...)
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. , A and B, upper right) relative to the high level of KLP67A visible in early embryos (the stage 1 embryo in Fig. , 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. . 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.
Figure 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 (more ...)