From the Department of Biochemistry and Molecular
Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada and
the
Department of Anatomy and Cell Biology, Queen's University,
Kingston, Ontario K7L 3N6, Canada
Received for publication, December 23, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Conventional kinesin I motor molecules are
heterotetramers consisting of two kinesin light chains (KLCs) and two
kinesin heavy chains. The interaction between the heavy and light
chains is mediated by the KLC heptad repeat (HR), a leucine zipper-like motif. Kinesins bind to microtubules and are involved in various cellular functions, including transport and cell division. We recently
isolated a novel KLC gene, klc3. klc3 is the
only known KLC expressed in post-meiotic male germ cells. A monoclonal
anti-KLC3 antibody was developed that, in immunoelectron microscopy,
detects KLC3 protein associated with outer dense fibers (ODFs), unique structural components of sperm tails. No significant binding of KLC3
with microtubules was observed with this monoclonal antibody. In
vitro experiments showed that KLC3-ODF binding occurred in the
absence of kinesin heavy chains or microtubules and required the KLC3
HR. ODF1, a major ODF protein, was identified as the KLC3 binding
partner. The ODF1 leucine zipper and the KLC3 HR mediated the
interaction. These results identify and characterize a novel
interaction between a KLC and a non-microtubule macromolecular structure and suggest that KLC3 could play a microtubule-independent role during formation of sperm tails.
Kinesin light chains
(KLCs)1 are components of the
conventional kinesin motor molecule that consists of two kinesin heavy
chains (KHCs) associated with two KLCs. Kinesins bind to and move along microtubules, powering the transport of proteins,
macromolecules, and organelles (reviewed in Refs. 1-5).
Although extensive work has been done on elucidating the structure and
function of KHCs, relatively little is known about light chains. KLCs
have been suggested to bind cargo and to regulate the activity of KHCs. Clues as to possible functions and the domains involved came from sequence comparisons and genetic analysis of KLCs that were cloned from
Caenorhabditis elegans (6), sea urchin (7), squid (8), Drosophila (9, 10), and mammals (11-16). In mouse, KLC1, a largely neuronal form of KLC, and the ubiquitous KLC2 have been identified (12). KLCs share an N-terminal heptad repeat (HR), reminiscent of a leucine zipper motif, that is involved in binding to
KHCs (10, 17). The KLC middle region is highly conserved and consists
of tandem tetratricopeptide repeats that, in other proteins, mediate
protein interactions (18). Their role in KLC function may be similar.
The highly variable C-terminal region of KLCs has been proposed to bind
cargo such as organelles and macromolecules; this is supported by
direct binding studies in which rat KLC1b was shown to bind to
mitochondria (13) and by the use of specific antibodies that bind to
KLC and block its interaction with organelles (14). As mentioned, KLCs
may also regulate KHC and keep it in an inactive state by preventing
the active conformation of KHC (11). This was supported by the recent analysis of KLC1 knockout mice; mutant mice were small and exhibited motor disabilities (19). Also, in these mice, a pool of KHC KIF5A was mislocalized to the peripheral
cis-Golgi.
We recently reported the isolation of a novel KLC gene,
klc3, which contains a conserved HR motif that mediates
binding to KHCs as well as five tetratricopeptide repeats (20). In
association with KHC, KLC3 binds to microtubules in an
ATP-dependent manner. KLC3 is expressed in several tissues,
including testis and brain; analysis of different male germ cells
demonstrated that spermatids are the major site of KLC3 expression.
Interestingly, male haploid germ cells do not detectably synthesize
KLC1 and KLC2, as determined by reverse transcription-PCR, suggesting
that KLC3 probably carries out its function in transport and other
motor-based processes and/or that other spermatid KLCs remain to be
identified. This is in agreement with the observation that male KLC1
knockout mice are fertile (19). Kinesin-related proteins (KRPs), which
do not contain light chains, have also been described in rat testis (21, 22), and two KRPs have sequence similarity to the previously described BimC subfamily of KRPs involved in mitosis (23). The other
KRPs expressed in testis had no homology to known kinesins and likely
represent novel family members. The KIF3 KRPs were also detected in
testis (24); KIF3A and KIF3B form heterodimers that function as
microtubule-based (+)-end transporters of membranous organelles (25). A
KIF3C knockout mouse is viable and apparently normal (26). Finally,
kinesin II, a member of the KIF3 family, is expressed in developing
rat, sea urchin, and sand dollar sperm tails (27, 28). Because kinesins
and KRPs transport cargo on microtubule tracks, it is interesting that
rat testicular KRP2 and KRP6 are associated with spindle microtubules
(22). Together, these data suggest that conventional kinesins as well
as KRPs play a role in spermatid flagellar transport.
Spermatogenesis is characterized by continuous proliferation and
differentiation of germ cells. Upon completion of meiosis, haploid
cells called spermatids emerge and differentiate into spermatozoa
through a process known as spermiogenesis. Major morphological changes
occur at this stage, including nuclear condensation and formation of
the sperm tail, which contains unique structures not present in cilia
and flagella, viz. outer dense fibers (ODFs) and the fibrous
sheath (FS). The sperm tail can be subdivided into distinct regions,
including the midpiece, which contains all mitochondria surrounding
the ODFs and axoneme, and the principal piece, which contains
the FS, ODFs, and axoneme. Several ODF components and associated
proteins were recently cloned, and we demonstrated that they interact
specifically via leucine zipper motifs (29-31). The function of ODFs
remains unknown, but might be manyfold, including strengthening of the
long sperm tail, a function in elastic recoil; linking of the FS and
mitochondrial sheath to the axoneme; and regulation of motility.
To investigate the localization of KLC3 in developing spermatids, we
raised monoclonal and polyclonal antibodies against KLC3. Using the
monoclonal antibodies (mAbs), we discovered the association of KLC3
with ODFs in elongating spermatids, a structure devoid of microtubules,
and details of this association were explored. The molecular basis of
the ODF association is the ability of KLC3 to bind to the major ODF
protein ODF1.
Antibody Production and Immunocytochemical Analysis of KLC3
Expression in Testis
A fusion protein containing maltose-binding protein (MBP) linked
to KLC3 (20) was produced by inducing transfected TB1 bacteria with
isopropyl- Adult male Sprague-Dawley rats were anesthetized, and their testes and
epididymides were fixed by retrograde perfusion through the abdominal
aortas either with standard Bouin's fixative for histology or with
0.8% glutaraldehyde and 4% paraformaldehyde in 0.1 M
phosphate-buffered saline containing 50 mM lysine (pH 7.4)
for ultrastructure. After perfusion, the tissue designated for electron
microscopy was razor-cut into 1-mm2 blocks, immersed in the
respective fixative for 1 h, washed extensively with buffer, and
processed for Lowicryl embedding. The issue designated for light
microscopy was razor-cut into 3-4-cm2 blocks, immersed in
Bouin's fixative overnight, washed several times over 1 day with 70%
ethanol, and processed for paraffin embedding.
Immunohistochemistry--
After an extensive wash with 70%
ethanol, the tissue blocks were dehydrated and embedded in paraffin by
standard procedures. Five-µm sections were deparaffinized,
hydrated through a graded series of ethanol concentrations, and
immunostained as previously described (32).
Immunoelectron Microscopy--
Processing of tissues for
Lowicryl K4M embedding followed a standard protocol used in our
laboratory (33). Lowicryl-embedded ultrathin sections of testes and
epididymides were mounted on 200-mesh Formvar-coated nickel grids,
transferred, and floated tissue side down on 10-20-µl drops of the
following solutions: 10% goat serum in 20 mM
Tris-HCl-buffered saline (TBS) (pH 7.4), 15 min; anti-KLC3 mAb diluted
1:10 in TBS, 1 h; TBS containing 0.1% Tween 20, 5 × 5 min;
10% goat serum in TBS, 15 min; colloidal gold (10 nm)-conjugated goat
anti-mouse IgG diluted 1:20 in TBS, 45 min; TBS containing Tween 20, 3 × 5 min; and distilled H2O, 2 × 5 min. The
sections were then counterstained with uranyl acetate and lead citrate
and examined by electron microscopy. Controls consisted of replacing
the primary antibody (anti-KLC3) step with TBS or mAbs raised against
other proteins that were diluted 1:10 in TBS.
KLC3 and ODF1 Plasmid Constructs
The KLC3 C-terminal deletion (KLC3 KLC3-ODF Binding Assays
In Vitro KLC3-ODF Binding Assay--
ODFs were isolated from rat
epididymal spermatozoa as described by Oko (35). Vectors
containing KHC, KLC3, and the KLC3 ODF Western Blot Binding Assay--
To identify proteins present
in purified ODFs or the FS that may bind KLC3, a modified Western blot
binding assay was carried out. Proteins from fractionated male germ
cells obtained by centrifugal elutriation (36, 37) or from ODFs and the
FS were separated on 10-15% gradient SDS-polyacrylamide gels and
transferred to NitroPlus membranes. Membranes were blocked and washed
as described above and were then incubated in Western blot
binding buffer (30) containing 1 mM ATP and 1000 cpm/ml
35S-labeled KLC3 protein, produced by in vitro
translation, for 16 h at 4 °C. Blots were washed three times
and exposed to Biomax film.
Yeast Two-hybrid KLC3 Interaction Assays--
The use and
construction of yeast two-hybrid plasmids, cDNA expression vectors
based on pGAD (containing the GAL4 activating domain) and pGBT9
(containing the Gal4 DNA-binding domain), and cell lines have been
described previously (29). The interaction between KLC3 and
ODF1, ODF2, and mutants was analyzed in yeast as
described above. Colonies that grew on His Western Blot Analysis of Purified ODFs--
Extracts prepared
from purified ODFs were separated by electrophoresis on
SDS-polyacrylamide gels and transferred to Hybond-P polyvinylidene
difluoride membranes (Amersham Biosciences). Specific proteins on blots
were analyzed by incubation with antisera to ODF1, KLC3 Associates with Spermatid ODFs--
We recently cloned and
characterized a novel gene, klc3, which has the
distinguishing structural and functional features of KLC proteins (20).
Interestingly, we found that spermatids express only KLC3, not the
other known light chains KLC1 and KLC2. This suggested that KLC3 might
carry out roles specific to the process of spermiogenesis in addition
to a general function in transport. To approach this possibility, we
generated affinity-purified polyclonal anti-KLC3 antibodies as well as
monoclonal anti-KLC3 antibodies (20), both of which specifically
recognized the 58-kDa KLC3 protein, as shown in Fig.
1; polyclonal and monoclonal anti-KLC3 antibodies recognized KLC3 in elutriated elongating spermatids (lanes 1 and 4, respectively) as well as in
epididymal sperm (lanes 3 and 5, respectively).
No protein was observed in early spermatocytes (lane 2) or
liver (lane 6), which does not produce klc3
mRNA (20). These antibodies were next used in immunocytochemistry
of rat testis. Surprisingly, the polyclonal and monoclonal antibodies displayed overlapping but distinct KLC3 expression patterns during spermiogenesis at the light microscopic level (as shown in Figs. 2 and 3). The results of these studies
are summarized in Fig. 4. Note that rat spermiogenesis is divided in 19 distinguishable steps of development.
Fig. 2 shows the results of immunocytochemistry using affinity-purified
polyclonal antibodies and illustrates that KLC3 protein expression was
first detectable in step 8 round spermatids (stage VIII of the
seminiferous epithelium) (middle and lower
panels). KLC3 immunostaining appeared to peak in the cytoplasm of
elongating spermatids at stages XIV-I, thereafter gradually
diminishing from this region (steps 16-19, stages II-VIII), but at
the same time becoming prominent in the tails of maturing elongated
spermatids. Note that, initially, KLC3 staining in the tail was light
(arrows), but later became very dense
(arrowheads) along the entire tail. This suggests that KLC3
associates with structures that run the length of the tail,
viz. the axoneme and/or ODFs. Fig.
3 shows the results of immunocytochemical
analyses using mAb B11. KLC3 labeling was first detectable in step 14 spermatids (Fig. 3A). Cytoplasmic staining became prominent
from step 15 spermatids onwards and at later steps became concentrated
in the midpiece area of elongated sperm tails (Fig. 3B).
Fig. 3C (and inset) shows cross-sections of
midpieces, and Fig. 3D (and inset) shows
longitudinal midpiece sections, both exposures revealing a strong KLC
immunoreactivity in mature spermatids to be exfoliated. This result
indicates that KLC3 proteins detected by the mAb are present in
association with structures present in the sperm tail midpiece
(e.g. the ODFs and/or mitochondrial sheath), but not the
axoneme or FS. The upper panel of Fig.
4 summarizes the period of spermatid
development showing KLC3 staining as detected by the polyclonal and
monoclonal antibodies; the lower panel indicates
schematically KLC3 staining in the cytoplasm and tail structures
observed using the mAb.
Localization of KLC3 to ODF Surfaces--
Because KLC3
immunoreactivity appeared concentrated in the midpieces of elongated
spermatids, we used the mAb in a more precise ultrastructural analysis
of KLC3 localization in spermatid tails. The results shown in Fig.
5 reveal a novel association pattern for
KLC3 in mature step 19 elongated spermatids. Fig. 5 (A and B) shows cross-sections, and Fig. 5C shows a
longitudinal section through rat sperm. The mAb detected KLC3
predominantly in association with the surface of ODFs, but not with the
axoneme, confirming the light microscopic observations with this
antibody. The KLC3 immunogold label was observed between adjacent ODFs
and between ODFs and surrounding mitochondria (examples are shown in
Fig. 5D). Quantitation of gold label in all micrographs
indicated that labeling was not significant outside of this designated
region, including sites within the ODFs, FS, or axoneme. In mature
sperm, microtubules have not been shown to be present between adjacent ODFs or between mitochondria and ODFs (38). Based on these results, we
conclude that mAb B11 detects KLC3 proteins that display a novel
subcellular localization in elongating spermatids, and we proceeded to
explore the possibility of a binding association of KLC3 with the
macromolecular ODF structure.
The HR of KLC3 Is Involved in ODF Interaction--
An in
vitro ODF binding assay was developed to study the molecular basis
of the novel KLC3-ODF association observed in elongating spermatids. In
preparation, ODFs were isolated and purified from mature rat epididymal
spermatozoa using gradient centrifugations as described (35). Purified
ODFs were examined by Western blot analysis to confirm that they were
completely devoid of endogenous
Next, in vitro translated 35S-radiolabeled KLC3
was added to purified ODFs in the presence of 0.5% Sarkosyl. After
binding, ODFs were pelleted through a sucrose cushion, washed and
repelleted several times, and analyzed by SDS-PAGE. The results show
that wild-type KLC3 (58 kDa) could bind to purified ODFs in the absence of detectable KHC or microtubules (Fig.
7, compare lanes 1 and 5), as evidenced by its presence in ODF pellets. To analyze
the specificity of this assay, we next tested binding of an unrelated protein (p21ras) to purified ODFs. The Ras oncoprotein failed
to bind (compare lanes 4 and 8). To further
address specificity, we mixed in vitro translated
radiolabeled KLC3 and KHC proteins and subjected them to ODFs; in
comparison with KLC3, KHC showed virtually no binding (lanes
9 and 10). We next exploited this binding assay to
delineate KLC3 sequences involved in ODF binding; deletion mutants were constructed in the HR and C-terminal regions of KLC3 and analyzed in
the ODF binding assay. The results show that the 52-kDa KLC3 Western Blot Overlays Show Binding of KLC3 to a 27-kDa ODF
Protein--
ODFs contain several major proteins as well as a number
of other integral proteins. We have previously cloned and characterized two major ODF proteins, ODF1 (27 kDa) (39) and ODF2 (84 kDa) (29). To
identify which ODF protein(s), if any, bind to KLC3, we carried out
Western blot overlay assays using radiolabeled KLC3 as a probe;
extracts were prepared from purified spermatocytes and elongating
spermatids as well as from isolated purified ODFs and the FS. Note
that, whereas elongating spermatids produced ODF and FS proteins,
spermatocytes did not. Extracted proteins were separated by SDS-PAGE,
transferred to filters, and probed with 35S-radiolabeled
wild-type KLC3. We also carried out this assay using the
35S-radiolabeled KLC3 Specific KLC3-ODF1 Interaction Is Mediated by Leucine Zipper
Motifs--
ODF1 is a multifunctional protein that contains an
N-terminal leucine zipper motif that specifies binding to several ODF
and ODF-associated proteins (29-31) as well as a conserved CGP repeat in the C terminus that we recently showed binds to a novel RING finger
protein, OIP1 (40). To prove that ODF1 can bind KLC3 and to delineate
regions in ODF1 involved in this binding, we used two different assays:
the yeast two-hybrid system and a cell-free GST-ODF1 pull-down assay.
In addition, because the HR domain resembles a leucine zipper motif,
and because we had demonstrated that the major ODF and ODF-associated
proteins interact using leucine zipper motifs, one possibility was that
the KLC3 HR domain interacts with the ODF1 leucine zipper motif.
First, interaction experiments were
carried out in yeast using selection for growth in the absence of His
and activation of the
To confirm and extend the yeast results, we incubated different
GST-ODF1 fusion proteins (34) with 35S-radiolabeled KLC3.
Bound KLC3 was eluted and analyzed by SDS-PAGE and autoradiography, and
binding was quantitated. The results are shown in Fig.
10. Fig. 10A shows
schematically the GST-linked ODF1 fragments that were used in this
analysis as well as the approximate locations of the leucine zipper
motif and the CGP repeats. Fig. 10B shows a
Coomassie-stained gel for quantitation of the amount of GST fusion
proteins used in the binding reactions. Fig. 10C shows the
autoradiogram of bound KLC3. For quantitation of binding results, the
bands in Fig. 10C were normalized for input GST fusion
proteins (shown in Fig. 10B). These results show that KLC3
did not bind GST, as expected. KLC3 bound efficiently in vitro to the ODF1 N-terminal half, but not to the ODF1 C-terminal half or a deletion variant of the C-terminal half (GST-ODF1 Kinesins are heterotetrameric mechanoenzymes that consist of two
heavy chains and two light chains (4, 5). They are abundant and have
been detected in virtually all cell types. Kinesins bind to and move
along microtubules toward the (+)-end with few exceptions, such as the
KRP NCD (41). Most work has concentrated on the motor
domain-containing KHC proteins, which share motifs with the myosin head
(42). The globular motor domain is linked via First, as a component of kinesin, KLCs mediate the interaction between
microtubules, kinesin, and membrane surfaces, including vesicles (9),
microsomes (48), mitochondria (13), and the trans-Golgi
network (49). Several experiments support such a role. In
Drosophila KLC mutants, large aggregates ("organelle jams") accumulate in axons, resulting from a block in axonal
transport (9). Also, anti-KLC antibodies inhibit fast axonal transport without affecting microtubule binding or ATPase activity in
vitro and cause release of purified membrane vesicles from kinesin
(14). Rat KLC1b binds via its C terminus to mitochondria in
cultured CV1 cells and human skin fibroblasts (13). The divergent C
terminus of KLC has been proposed to function as an attachment site for organelles to be transported along microtubules. Second, KLC appears to
regulate kinesin activity. Upon binding of KHC to KLC, KHC is
released from microtubules and is kept in an inactive state by
interaction between the tail and motor domains of KHC. Verhey et
al. (11) hypothesized that cargo binding triggers a conformational change resulting in microtubule binding of the activated kinesin. As
described in the Introduction, several kinesins and KRPs have been
detected in sperm, including kinesin associated with the manchette, a
transient microtubule structure (50); KRPs (21, 22); kinesin II, a
KIF3-based motor molecule (27); and KAP3 (51). It is important to note
that kinesin II, which was found in the midpiece, consists of two
different KIF3 motor proteins linked to the dynactin component Glued
(52, 53) and lacks conventional KLC. From morphogenetic studies, it
appears that a flagellar transport mechanism must exist. First, during
post-meiotic differentiation of spermatids, a membrane-bound
constriction point forms called the annulus, which separates the narrow
and long periaxonemal compartment from the bulk of the cytoplasm
(i.e. cytoplasmic lobe). Proteins needed to form specialized
components of the forming sperm tail (e.g. the FS and ODFs)
must thus pass through the annulus. Second, the FS forms during
spermiogenesis in a distal-to-proximal orientation; thus, FS proteins
must be transported to the tip of the periaxonemal compartment to begin assembly. The axoneme, which represents the first tail structure to be
formed during spermiogenesis, links the periaxonemal compartment with
the cytoplasmic lobe through the annulus and thus likely provides the
molecular basis for microtubule-based flagellar transport.
KLC3 Protein Can Bind to ODFs--
We have reported that the
recently discovered kinesin light chain KLC3 can associate with ODFs,
one of two unique macromolecular structures in mammalian spermatozoa,
the other one being the FS. We discovered, in immunocytochemistry
assays using anti-KLC3 mAb B11, that KLC3 protein is present in
elongating spermatids from stage XIV onwards and appears to localize to
the sperm tail midpiece. Immunoelectron microscopy with the mAb showed
that, in mature sperm, KLC3 associates with the surface of ODFs and is
present in the space between adjacent ODFs and between mitochondria and ODFs, which are devoid of tubulins (38). KLC3 is not present in the
medulla of ODFs, indicating that KLC3 is not an integral ODF protein
like ODF1 (36, 54) and ODF2 (29, 55). Indeed, Western blot analysis of
highly purified ODFs failed to detect KLC3 (Fig. 6). The same
experiment indicated that KHC is also not an integral ODF protein, and
we also showed that KHC has low affinity (if any) for purified ODFs.
However, we do not know if KHC is present on ODFs in mature sperm
either as part of a kinesin or as an individual protein. Be that as it
may, the presence of KLC3 on ODFs occurs in the absence of
microtubules. The mAb failed to detect KLC3 label over the axoneme or
over the manchette, a microtubule-rich transient structure in
elongating spermatids that binds several microtubule-associated
proteins (30, 56). We conclude that the monoclonal KLC3 epitope
detected in association with the ODFs is not detectable in the
association of KLC3 with microtubules in spermatids. Together with our
observation that KLC3 can bind in vitro to ODFs in the
absence of microtubules, these results suggest the possibility that
KLC3 may be able to carry out a microtubule-independent role in
spermiogenesis (see below).
Leucine Zipper-like Repeats Mediate KLC3-ODF1 Binding--
We
investigated the mechanism that KLC3 employs to bind to ODFs. First, a
new ODF binding assay showed that the HR domain of KLC3 is involved in
the ODF association. This is interesting because, in kinesin-based
cargo transport, the KLC HR domain interacts with KHC, and cargo
associates with the variable KLC C terminus or part of the
tetratricopeptide repeats (13, 57). We found that deletion of the C
terminus has no effect on ODF binding. Our findings imply that ODFs
cannot be categorized as regular cargo for transport. Second, our
experiments uncovered that KLC3 binds directly to ODF1 and that it
employs its HR sequence in this interaction. Our data predicted that
the leucine zipper of ODF1 is involved in KLC3 binding. Indeed,
deletion of the ODF1 leucine zipper significantly reduced binding
(20-fold) to KLC3. We had previously determined that the ODF1 leucine
zipper is crucial for the association with ODF2 (29); SPAG4, a
spermatid-specific axoneme-binding protein (30); and SPAG5 (31, 58).
The specificity of these leucine zipper-mediated interactions is
underscored by our observation that KLC3 does not bind to ODF2 even
though ODF2 contains two functional leucine zipper motifs (29, 59).
Role for KLC3 in Organization of Structural Components of the Sperm
Tail--
Based on the observation that KLC3 is the only one of three
known light chains to be expressed in spermatids, we had suggested previously (20) that, in addition to its ability to carry cargo down
the sperm tail axoneme in the context of a kinesin-microtubule interaction, KLC3 must fulfill unique spermatid-specific roles. Indeed,
the mAb used in this study indicates that KLC3 localizes to ODFs,
suggestive of a spermatid-specific role. What is the nature of this
binding and what could such a role(s) be?
The results described here suggest the intriguing possibility that KLC3
proteins can bind to spermatid ODFs in a microtubule-independent manner. The data in support of this possibility are the following. (i)
The areas surrounding ODFs where KLC3 peptides are detected by mAbs in
mature sperm are devoid of microtubules; (ii) the ODF preparations used
in the in vitro binding assays do not contain
A role for KLC3·ODF complexes must take into account the development
of major elongating spermatid structures. One intriguing possible role
for KLC3·ODF complexes was suggested by the mAb immunocytochemistry
experiments. A redistribution of mitochondria from the periphery of
spermatids to the axonemal region just below the sperm head, the future
midpiece, occurs in step 15-16 spermatids, a developmental stage that
coincides with high level expression of KLC3 and a change in
localization of KLC3 from a general cytoplasmic one to one in the sperm
tail midpiece. In agreement, we observed KLC3 label between ODFs and
mitochondria. We found that KLC3 can bind to and cluster mitochondria
and that this activity depends on a region different from the HR (data
not shown). In the context of a possible role for KLC3 in mitochondrial
ODF interactions, the following interesting observation has been made.
hpy mice, which lack normal axonemal development
during spermiogenesis, contain immature spermatids that harbor
aggregates of mitochondria clustered around pieces of ODFs (60). This
demonstrates that normal axonemal morphogenesis is a prerequisite for
tail development, and it also indicates that, in the absence of the
axoneme, mitochondria can still redistribute to ODFs. We propose that
KLC3 might be involved in aspects of the mitochondrial redistribution.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside as described (29). Bacteria were lysed by sonication. After centrifugation, the MBP-KLC3 fusion protein was purified from the bacterial extract using
amylose-agarose columns and eluted using maltose. Eluted fusion protein
was analyzed by SDS-PAGE. For mAb production, 6-week-old BALB/c
mice were injected with 10 µg of MBP-KLC3 fusion protein. For
hybridoma production, SP2/MIL-6 myeloma cells (grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum) were
fused to spleen cells. mAb production was analyzed by
immunofluorescence using frozen rat testicular sections and Western
blot assays using NitroPlus membranes (Micron Separations Inc.,
Westboro, MA) onto which MBP-KLC3 had been transferred. Polyclonal
antibodies were produced as follows. New Zealand White rabbits were
injected with purified MBP-KLC3, initially with complete Freund's
adjuvant and subsequent injections with incomplete Freund's adjuvant.
Serum was collected and tested for KLC3 recognition by Western blot
analysis as described above. Affinity-purified polyclonal anti-KLC3
antibody was isolated as described (30) by incubation of total
antibodies with membrane-immobilized MBP-KLC3 protein, washing of
membrane strips, and elution of bound antibodies.
C) was created by PCR using
forward primer C1478 (5'-CGCTAAGTGGACTGGCTGCAG-3'), reverse primer
C1174 (5'-GCTGAGGATCTCCTTGTATAGCTCC-3'), and pBS(ATG)KLC3 (20) as a
template. The deletion mutant was cloned into the pGAD424 vector for
use in yeast. Wild-type KLC3- and KLC3
HR mutant-containing plasmids
(20) and all ODF1-containing plasmids (29, 34) have been described previously.
HR deletion mutant have been
described previously (20). To analyze binding of KLC3 protein to
purified ODFs, KLC3, KLC3
HR, KLC3
zip, or KHC was transcribed
in vitro and translated in the presence of
[35S]cysteine using the TNT reticulocyte
transcription and translation system (Promega). Radiolabeled proteins
were incubated with purified ODFs at 30 °C for 15 min. ODFs were
pelleted at 30,000 rpm for 15 min at 22 °C. Supernatants were saved
for SDS-PAGE analysis. Two subsequent washing/pelleting reactions were
performed. Aliquots of both supernatants and pellets were boiled in SDS
sample buffer and analyzed by electrophoresis on 10%
SDS-polyacrylamide gels, and the gels were dried and exposed to Biomax
film (Eastman Kodak Co.).
plates
were tested for
-galactosidase expression on membranes as described
previously (29).
-tubulin (Sigma),
KHC (Chemicon International, Inc.), and KLC3, followed by horseradish
peroxidase-coupled secondary antibodies. Blots were developed using
chemiluminescence (LumiGLO chemiluminescent substrate system;
Kirkegaard & Perrie Laboratories, Inc., Gaithersburg, MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Specificity of polyclonal and monoclonal
anti-KLC3 antibodies. Protein extracts prepared from elutriated
rat spermatids (lanes 1 and 4), epididymal sperm
(lanes 3 and 5), spermatocytes (lane
2), and liver (lane 6) were analyzed by Western blot
assays using affinity-purified polyclonal and monoclonal anti-KLC3
antibodies. Note that both antibodies recognized KLC3. Molecular mass
markers are indicated to the right.
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Fig. 2.
Immunocytochemistry using polyclonal
anti-KLC3 antibodies. Rat testicular sections were
prepared, and KLC3 was visualized using affinity-purified polyclonal
anti-KLC3 antibodies. Sections were counterstained with methylene blue.
The Roman numerals refer to the stage of the cycle of the
rat seminiferous epithelium of individual cross-sections. Note that
KLC3 localized to the cell body of round spermatids at early stages of
spermiogenesis and to the forming sperm tail at later stages.
Arrows indicate lightly stained tails, and
arrowheads indicate prominently stained tails. In some
stages, KLC3 was also expressed in Sertoli cells in a pattern of
streamers. Scale bars = 40 µm.
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Fig. 3.
Immunocytochemistry using monoclonal
anti-KLC3 antibodies. Rat testicular sections were analyzed as
described in the legend to Fig. 1, except using anti-KLC3 mAb B11.
Immunostaining was first observed in the cytoplasm of step 14 spermatids at stage XIV (A) and peaked in step 15-16
spermatids at stages I-III (B). KLC3 immunostaining
could be observed in the midpieces of step 18-19 spermatids in
cross-sections (C, inset) and in longitudinal
sections (D, inset). Scale bars = 40 µm.
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Fig. 4.
KLC3 expression during spermiogenesis.
Shown are schematic representations of timing (upper panel)
and pattern of KLC3 expression (lower panel) in spermatids
during spermiogenesis. Shown are spermatids at different developmental
stages ranging from steps 1 to 19 (mature); levels and localization of
KLC3 as detected by mAbs are indicated in shades of gray.
KLC3 is first detected in step 12 spermatids. KLC3 protein accumulates
in the midpiece at later stages. The midpiece (m) and head
(h) are indicated.
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Fig. 5.
Immunoelectron microscopic analysis of KLC3
distribution in sperm tails. Ultrastructural analysis of KLC3
localization in sperm tails was carried out using immunogold-labeled
anti-KLC3 mAb B11. A and B, cross-sections
through midpieces of mature sperm tails. Note that immunogold label was
associated with the surface of ODFs. C, longitudinal section
through the midpiece of a mature sperm tail. Label was often present
between the mitochondrial sheath and the ODFs (arrows point
to examples). smr, submitochondrial reticulum;
ax, axoneme; odf, outer dense fibers;
m, mitochondria. D, examples of gold label in
association with ODFs. Scale bars = 0.1 µm.
-tubulin and KHC, which would have
complicated the binding assay interpretations, and we tested them for
the presence of KLC3. Fig. 6 shows that
purified ODFs did not contain detectable
-tubulin or KHC, indicating
that the ODF preparations were not contaminated with axonemal
components. In addition, we did not detect KLC3 in purified ODF
preparations, indicating that, as suggested by the immunoelectron
microscopic data, KLC3 is an ODF-associated protein rather than an
integral ODF protein and is lost in the ODF isolation procedures.
Controls for all three proteins were positive. Brain expressed KHC;
microtubule preparations contained
-tubulin; and testis expressed
KLC3, as expected.
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Fig. 6.
Purified ODFs do not contain KHC or
-tubulin. To investigate the presence of KHC,
-tubulin, and KLC3 in purified ODF preparations, Western blot
analysis was done comparing ODFs with the indicated positive controls.
The antisera used in this analysis are indicated below the lanes and
include anti-KHC (
KHC), anti-
-tubulin
(
tub), anti-KLC3 (
KLC3), and, as positive
control for ODF, anti-ODF2 (
ODF2). odf,
purified ODFs; brain, total brain extract; brain
mt, purified brain microtubules; sperm, sperm tails.
Note that ODFs contained ODF2 as expected, but not any of the other
proteins analyzed.
HR mutant could not bind to ODFs (lane 6). Deletion of the
variable C-terminal sequence in the 49-kDa KLC3
C mutant had no
significant effect on ODF binding (compare lanes 3 and
7). These results indicate that the HR domain of KLC3 is
involved in ODF interaction. This is interesting in light of the fact
that the HR mediates KLC-KHC binding in the context of a kinesin
complex.
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Fig. 7.
KLC3 can bind in vitro to
isolated ODFs. Purified rat ODFs were incubated with in
vitro translated radiolabeled wild-type KLC3 (WT), the
KLC3 HR mutant (
HR), the KLC3
C mutant
(
C), and p21ras (RAS), which was
included as a nonspecific control for ODF binding. After repeated
washings and pelleting steps, the amount of bound KLC3 in the final
pellets was analyzed by SDS-PAGE and autoradiography. Lanes
1-4 (translations) show the input amounts of in
vitro translated radiolabeled KLC3 variants and p21ras in
the different binding reactions. Lanes 5-8 (ODF
pellets) show the results from analysis of radiolabeled proteins
in the washed ODF pellets. Radiolabeled KLC3 and KHC were mixed and
incubated with purified ODFs (T; lane 9). Bound
protein was analyzed (P; lane 10). Note that,
whereas wild-type KLC3 and KLC3
C bound efficiently to pure ODFs, a
mutation in the HR domain (KLC3
HR mutant) abolished binding.
HR deletion mutant as a control for
nonspecific binding because this mutant did not bind ODFs (see Fig. 7).
The results of the binding experiments are shown in Fig.
8 (A and B), and
the corresponding Coomassie stains of these gels are also shown (Fig.
8, C and D). The results show that wild-type KLC3 could bind to a 27-kDa ODF protein present in spermatids (Fig. 8A, lanes 1 and 2), but not to any
proteins from spermatocytes (lane 3). Deletion of the HR
domain abolished binding to this spermatid protein (Fig. 8B,
lanes 6 and 7). It is also shown that wild-type
KLC3 bound specifically to a 27-kDa protein in purified ODFs; the
detected band (Fig. 8A, lane 4) overlapped
exactly with the ODF1 protein (Fig. 8C, lane 4).
Together, these results strongly suggest the possibility that KLC3 can
bind to the major ODF protein ODF1 (see below). Fig. 8A also
shows that KLC3 did not bind to the 84-kDa ODF2 protein (lane
4) or to any FS proteins (lane 5). These data
suggest that ODF1 is a strong candidate for a KLC3-interacting ODF
protein and confirmed our observation that the HR domain is involved in
KLC3-ODF binding.
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Fig. 8.
Interaction of KLC3 with ODF protein. To
identify ODF proteins that could associate with KLC3, Western blot
overlay experiments were carried out using the indicated radiolabeled
proteins (wild-type (wt) KLC3 and KLC3 HR) as probes.
Extracts were prepared from purified early spermatids (es;
lanes 1, 2, 6, and 7),
purified pachytene spermatocytes (p; lanes 3 and
8), purified ODFs (odf; lanes 4 and
9), and purified FS (fs; lanes 5 and
10). Protein extracts were separated by SDS-PAGE,
transferred to filters, and incubated with the indicated probes. After
washes, filters were exposed. A and B show
autoradiograms of Western blot binding experiments, and C
and D show the corresponding Coomassie staining patterns.
Note that the 27-kDa ODF1 protein was present in purified ODF
preparations.
-galactosidase reporter gene as indicators of
protein interaction. The results of yeast two-hybrid assays are
shown in Fig. 9 and Table I. The results
obtained in the His
selection and LacZ activation
assays were identical. These data demonstrate that (i) KLC3 can bind to
ODF1, but not to ODF2, in agreement with the suggestions from the
Western blot overlay assays; (ii) KLC3 associates with the N-terminal
half of ODF1 (ODF1NT), but not with the C-terminal half (ODF1CT); and
(iii) deletion of the KLC3 HR domain abolishes binding to ODF1, whereas
deletion of the KLC3 C terminus does not affect binding. The results
also show that the first 100 amino acid residues of ODF1 (ODF1NT100) suffice for KLC3 interaction. In conclusion, the yeast data demonstrate that KLC3 and ODF1 can interact directly.
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Fig. 9.
ODF1 can specifically associate with KLC3 in
yeast. The binding of KLC3 to ODF1 was analyzed in yeast. KLC3
expression constructs were based on pGAD424, and ODF1 expression
constructs were based on pGBT9 as described previously (20, 29). Growth
in the absence of His was tested in yeast strain HF7c (upper
panels). In His growth assays, the plasmid
combinations indicated in the upper right panel were
introduced in yeast strain HF7c, and colonies containing both plasmids
were selected and grown as shown in the upper left panel.
Activation of the
-galactosidase reporter gene was tested in yeast
strain SFY256 (lower panels). For each combination, four
yeast SFY256 colonies containing the indicated DNAs were plated as
small horizontal streaks, grown, and transferred to filters before
carrying out the LacZ assay. ODF1NT and ODF1CT contain the N-
and C-terminal halves of ODF1, respectively. wtKLC3,
wild-type KLC3.
KLC3 binding to ODF1 in yeast
CT) that
removes most of the CGP repeats, in agreement with the results in
yeast. Importantly, deletion of the ODF1 leucine zipper
(GST-ODF1NT-(25-147)) significantly decreased KLC3 binding to 5% of
wild-type KLC3 binding. The first 100 amino acid residues in the ODF1
N-terminal half (GST-ODF1
NT) bound KLC3 with the same efficiency as
wild-type KLC3. Together, the binding experiments positively identify
ODF1 as the ODF protein that binds KLC3. Moreover, we have shown that this interaction is mediated by the ODF1 leucine zipper motif.
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Fig. 10.
The ODF1 leucine zipper is involved in KLC3
binding. To analyze ODF1 sequences that mediate binding to the HR
domain of KLC3, GST-ODF1 fusion protein pull-down assays were carried
out. A shows the different ODF1 fragments that were linked
to GST and tested for their ability to bind to KLC3. These fusion
proteins were expressed in bacteria, purified, and incubated with
in vitro translated radiolabeled KLC3. B shows
the fusion proteins used in these experiments after separation by
SDS-PAGE and staining with Coomassie. C shows the
corresponding autoradiogram. The bands in C were quantitated
and normalized for the different amounts of GST-ODF1 proteins used in
the experiments (see B), resulting in the relative binding
numbers shown below the autoradiogram (binding to ODF1NT was set
arbitrarily at 100%).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets to a
coiled-coil tail domain responsible for dimerization. The C terminus of
KHC interacts with the HR domain in the N terminus of KLC (43). Because
kinesins and KRPs bind microtubules, it was proposed and later
demonstrated that they function in movement of organelles associated
with axons, the axoneme, and mitotic spindles (44-46). Kinesins and
KRPs are also involved in spindle assembly and maintenance, attachment
of microtubules to chromosomes, and chromosome movement (47). In
contrast to KHC and KRPs, little is known about the interacting partner
of conventional kinesins, KLC. Two roles have been postulated for
KLCs.
-tubulin;
(iii) KLC3 can bind directly to ODF1 in yeast; and (iv) ODF1 and
purified ODFs bind to the HR motif in KLC3, a motif normally used for
binding KHC to form kinesins, not for interacting with cargo. Occupancy
of the KLC3 HR motif by ODF1 will likely exclude KHC from binding to
the same sequence. Indeed, preliminary experiments failed to detect a
KLC3 complex containing both ODF1 and KHC (data not shown). Based on
these and previous results, we propose that, in spermatids, distinct
KLC3·KHC kinesin complexes as well as KLC3·ODF1 complexes exist.
The former likely act in paradigm kinesin-mediated cargo transport,
whereas the latter may carry out a spermatid-specific function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. X. Shao for expert advice on the yeast two-hybrid system and H. Orchard for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Canadian Institutes of Health Research (to R. O. and F. A. v. d. H.) and from the Natural Sciences and Engineering Council of Canada (to R. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported in part by a studentship from the Alberta Cancer Foundation.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Calgary, 330 Hospital Dr. N. W., Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3323; Fax: 403-283-8727; E-mail: fvdhoorn@ucalgary.ca.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M213126200
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ABBREVIATIONS |
---|
The abbreviations used are: KLCs, kinesin light chains; KHCs, kinesin heavy chains; HR, heptad repeat; KRPs, kinesin-related proteins; ODFs, outer dense fibers; FS, fibrous sheath; mAbs, monoclonal antibodies; MBP, maltose-binding protein; TBS, Tris-HCl-buffered saline; GST, glutathione S-transferase.
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