At low levels of overexpression, CLIP-170, when fused to GFP, was shown to associate specifically with the ends of growing MTs (Perez et al. 1999). This behavior, which is visualized as movement of “comet-like” dashes, has now been documented for a variety of other proteins; it has been termed “plus-end tracking,” and the proteins that show it are called “+TIPs” (Schuyler and Pellman 2001). Different mechanisms account for plus-end tracking (for recent review, see Akhmanova and Hoogenraad 2005). In mammalian cells, CLIP-170 displays “treadmilling”; that is, it associates with the freshly polymerized distal end of a growing MT and dissociates from the “older” MT lattice, while it remains immobile with respect to the MT (Perez et al. 1999). However, the CLIP-170 homologs in budding and fission yeast, called Bik1p and Tip1p, respectively, are delivered to MT ends by kinesin motors (Busch et al. 2004; Carvalho et al. 2004).

The presence of CLIP-170 at the tips of growing MTs has focused attention toward the role of this protein in the regulation of MT dynamics. Budding yeast Bik1p has a MT-stabilizing function (Carvalho et al. 2004), and fission yeast Tip1p acts as anti-catastrophe factor, preventing the premature conversion of MT growth to shrinkage in regions other than the cell ends (Brunner and Nurse 2000). Using a dominant-negative approach, it was shown that in CHO cells CLIP-115 and CLIP-170 have redundant roles as MT rescue factors, that is, they prevent MTs that have undergone catastrophe at the cell periphery from shrinking back to the centrosome (Komarova et al. 2002). This function is supported by in vitro studies, in which the dimeric head domain of CLIP-170 promotes MT rescues and induces the formation of curved oligomers of purified tubulin (Arnal et al. 2004). Together these data underscore the role of CLIPs as “positive” regulators of MT growth.

CLIP-170 interacts directly with the CLASPs, which are also +TIPs (Akhmanova et al. 2001), and with IQGAP1, a Rac1/Cdc42-binding protein (Fukata et al. 2002). These interactions may play a role in regulating MT dynamics in specific cell areas, such as the leading edge of motile fibroblasts. The Drosophila melanogaster homolog of CLIP-170, D-CLIP-190 (Lantz and Miller 1998), was shown to associate with myosin VI, indicating that D-CLIP-190 might be involved in linking the actin and MT networks. CLIP-170 is also involved in the dynein-dynactin pathway: It binds to LIS1, which is a regulator of dynein function (Coquelle et al. 2002; Tai et al. 2002). In addition, it interacts directly with the dynactin component p150^Glued and contributes to targeting of the dynactin complex to the growing MT ends (Goodson et al. 2003; Lansbergen et al. 2004). In mitotic cells, CLIP-170 was shown to accumulate at prometaphase kinetochores, where it colocalizes with dynein, dynactin, and LIS1 (Dujardin et al. 1998; Coquelle et al. 2002; Tai et al. 2002), suggesting a function for this protein in mitosis as well as in interphase. Consistent with this distribution, a recent study using RNA interference (RNAi) revealed an essential role for CLIP-170 during mitosis in HEp-2 cells (Wieland et al. 2004).

While these studies have provided clues about cellular functions of CLIP-170, the role of this protein in the whole animal has never been addressed. In order to investigate the in vivo function of murine CLIP-170, we generated CLIP-170 knockout (KO) and GFP-CLIP-170 knock-in (KI) mice. CLIP-170 KO mice are viable, yet male KO mice produce abnormal sperm, which lack a typical, hook-shaped head morphology. We therefore focused on the role of CLIP-170 in spermatogenesis and discovered a novel, testis-specific isoform of CLIP-170. Antibody staining reveals a differentiation-stage-dependent distribution of CLIP-170 in germ cells of the testis. Using GFP-CLIP-170 KI mice, we performed live analysis in testis tubules. Dynamic, plus-end-tracking GFP-CLIP-170 is detected in spermatogonia. During spermiogenesis, GFP-CLIP-170 associates with centrosomes, spermatid manchettes, and the manchette rings. Bleaching studies reveal that at these later stages, GFP-CLIP-170 is mainly detected in immobile structures. Thus, CLIP-170 can act both as a +TIP and as a structural protein and is essential for normal spermatogenesis.

Figure 1. Genomic organization and expression of the gene encoding CLIP-170. (A) Genomic organization of the gene encoding CLIP-170. The position of common (1-24) and alternatively spliced (+11, +35, LORF) exons and transcription initiation sites (1, alt1, te) (more ...)

A short, testis-specific CLIP-170 cDNA has been described, which encodes a C-terminal isoform of CLIP-170, called CLIP-50 (Tarsounas et al. 2001). The 5′-end of this cDNA is located upstream of exon 16, and it is collinear with CLIP-170 clone BC007191 from exon 16 to exon 24. The 5′-end of another mouse testis cDNA (accession no. BB616336) is located ~14 kb downstream of exon 1. These results suggest that in the testis, multiple transcription initiation sites exist in the CLIP-170 gene.

Surprisingly, we found a sequence of >2 kb between exons 15 and 16 (Fig. 1A) that is highly conserved between man and mouse and is surrounded by consensus splice acceptor and donor sites. When translated, this domain, which has a high potential to form coiled-coils, does not interrupt the CLIP-170 coding sequence. We called this putative novel exon LORF (long open reading frame). By using RT-PCR, we confirmed that the LORF exon is abundantly expressed in the testis, but it is also present in other tissues (data not shown).

Northern blot analysis in various tissues from mouse, rat, and human revealed ubiquitous CLIP-170 expression, with varying mRNA levels (Fig. 1B-E). In all three organisms, CLIP-170 transcripts in brain are somewhat shorter than the common mRNAs in other tissues, consistent with the smaller brain-specific splice variant (Akhmanova et al. 2001). In rat and mouse testis, multiple mRNAs are present, ranging in size from ~2 to ~7 kb (Fig. 1B,D,E). The shortest testis transcript likely corresponds to the CLIP-50 mRNA (Tarsounas et al. 2001), since it does not hybridize to the 5′ part of CLIP-170 cDNA (Fig. 1E, upper panel). Hybridization of the mouse Northern blot with a LORF-specific probe revealed that the longest testis transcript contains the LORF exon (Fig. 1E, lower panel). Taken together, these data suggest that several CLIP-170 isoforms are produced from the Rsn gene. Most notably, inclusion of the LORF exon extends the CLIP-170 coding sequence with ~87 kDa.

Figure 2. Generation of CLIP-170 KO and GFP-CLIP-170 KI in alleles. (A) Targeting strategy of CLIP-170 KO and GFP-CLIP-170 KI alleles. A segment of the murine Rsn gene is shown, with the ATG-containing exon and diagnostic restriction enzyme sites. The 5′-end (more ...)

Targeting efficiency in ES cells with the GFP-loxP-pMC1neo-loxP cassette and 5.5 kb of homologous upstream and downstream sequences was 2%. Targeted ES cells were transfected with a plasmid expressing Cre recombinase, to delete pMC1neo. One targeted ES cell clone, as well as one clone with correctly excised PMC1neo, were injected into blastocysts to allow germline transmission of the mutated alleles. Correct integration and excision were verified by Southern blotting and PCR analysis, using upstream and downstream probes (Fig. 2B; data not shown).

We bred the modified CLIP-170 alleles back to the C57BL/6 background and generated heterozygous and homozygous CLIP-170 KO and KI mice. Northern blot analysis showed that the insertion of the GFP-loxP-pMC1neo-loxP cassette results in absence of normal CLIP-170 mRNA in several KO tissues (Supplementary Fig. 1A,B; data not shown). These results were confirmed by RT-PCR (Supplementary Fig. 1C,D). Consistent with these data, we did not detect CLIP-170 by Western blot in brain lysates from homozygous CLIP-170 KO mice (Fig. 2C; Supplementary Fig. 1E).

Northern blot analysis further showed that expression of the testis-specific CLIP-50 transcript was not affected by the targeting procedure (Supplementary Fig. 1B). Western blot analysis confirmed that CLIP-50 is, indeed, expressed in testis extracts of wild-type, CLIP-170 KO, and CLIP-170 KI mice (Supplementary Fig. 1E). Thus, the targeting strategy does not perturb CLIP-50. In addition, whereas in most KO tissues, including brain, heart, skeletal muscle, mouse embryonic fibroblasts (MEFs), and testis, no CLIP-170 could be detected by Western blot (Fig. 2C-F; Supplementary Fig. 1E,F), in lung and in embryonic extracts residual CLIP-170 was observed with two different CLIP-170-specific antibodies (Fig. 2D; data not shown). We confirmed the existence of a CLIP-170 mRNA with a correctly sized 5′-end in KO lung using RT-PCR (Supplementary Fig. 1D). Therefore, insertion of the GFP-loxP-pMC1neo-loxP cassette into the CLIP-170 gene does not result in ablation of protein expression in all tissues. We conclude that the CLIP-170 KO allele is a hypomorphic one.

In the GFP-CLIP-170 KI mice, a protein of ~200 kDa was produced instead of CLIP-170, which was present at similar levels compared with wild-type protein (Fig. 2C,D; Supplementary Fig. 1E,F). Equal expression levels of CLIP-170 and GFP-CLIP-170 are particularly apparent in heterozygous samples. CLIP-115 levels are not altered in brain lysates from CLIP-170 KO or GFP-CLIP-170 KI mice (Fig. 2C, left panel). Thus, reduction in the level of CLIP-170 does not lead to increased CLIP-115 expression. Conversely, in CLIP-115 KO mice, CLIP-170 is not up-regulated (Hoogenraad et al. 2002).

To characterize the novel ~240-kDa LORF-containing isoform of CLIP-170, we raised rabbit polyclonal antibodies (LORF#00 and #01) against a bacterially produced “LORF” protein. As shown in Figure 2E, the LORF antibodies specifically recognize ~240-kDa CLIP-170 and ~270-kDa GFP-CLIP-170 isoforms in testis extracts from wild-type and CLIP-170 KI mice, respectively. These data demonstrate that the LORF exon is translated in the testis. The LORF-CLIP-170-specific signal increases upon maturation of the testis, in particular from week 3 to 4 of post-natal life (Fig. 2F). In this developmental window, the testis becomes populated with spermatids, indicating that this CLIP-170 isoform has a function in spermiogenesis rather than (pre-)meiotic stages of spermatogenesis.

Figure 3. Cellular analysis of the CLIP-170 deletion and GFP-CLIP-170 insertion. (A-F) CLIP-115 and dynactin distribution in wild-type (A-C) and CLIP-170 KO MEFs (D-F). CLIP-115 (green) and dynactin (red) were detected with antibodies #2238 and antibodies against (more ...)

We next examined the distribution and behavior of GFP-CLIP-170 in homozygous KI MEFs. We first performed an immunoprecipitation with anti-GFP antiserum to pull down GFP-CLIP-170 and associated proteins from cultured MEFs. Results of this experiment (presented in Supplementary Fig. 1G) showed that no other GFP fusion protein is detected but full-length GFP-CLIP-170 and that this protein interacts with CLASP2 and p150^Glued, as was previously shown for CLIP-170 (Akhmanova et al. 2001; Lansbergen et al. 2004). In fixed cells GFP-positive dashes are localized at the distal ends of MTs and some staining is observed in the cytoplasm (Fig. 3G-I), in line with published data (Rickard and Kreis 1990; Perez et al. 1999). The GFP-positive signals overlapped with anti-CLIP-170 antibody patterns (Supplementary Fig. 1H-J). We derived keratinocytes from homozygous CLIP-170 KO mice and transfected these with Cre recombinase to generate a mixed culture of GFP-CLIP-170 KI and CLIP-170 KO cells. Only in the KI keratinocytes did we detect significant levels of p150^Glued at MT tips (Fig. 3J,K), indicating that GFP-CLIP-170 is able to direct the localization of the dynactin complex to MT plus-ends.

The behavior of GFP-CLIP-170 was further analyzed in living homozygous KI MEFs and glial cells. We observed comet-like dashes in these cell types (Fig. 3L; Supplementary Movie 1), which moved with an average velocity of 0.57 ± 0.11 μm/sec in glia (60 dashes analyzed in 10 cells) or 0.50 ± 0.08 μm/sec in MEFs (25 dashes analyzed in three cells; in both cases the standard deviation is indicated). This velocity is similar to the values obtained by us in cells overexpressing low levels of various +TIPs (Akhmanova et al. 2001; Stepanova et al. 2003). Treatment of cells with low doses of nocodazole and taxol immediately abolished the dynamic dashes in GFP-CLIP-170 KI cells (data not shown). Taken together, these data suggest that GFP-CLIP-170, expressed from the modified Rsn allele, recapitulates the behavior and functionality of CLIP-170 in cultured cells and can be used as a tool to analyze the distribution and dynamics of CLIP-170 in vivo. This conclusion is in line with previous observations, that an overexpressed GFP-CLIP-170 fusion protein is functional in cultured cells (Perez et al. 1999; Komarova et al. 2002; Stepanova et al. 2003).

Comparison of hematoxylin/eosin (H/E)-stained testis sections of CLIP-170 KO, KI, and wild-type mice showed no obvious morphological abnormalities in the size or organization of the testis tubules or the interstitium (Fig. 4A-C). Detailed examination of these sections, however, indicated that KO mice produce abnormal sperm. Analysis of sperm from the epididymis demonstrated that the CLIP-170 KO mice produce normal sperm numbers and that spermatozoa are motile, but that all sperm have an abnormal morphology; 100% display severe head abnormalities, whereas 20% of these show combined head and tail defects (Table 1, two mice were analyzed per genotype; see also Fig. 4D-F). The main characteristic of the abnormal head is a lack of the typical hook-shaped appearance (Fig. 4E), suggesting a defect in nuclear shaping (Russell et al. 1991). This may explain the fertility problems of male homozygous CLIP-170 KO mice. The sperm morphology was completely normal in GFP-CLIP-170 KI animals (Fig. 4F), indicating that GFP-CLIP-170 can substitute for the function of endogenous CLIP-170 in vivo.

Figure 4. Testicular analysis of the CLIP-170 deletion and GFP-CLIP-170 insertion. (A-C) H/E-stained 7-μm sections of the testes from wild-type, CLIP-170 KO, and GFP-CLIP-170 KI mice. Bar, 50 μm. (D-F) Sperm preparations, isolated from the epididymis (more ...)

Table 1. Reproductive organ weights and sperm counts of wild-type, CLIP-170 KO, and GFP-CLIP-170 KI mice

Anti-LORF antibodies produced no specific staining in CLIP-170 KO sections (data not shown), while they reacted strongly with the elongating spermatids of the wild-type and GFP-CLIP-170 KI mice (Fig. 4O-R; data not shown). However, these antibodies did not stain spermatogonia well, which was particularly obvious when anti-GFP and anti-LORF signals were compared in the same sections of GFP-CLIP-170 KI testes (Fig. 4O-Q). Thus, the longest isoform of CLIP-170 preferentially localizes to post-meiotic germ cells, consistent with the Western blot results of Figure 2F. Taken together, these data suggest that CLIP-170 is an important component of male germ cells, with a prominent localization in spermatids.

Figure 5. CLIP-170 and GFP-CLIP-170 distribution in wild-type and KI testis. (A,B) Unstained cryosections from GFP-CLIP-170 KI mice. Bars: A, 40 μm; B, 8 μm. (C-K) Localization of GFP-CLIP-170 to the centrosomes and the spermatid manchette. Testis (more ...)

In normal mice, the manchette begins to form in step 8 spermatids, when the shape of the nucleus changes from spherical to slightly elongated. In step 9 the nucleus becomes even more elongated, and the manchette runs parallel to the nuclear envelope. Observations by fluorescent microscopy revealed that in CLIP-170 KO mice, developing manchettes with EB3-positive rings are present in step 8-9 spermatids (Fig. 6A-F). At later stages (steps 11-13), when manchettes undergo a transition from a relatively symmetric cylindrical MT array to an asymmetric configuration (Fig. 5Q), striking abnormalities were observed in the distribution of tubulin, EB3 (Fig. 6G-I; see also Supplementary Movie 3, which shows a 3D representation), and dynactin (Fig. 6J,K), which were often present only on one side of the nucleus. This correlated with defects in the morphology of the nuclei that failed to acquire their normal slender shape with a curved rostral end, but instead had an irregular, often club-like appearance (Fig. 6H,K).

Figure 6. Immunofluorescent analysis of CLIP-170 KO testis. (A-I) Paraffin-embedded testis sections from CLIP-170 KO mice were incubated with antibodies against β-tubulin (red) (A-C), or β-tubulin (red) and EB3 (green) (D-I) and counterstained with (more ...)

Electron microscopy (EM) analysis showed that in wild-type step 8 spermatids, the onset of manchette formation was clearly demarcated by a narrow belt of MTs that covered approximately half of the nucleus, with the acrosome spanning the other half (Fig. 7A). In CLIP-170 KO mice, abnormal manchette features could be observed already at this stage: The belt of MTs did not span the nucleus properly, and MTs were present in aberrant areas such as within the acrosome and the nucleus (Fig. 7B). Steps 9-13 in KO mice presented absent or greatly reduced manchette MTs along the nuclear envelope, ectopic MTs along regions of the nucleus that normally do not display manchettes, or MTs at one side of the nucleus only (Supplementary Fig. 3A; data not shown). In addition, manchette MTs from KO mice were frequently localized in membrane-bound regions that seemed to lie within spermatid nuclei (see arrows in Fig. 7B,D; Supplementary Fig. 3B). From step 11 onward, MTs were organized in a regular fashion in elongating spermatids from wild-type and GFP-CLIP-170 KI mice (Fig. 7C,E; Supplementary Fig. 3C), but they were severely disorganized in sections from KO mice (Fig. 7D,F). This disturbed manchette formation seemed to influence the nuclear shaping, resulting in a more rounded nucleus than in wild-type animals (Fig. 7, cf. C and D; Supplementary Fig. 3B). In addition, MTs in CLIP-170 KO mice were often aggregated in large cytoplasmic clusters (Supplementary Fig. 3D). Finally, in step 16 spermatids, the final stage before sperm release from Sertoli cells, the nuclei in the CLIP-170 KO testis were highly irregular in shape (Supplementary Fig. 3F).

Figure 7. Ultrastructural morphology of the testis of wild-type and CLIP-170 KO mice. (A) Step 8 spermatid from a wild-type mouse with manchette MTs (indicated by arrowheads) located in close apposition to the nucleus in the area not occupied by the acrosome (indicated (more ...)

Previous EM studies demonstrated that manchette MTs are connected by cross-bridges (Wolosewick and Bryan 1977; see also Fig. 7E; Supplementary Fig. 3C). We measured the distance between MTs at sites of cross-bridges in transverse sections from wild-type, KO, and KI mice (Table 2). We restricted our analysis to regions where regularly spaced MTs could be detected. Since MT organization is severely affected in CLIP-170 KO spermatids (Fig. 7), such sites are rarely seen, compared with wild-type or GFP-CLIP-170 KI sections. The data in Table 2 show that average cross-bridge size is very similar in wild-type and CLIP-170 KO spermatids; this distance correlates well with previous results (Wolosewick and Bryan 1977). Surprisingly, cross-bridges in GFP-CLIP-170 KI spermatids are slightly smaller, the size difference being highly significant (P < 0.005; ANOVA, single-factor analysis). We also counted more cross-bridges per MT in cross-sections in the GFP-CLIP-170 KI spermatids, than in wild-type and KO mice (Table 2). Together, these results indicate that CLIP-170 is not an essential cross-bridge component; however, given the disorganized manchettes in the CLIP-170 KO and the smaller size and increased number of cross-bridges in GFP-CLIP-170 KI mice, we propose that CLIP-170 influences the formation of cross-bridges between manchette MTs.

Table 2. Size and number of MT cross-bridges in spermatid manchettes

The distribution of GFP-CLIP-170 was examined by scanning tubules longitudinally and acquiring optical sections through the tubule. Bright signals were observed in the cytoplasm of cells that formed syncytia close to the basal lamina. The number of positive cells within a syncytium differed (e.g., Fig. 8, cf. A and B). The GFP-CLIP-170-positive cells in Figure 8A and B (which are still images of Supplementary Movies 4, 5) have oval nuclei with a diameter of ~7 μm (18 nuclei counted). In combination with Hoechst nuclear staining and numbers of cells in syncytia, we identified these cells as type B spermatogonia and pre-leptotene primary spermatocytes (Vigodner et al. 2002). In addition, we detected strong GFP signals closer to the lumen of the tubule, in elongating spermatids (Fig. 8C,D; Supplementary Movies 6, 7). We did not observe any GFP-CLIP-170 in Sertoli cells.

Figure 8. Behavior of GFP-CLIP-170 in live testis tubules. Testis tubules were dissected from GFP-CLIP-170 KI mice (in some cases treated with Hoechst) and analyzed with a confocal/multiphoton microscope. (A) Low-magnification view of GFP-CLIP-170 distribution. (more ...)

Cells that expressed somewhat lower levels of GFP-CLIP-170 were located very near to the basal lamina. Nuclear sizes in these cells ranged from 9 × 10 μm to 12 × 15 μm. Based on these observations and Hoechst staining, these cells were identified as undifferentiated (e.g., Fig. 8F) and differentiated (e.g., Fig. 8E) type A spermatogonia (see also Supplementary Movies 8, 9). Starting at undifferentiated A-paired spermatogonia (Fig. 8F), the male germline cells form syncytia, connected by intercellular cytoplasmic bridges. GFP-CLIP-170 nicely visualizes these bridges.

All signals were specific and not due to autofluorescence, since imaging of wild-type testis tubules with the same settings revealed no green fluorescent signal (data not shown). Thus, GFP-CLIP-170 is expressed in a highly specific pattern in germ cells at different steps of spermatogenesis.

We used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) to further examine the dynamic properties of GFP-CLIP-170. In cells with predominantly plus-end-tracking GFP-CLIP-170, a fast recovery of fluorescence in the bleached area was observed, which was complete after ~1 min (Fig. 8F,H; Supplementary Fig. 4A,B). Fluorescence recovery was accompanied by fluorescence loss in nonbleached areas of the same cell. When cells with dynamic GFP-CLIP-170 were completely bleached, very slow fluorescence recovery was observed (data not shown), indicating that GFP-CLIP-170 can pass through intercellular bridges from one cell of the syncytium to another.

In pre-leptotene spermatocytes with high GFP-CLIP-170 signal, FRAP-FLIP analysis revealed recovery of fluorescent signal in the bleached area in ~5 min, which was accompanied by loss in fluorescent signal in nonbleached regions of the same cell (data not shown). In contrast, analysis in elongating spermatids showed relatively immobile GFP-CLIP-170 during the course of the experiment (Fig. 8G,I; Supplementary Fig. 4C-F; Supplementary Movies 10, 11). These data indicate that GFP-CLIP-170 becomes immobile as male germ cells differentiate.

CLIP-170 is the founding member of the +TIP group of MT-associated factors (Perez et al. 1999). Since nothing is known about the in vivo function of mammalian CLIP-170, we wished to address this using gene-targeting technology. The documentation of dominant-negative properties of a C-terminal CLIP-170 mutant (Komarova et al. 2002) and the finding of CLIP-50 (Tarsounas et al. 2001) suggest that complete inactivation of CLIP-170 function can only be achieved by deleting the whole gene. However, since the Rsn gene spans ~100 kb, deletion carries the risk of removing (regulatory regions of) other genes. We therefore devised a strategy, whereby we first generated a CLIP-170 KO allele by inserting a GFP-loxP-neo-loxP cassette in front of the first ATG codon. Subsequently, Cre-mediated excision of the neo-gene (via the loxP sites) generated the GFP-CLIP-170 KI allele.

The CLIP-170 KO allele turned out to be leaky in the adult lung and the embryo. It is possible that residual amounts of CLIP-170 are sufficient to carry KO mice through life. However, we emphasize that in the majority of the tissues investigated, including MEFs, we did not detect any CLIP-170, although there could exist small quantities of CLIP-170 that are below our levels of detection. Consistent with a mild phenotype in mice, cultured CLIP-170 KO MEFs do not show any obvious abnormalities under normal culture conditions. This is in line with RNAi studies that we performed in HeLa cells (Lansbergen et al. 2004). CLIP-115 might take over part of the function of CLIP-170 in cultured fibroblasts, since this protein can act as a MT rescue factor in CHO cells (Komarova et al. 2002). Our results in CLIP-170 KO MEFs do not concur with the mitotic phenotype of CLIP-170 knock-down reported in HEp-2 cells (Wieland et al. 2004). One explanation, which may account for these apparently contradictory results, is that redundant pathways normally exist in mammalian mitosis, one of which requires CLIP-170. In HEp-2 cells, only this pathway would be operative and hence, knock-down of CLIP-170 causes a severe defect. In cultured CLIP-170 KO MEFs (and perhaps in mice), redundant pathway(s) would still be functional and guide cells through mitosis. Interestingly, treatment of cultured ovarian cells with antisense oligonucleotides against restin revealed differential effects on cell proliferation in different cell types (Ho et al. 2003). More studies are required to address this issue.

The fact that male CLIP-170 KO mice are subfertile focused our attention on CLIP-170 function in male germ-cell development. We show that CLIP-170 is expressed in two waves during spermatogenesis, the first beginning in undifferentiated spermatogonia and lasting until the primary spermatocyte stage. Future studies will address possible role(s) of CLIP-170 at this stage, for example, in the maintenance of large syncytia capable of migration through the blood-testis barrier. The second wave of CLIP-170 expression occurs in spermiogenesis. We detected CLIP-170 very conspicuously on the spermatid manchette and the manchette ring. Consistent with this distribution, absence of CLIP-170 severely affects formation and/or maintenance of the spermatid manchette in KO mice.

The manchette consists of regularly spaced MTs that are, on one side, embedded in the manchette ring (Russell et al. 1991). It is likely that the MT-binding properties of CLIP-170 are relevant for its function in shaping the manchette. On the other hand, CLIP-170 interacts with the dynactin complex through p150^Glued (Lansbergen et al. 2004). Arp1, which is another dynactin component, was shown to be associated with the fuzzy material that links manchette MTs to the nuclear envelope (Fouquet et al. 2000). A similar localization has been described for dynein (Yoshida et al. 1994). In CLIP-170 KO mice, dynactin distribution is perturbed. In EM micrographs from CLIP-170 KO mice, where we observed manchette MTs close to the nucleus, these MTs appeared to be linked to the nuclear envelope and the fuzzy material was present. Still, it could be that lack of CLIP-170 mislocalizes the dynein-dynactin complex and that this is an underlying cause for the dissociation of manchette MTs from the nucleus.

Nuclear transformation into the characteristic hook-like shape depends on a functional manchette (Russell et al. 1991), and manchette abnormalities, such as those observed in the azh (abnormal spermatozoon head shape) mutant, correlate with sperm head deformities (Hugenholtz et al. 1984). The results in CLIP-170 KO mice, which produce sperm with abnormally shaped heads, confirm this conclusion. Interestingly, the azh mutation is due to a deletion in the Hook1 gene, which encodes a protein resembling CLIP-170: Hook1 is a dimer with an N-terminal MT-binding domain, which is separated by a coiled-coil region from an organelle interaction domain (Mendoza-Lujambio et al. 2002).

CLIP-50 is a short testis-enriched C-terminal isoform of CLIP-170 (Tarsounas et al. 2001). Antisera raised against this protein should recognize CLIP-170, LORF-CLIP-170, and CLIP-50 and cannot distinguish between these proteins in immunocytochemistry studies in wild-type mice (Tarsounas et al. 2001). We show that in our CLIP-170 KO mice, the expression of CLIP-50 is not affected. We observe weak staining with #2360 antibodies in spermatids of these mice, while spermatids of wild-type mice are brightly stained by the same antibodies. These results indicate that CLIP-50 is a minor CLIP-170 isoform localized to spermatid manchettes. The functional relevance of CLIP-50 remains unclear.

Present and previous (Perez et al. 1999) results strongly suggest that GFP-CLIP-170 and CLIP-170 have very similar properties. In KI mice, GFP-CLIP-170 is expressed at similar levels compared with CLIP-170 in all tissues examined. Although tagged with GFP, the same isoforms are detected in homozygous KI mice as in wild-type animals. In addition, GFP-CLIP-170 can substitute for CLIP-170 in cultured cells, and the GFP-CLIP-170 KI mice are fertile, their spermatozoa have a normal hook-shaped appearance, and, with the exception of cross-bridge size and number, spermatid manchettes appear completely normal. In sections from KI testis, we detected GFP-CLIP-170 with anti-CLIP-170 and anti-LORF antisera, and the distribution of p150^Glued and EB3 was normal. Taken together, these data strongly suggest that GFP-CLIP-170 can functionally replace CLIP-170 in vivo in the testis. Thus, the dynamic behavior of GFP-CLIP-170 in live testis tubules likely recapitulates that of CLIP-170.

Time-lapse imaging in testis tubules shows plus-end-tracking GFP-CLIP-170 in spermatogonia. Bleaching studies in spermatogonia show that GFP-CLIP-170 is mobile, in line with the dynamic behavior of the protein. As the spermatogonia differentiate, GFP-CLIP-170 signal becomes initially more intense, and a gradual loss of plus-end-tracking GFP-CLIP-170 is observed. Bleaching studies confirm that GFP-CLIP-170 becomes less mobile. However, it is not completely immobile, and the protein even appears to pass from one cell of the syncytium to another.

In elongating spermatids, CLIP-170 is localized to the centrosomes and manchettes. The presence of γ-tubulin at the spermatid centrosome (Fouquet et al. 1998) suggests that the minus-ends of MTs may be nucleated at this location. Consequently, MT plus-ends could be located near the manchette ring, consistent with a prominent localization of +TIPs (CLIP-170, EB3, and dynactin) at this site. However, the manchette appears to be a stable structure; it lasts several days and is made of stabilized MTs, which contain detyrosinated α-tubulin (Fouquet et al. 1997) and which are interconnected by cross-bridges. In agreement with manchette stability, both the time-lapse imaging and bleaching studies demonstrate that GFP-CLIP-170 is largely immobile in spermatids. It is therefore possible that CLIP-170 is involved in stabilization of manchette MTs.

Interestingly, high levels of overexpressed CLIP-170 induce the formation of stable MT bundles in cultured cells, with MT plus-ends assembled as patches in the cell periphery (e.g., Pierre et al. 1994). This was considered to be an artifact of overexpression, but it is noteworthy that relatively high levels of GFP-CLIP-170 in testis correlate with immobile protein and the presence of stable MTs. A structural, rather than a dynamic, function of CLIP-170 in spermatids correlates with the expression of a longer isoform, strongly extended in the coiled-coil region by inclusion of the LORF exon. LORF-encoded sequences could allow new protein-protein interactions of CLIP-170. Alternatively, LORF could alter the folding properties of CLIP-170, thereby generating a protein with different MT-binding and/or other protein-binding characteristics.

Although manchettes are stable structures, injection of the MT-stabilizing agent taxol in rat testis disorganizes the MTs of the manchette (they are occasionally even enveloped by the nucleus) (Russell et al. 1991). Therefore, proper control of MT dynamics seems essential for normal manchette formation and sperm head shaping. Our data emphasize a role for CLIP-170 in formation and/or maintenance of the spermatid manchette. This function may be more static and structural than would be expected on the basis of studies of the dynamics of CLIP-170 in cultured somatic cells. This might reflect an exceptional organization of MT dynamics during spermatogenesis, which, with the use of GFP-CLIP-170, has been visualized for the first time.

Antibodies against the CLIP-170 LORF exon (nucleotides 122672290-122670023 on mouse chromosome 5, version NCBI m32) were prepared as described (Hoogenraad et al. 2000), using a GST-fusion protein approach. We also used rabbit polyclonal antibodies #2360 against CLIP-170 (Coquelle et al. 2002), #2221 against CLIP-115 and CLIP-170, #2238 against CLIP-115 (Hoogenraad et al. 2000), #02-1005-07 against EB3 (Stepanova et al. 2003), and anti-GFP antibodies (Abcam). Mouse monoclonal antibodies were against β-tubulin (Sigma), p150^Glued and p50 (BD Biosciences), actin (Chemicon), and GFP (Santa Cruz, Roche). Secondary antisera were alkaline phosphatase-labeled anti-rabbit and anti-mouse antibodies (Sigma), FITC-labeled goat anti-rabbit antibody (Nordic Laboratories), and Alexa 594-labeled anti-mouse antibody (Molecular Probes).

For H/E staining, we fixed testis from adult mice in Bouin solution (overnight at 4°C), washed and dehydrated material using ethanol and xylene, and embedded the testis in paraffin. Sections of 7 μm were cut, mounted on SuperFrost Plus slides (Menzel-gläser), rehydrated, and stained with H/E.

For immunofluorescence analyses, testes were fixed in 4% paraformaldehyde in PBS overnight at 4°C, and treated as above. Before immunostaining, sections were treated with the microwave (three times for 5 min, 750 W) in 10 mM NaCitrate buffer (pH 6.0) to expose antigens and then stained using standard procedures. Alternatively, testis cryosections were fixed in 4% paraformaldehyde in PBS for 20 min and used directly for immunofluorescent staining.

The fluorescence live imaging of cultured somatic cells was performed as described (Stepanova et al. 2003). For imaging and FRAP analysis of testis tubules, testes were decapsulated and the tissue was immersed in 20 mL of Dulbecco's phosphate-buffered saline (PBS) containing 0.9 mM Ca²⁺, 0.5 mM Mg²⁺, 8 mM DL-lactic acid, and 5.6 mM glucose (PBS⁺), in the presence of collagenase (1 μg/μL; Roche) and hyaluronidase (0.5 μg/μL; Roche) in a siliconated 100-mL Erlenmeyer flask. This flask was incubated in a 33°C waterbath, in air, and shaken at 90 cycles/min for 15-20 min with an amplitude of 20 mm. The incubation was terminated when the mild enzymatic digestion had resulted in dissociation of the interstitial tissue and disengagement of testis tubules. The tubules were separated from the interstitial cells by washing in PBS⁺ twice. Next, several tubules were placed in a 30-mm Petri dish, in PBS⁺ supplemented with 0.2% bovine serum albumin (BSA). When required, 5 μg/μL Hoechst 33342 (Molecular Probes) was added. In this incubation system, the Hoechst stain is taken up only by nuclei on the basal side of the Sertoli cell barrier (also called “blood-testis barrier”). This allows for identification of all cells on the basal side of this barrier: peritubular cells, Sertoli cells, spermatogonia, and pre-leptotene spermatocytes. Selected tubules of ~1 cm length were transferred into a drop of 30 μL of PBS⁺ with 0.2% BSA on a 24-mm coverslip in a live cell chamber. A 16-mm coverslip was placed on top, and this was overlaid with PBS-saturated mineral oil.

When applicable, the testis was injected through the rete testis with Hoechst 33342 and Trypan blue (Sigma) in 3-5 μL of PBS, 1 h prior to testis dissection, to allow spreading of the vital DNA stain throughout the adluminal compartment of the testis tubules and uptake by nuclei on the adluminal side of the Sertoli cell barrier. Trypan blue served as a marker for injected tubules. This method makes it possible to identify the germ cell types that have migrated through the Sertoli cell barrier.

The testis tubules were examined at 33°C, using a Zeiss LSM510NLO confocal/multiphoton set up, to allow simultaneous acquisition of phase-contrast, GFP, and Hoechst images. GFP-CLIP-170 movement ex vivo was analyzed as published (Stepanova et al. 2003). FRAP analysis was carried out with the 63× planapochromat (1.4 NA) oil immersion lens. Bleaching of an outlined region of interest (ROI) was done with four iterations of the 488-nm laser at full transmission (2.6 mW). Prior to and after bleaching, the laser was set at 2% transmission (55 μW). The Zeiss LSM software was used to measure pixel intensities inside different ROIs. Recovery values were normalized to the prebleach values for each ROI. Values were imported into Aabel (Gigawiz) for graphical representation and statistical analysis.

Quantification of cross-bridges was performed on electron micrographs with a final magnification of 160,000×. The numbers of cross-sectioned MTs and cross-bridges were established by counting them on 10 electron micrographs of each condition (wild type, KI, and KO). Subsequently, the size of all cross-bridges was measured. Statistical analysis was performed in Excel (Microsoft).

We thank Michiel de Ruyter for generating the anti-LORF antisera, Evelyn Wassenaar for dissection of testis tubules, Marja Ooms for help with immunohistochemistry, Jan de Wit for keratinocyte culture advice, An Langeveld for performing karyotype analysis, and Dick de Rooij (Utrecht University) for expert advice on male germ cell types. This work was supported by the Dutch Ministry of Economic Affairs (BSIK), the Netherlands Organization for Scientific Research (NWO Earth and Life Sciences and ZonMw), the Dutch Cancer Society, and an EEC integrated project on imaging.