| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Article |
Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
ABSTRACT
A-kinase anchor proteins (AKAPs) spatially restrict cAMP-dependent protein kinase by tethering it to various cellular structures. In the polarized sperm cell, various compartmentalized functions, such as motility generated by the flagellum, are modulated by cAMP-dependent protein kinase. This important regulatory enzyme is associated with AKAP4, the principal component of the fibrous sheath; AKAP4 is synthesized as a precursor, pro-AKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. To define the domains responsible for the intracellular distribution and assembly of AKAP4 into a macromolecular complex, various AKAP4-green fluorescent protein (GFP) constructs were introduced into somatic cell lines. The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in a diffuse cytoplasmic localization of the GFP fusion protein, suggesting that, the pro domain keeps the AKAP4 precursor unassembled in vivo until it is transported to the developing tail structure and incorporated into the fibrous sheath. When the mature AKAP4-GFP fusion protein was expressed, it localized in a punctate cytoplasmic pattern. Two domains critical for this punctate localization, T2a and T2b, are homologous to the T2-tethering domain of rat AKAP5 that is important for binding to the actin cytoskeleton in transfected HEK293 cells. In contrast to AKAP5, the distribution of AKAP4 was dependent on the microtubular cytoskeleton. The interaction of AKAP4 with the microtubular network provides evidence that the longitudinal columns of the fibrous sheath, which contain AKAP4, may interact directly with the outer microtubular doublets of the sperm axoneme.
gamete biology, gametogenesis, sperm, sperm motility and transport, spermatid
INTRODUCTION
Signaling complexes containing the ubiquitous cAMP-dependent protein kinase (PKA) are established in different microenvironments in the cell so that proteins can be phosphorylated in a spatially restricted manner. A-kinase anchor proteins (AKAPs) are a diverse family of proteins that compartmentalize protein phosphorylation by tethering PKA regulatory subunits to various cellular structures [1–3]. In doing so, AKAPs place PKA in close proximity to its upstream effector molecules and downstream targets, facilitating the localized propagation of second-messenger signals. All AKAPs contain tethering domains that bind the regulatory subunit of PKA [4]. These domains lack a clear consensus sequence, although all are predicted to form amphipathic helices. To position PKA in diverse cellular locations, AKAPs themselves must be targeted appropriately. In this regard, targeting domains have been identified in some AKAPs that mediate AKAP attachment to specific subcellular organelles and structures [5]. These targeting domains are not well characterized and, presumably, differ depending on the localization of a particular AKAP.
In the highly polarized sperm cell, various compartmentalized functions are regulated by PKA signaling. The flagellum, the unique structure responsible for motility, consists of the axoneme and a number of cytoskeletal elements that are localized in different regions of the tail. Among them is the fibrous sheath (FS), which is a tapering cylinder found only in the principal piece of the mammalian sperm flagellum, where it underlies the plasma membrane and circumscribes the outer dense fibers (ODFs) and the axoneme. It is composed of two longitudinal columns attached to microtubular doublets 3 and 8; numerous transverse ribs extend halfway around the sheath, bridging the two columns.
Consistent with the idea of PKA compartmentalization, several AKAPs have been identified in male germ cells and sperm [6–12]. Among them is the major structural protein of the FS, AKAP4, which is present in both the columns and ribs [6, 13]. During spermiogenesis, AKAP4 is synthesized as a precursor, pro-AKAP4, that is subsequently cleaved to the mature protein. Although AKAP4 binds both PRKAR1 (RI
) and PRKAR2A (RII) regulatory subunits of PKA [14, 15], we do not know how AKAP4 is positioned and assembled in the FS to enable it to tether the kinase. The establishment of signaling complexes during spermatogenesis is complicated by the absence of ribosomes in the developing flagellum. Because protein translation does not occur within the flagellum, components of the FS and other accessory structures must be transported from the cell body to various sites of assembly within the developing tail [16].
Assembly of the FS and other cytoskeletal structures that comprise the flagellum is a complex, poorly understood process that likely is dependent on specific protein-protein interactions. In this regard, AKAP4 may provide a framework for the transport and assembly of other proteins comprising the FS. Because spermatogenic cell lines are not available for studying the assembly of these cytoskeletal structures in vitro and it not feasible to transfect primary cultures of spermatids to follow flagellar biogenesis, we expressed various AKAP4-green fluorescent protein (GFP) constructs into somatic cell lines. Using this approach, we have defined specific domains of AKAP4 responsible for its intracellular localization.
MATERIALS AND METHODS
Generation of AKAP4 Constructs
Various regions of AKAP4 were amplified by PCR and subcloned into pEGFP-C2 (BD Biosciences) that allows mammalian expression from the CMV promoter. The GFP was present at the amino terminal end of the fusion proteins. Some AKAP4 regions also were subcloned into pEGFP-N2 to place GFP at the carboxyl terminus. After transformation, DNA was isolated from colonies, and the presence of each insert was verified by restriction digestion. The DNA constructs were sequenced to ensure that each insert was in-frame with GFP. Amino acid substitutions in the regions of AKAP4 that were homologous to the T2-targeting domain of AKAP5 were introduced by the Quik-Change Mutagenesis Kit according to the manufacturer's instructions (Stratagene).
Cell Culture and Transfection
Both NIH3T3 and HEK293 cells were seeded on coverslips in six-well plates at a density of 40 000 cells/well. Cells were grown in Dulbecco modified Eagle medium (DMEM; Invitrogen) with 10% fetal bovine serum and 30 µg/ml of gentamicin (Invitrogen) at 37°C with 5% CO2. Serum-free DMEM and FuGene-6 (Roche) were mixed. Then, DNA (1 µg) was added, with the ratio of FuGene to DNA ranging from 2:1 to 3:1. After incubating the mixture at room temperature for 45 min, the DNA-FuGene mixture was added to the cells. After culturing for 12 h, the cells were imaged without fixation for GFP expression. Mock transfections were performed in which water was substituted for the DNA construct.
Immunoblot Assays
After transfection, cells were cultured for 24–48 h. Boiling SDS sample buffer (without dithiothreitol [DTT]) was then added to transiently transfected cells. The cell suspensions were removed and sonicated on ice for three sets of 10 pulses per set at setting 60 on a Vibracell sonicator (Vibracell). The extracts were boiled for five minutes and then spun at 20 000 x g for 2 min to remove particulate material, and the protein concentrations were determined by a Micro BCA assay (Pierce). After adding DTT, proteins were separated by SDS-PAGE using a 10% polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore). The membrane was blocked in 5% gelatin (Sigma) in PBS containing 0.1% Tween-20 (PBST) overnight, washed, and probed with a polyclonal anti-GFP antibody (BD Biosciences) diluted 1:2000 in 3% BSA-PBST for 1 h. After washing, the membrane was incubated with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia) at a 1:5000 dilution in 3% BSA-PBST. The membrane was washed, developed using the ECL reagent (Amersham) according to the manufacturer's instructions, and exposed to radiographic film.
Extractability of AKAP-GFP from Somatic Cells
Cells were transfected with the various constructs, cultured, and harvested as described above. After washing once with PBS, the cells were extracted with PBS plus 0.1% Triton X-100 containing a protease-inhibitor cocktail (Roche) for 10 min at room temperature. The pellet and supernatant fractions were collected after centrifugation at 12 000 x g for 10 min. The pellet was resuspended in 1x sample buffer plus DTT, and 5x sample buffer plus DTT was added to the supernatant fraction. Equal amounts of supernatant and pellet were separated by SDS-PAGE and probed with anti-GFP.
Immunofluorescence Procedures
After transfection, cells were washed with PBS, and the coverslips were removed from the six-well plates. The coverslips were fixed either in 4% paraformaldehyde in 1x PBS (for -tubulin staining) or in 0.3% glutaraldehyde (for actin staining) for 15 min at room temperature. For staining with phalloidin, the unreacted aldehydes were quenched with 100 mM NaCl and 50 mM Tris-HCl (pH 7.5) for 5 min. Cells were washed with PBS, permeabilized with 0.01% Triton X-100 in PBS for 15 min, and blocked in 10% normal goat serum in PBS for 1 h at room temperature. For tubulin staining, the cells were washed with PBS, then incubated with an -tubulin monoclonal antibody (Sigma) at 1:100 in blocking solution for 1 h at room temperature. After washing, the cells then were incubated with Cy5-labeled anti-mouse antibody (Santa Cruz Biotech) at 1:100 in blocking solution and washed, and the coverslips were mounted on slides with Fluoromount-G (Southern Biotechnology Associates, Inc.). For actin staining, the cells were incubated with phalloidin CPITC (Sigma) at 1:1000 in PBS and incubated for 1 h at room temperature and then washed, and the coverslips were mounted. The cells were imaged using a Nikon TE 300 epifluorescence microscope; the images were collected using a MicroMax CCD camera (Roper Scientific) and MetaMorph software (Universal Imaging).
Nocodazole Treatment
After transfection with the appropriate AKAP4-GFP construct, cells were incubated for 12 h in medium with nocodazole (final concentration, 30 µM) and imaged. In some experiments, cells were washed with medium to remove the nocodazole and allowed to recover for 3.5 h before imaging.
RESULTS
The pro-AKAP4 (amino acids 1–840) is synthesized in the cell body of the spermatid and then transported to the developing flagellum [13]. Once there, most of the protein is cleaved to the mature AKAP4 (amino acids 180–840). A small amount of the pro region (either the cleaved peptide, amino acids 1–179, or the precursor) remains, although its role in sperm is not known. The studies to date have been performed with spermatogenic cells freshly isolated from testes. However, it has been difficult to analyze the molecular mechanisms of flagellar assembly, such as formation of the FS, because spermatogenic cell lines appropriate for studying this process in vitro do not exist. To examine AKAP4 assembly, we circumvented this limitation by transfecting NIH3T3 and HEK293 somatic cells with vectors expressing various constructs of AKAP4-GFP and by visualizing the fusion proteins using fluorescence microscopy (see Fig. 1 for a summary of the constructs). Results were identical regardless of the cell line that was used. This suggests that a particular pattern of the fusion protein localization was dependent on the intrinsic properties of the AKAP4 protein or its various truncated forms, not the cellular environment in which the fusion protein was expressed. Also, the positioning of GFP at either the N- or C-termini did not affect cellular localization, so only the data using the constructs in which the GFP was at the amino terminal end of the fusion protein are shown.
|
Expression of AKAP4-GFP Fusion Proteins
A number of AKAP4-GFP fusion constructs were generated. To confirm that the desired proteins were synthesized, cellular protein extracts were prepared from transfected cells and analyzed by immunoblotting with anti-GFP. Each recombinant protein migrated at its predicted size; proteins from some of the constructs are shown in Figure 2. The fusion protein containing the full-length AKAP4 precursor (amino acids 1–840; Mr 
97 000) plus GFP (Mr 30 000) had a Mr of approximately 130 000. Mature AKAP4 (amino acids 180–840; Mr 82 000) plus GFP was slightly smaller (Mr 110 000). The pro region of AKAP4 (amino acids 5–179; Mr 20 000) plus GFP had a Mr of approximately 55 000. Proteins from mock-transfected cells showed no immunoreactivity with the exception of two prominent and two minor, nonspecific bands that also were detected in all samples. Proteins from cells transfected with a construct expressing only GFP contained a prominent immunoreactive band at a Mr of approximately 30 000.
|
Pro Domain Is Responsible for Diffuse Cytoplasmic Localization of Pro-AKAP4
We hypothesized from our previous study that the role of the pro domain of the AKAP precursor is to keep the protein soluble during its transport from the cell body to the developing tail, where it is assembled into an insoluble complex [13]. To test this hypothesis, pro-AKAP4 (amino acids 1–840)-GFP was expressed in NIH3T3 cells. Under these conditions, a diffuse cytoplasmic fluorescence pattern was observed similar to that seen with GFP alone (compare Fig. 3, A and B). The exclusion of the fluorescence signal from the nuclei of these cells was expected, because neither AKAP4 nor GFP contain a nuclear localization signal. In contrast, when the full-length, mature AKAP4 (amino acids 180–840) was expressed, a distinctly punctate pattern was seen in the cytoplasm (Fig. 3C). The introduction of a construct encoding the pro domain of AKAP4 (amino acids 5–179) plus GFP into somatic cells resulted in a diffuse cytoplasmic localization similar to the pattern seen for pro-AKAP4-GFP (Fig. 3D). Cells that were mock-transfected with water instead of a plasmid DNA were not fluorescent (Fig. 3E).
|
These results suggested that the pro domain confers solubility to the AKAP4 precursor and that the mature protein possesses the necessary information for self-assembly into insoluble complexes. To test this hypothesis in our in vitro model, we transfected the pro-AKAP4-GFP, AKAP4-GFP, pro-GFP, and GFP constructs into HEK293 cells and tested the extractability of these proteins under conditions used previously [13]. In this study, we found that pro-AKAP4 is released into a soluble fraction when germ cells are extracted with 0.1% Triton X-100, whereas it remains particulate in caput and cauda sperm. The mature AKAP4 was particulate in both sperm samples. As shown in Figure 4, all three forms partitioned into the soluble and particulate fractions of somatic cells. However, the extractability of the pro domain fusion protein was similar to that of GFP, a purportedly soluble protein, in that each partitioned approximately equally into the soluble and particulate fractions. As expected from the in vivo studies [13], AKAP4-GFP remained more particulate after Triton X-100 extraction. We expected that the pro-AKAP4-GFP would be more extractable; however, it also remained more particulate after Triton X-100 extraction. Thus, these findings do not support the hypothesis proposed above that the pro domain alone confers solubility to the precursor, and they suggest that the mechanism of FS assembly may require other factors lacking in HEK293 cells.
|
Regions Within AKAP4 Are Responsible for the Punctate Appearance of Protein
As discussed above, when the full-length, mature AKAP4 (amino acids 180–840)-GFP protein was expressed, a punctate pattern was observed (Fig. 3C). Typically, a limited number (n = 1–5) of fluorescent foci were seen, and some of these foci appeared in the cytoplasmic extensions of the fibroblasts. When compared to the diffuse localization pattern observed when the pro domain was expressed, these results suggested that one or more sequence motifs in AKAP4 confer a punctate localization to the protein. To determine whether specific domains were responsible for the punctate localization, fragments of AKAP4 fused to GFP were expressed (the constructs and their localization patterns are summarized in Fig. 1).
A fusion protein containing the amino terminal 60% of AKAP4 (amino acids 180–569 of pro-AKAP4) had the appropriate molecular weight (Mr 62 000; data not shown) and appeared as a discrete fibrous structure (Fig. 5A). The assembly of this fusion protein into a fibrous pattern indicated that this region of AKAP4 was able to organize itself into a distinct cytoskeletal structure. However, when this region was truncated further to generate a fusion protein containing the amino-terminal end of mature AKAP4 (e.g., amino acids 180–239 of pro-AKAP4), a diffuse fluorescence was observed throughout HEK293 cells (Fig. 5B). This region contains a binding domain for PKA RI and RII [14, 15]. One AKAP4 construct that contained a region of the carboxyl end (amino acids 433–737) showed both a diffuse and punctate appearance in transfected cells, but all other constructs encompassing various carboxyl regions of AKAP4 were diffusely localized throughout the cytoplasm (Fig. 1). We conclude that neither the amino nor carboxyl ends of AKAP4 have a significant role in contributing to the punctate localization of the protein.
|
When central regions of AKAP4 were expressed as GFP fusion proteins (e.g., amino acids 327–451 of pro-AKAP4), the proteins localized in a punctate pattern (Fig. 5C). Multiple fluorescent foci were seen in cells with a pattern similar to that observed for the mature protein (amino acids 180–840). By analyzing a number of the short AKAP4 constructs that yielded a punctate localization pattern (e.g., amino acids 327–451 of pro-AKAP4) (Fig. 1), the presence of the region between amino acids 350–430 was determined to be a constant feature. This region contains two motifs that we previously identified as having homology to the T2 domain of rat AKAP5 [6]. The T1 and T2 domains of AKAP5 are essential for the intracellular targeting of AKAP5-PKA complexes to the cortical cytoskeleton of nonneuronal cells [5]. One domain of AKAP4, which we have termed T2a, spanned amino acids 353–366, whereas the second domain, T2b, contained amino acids 415–422 (Fig. 6E). The punctate localization pattern shown by AKAP4 persisted as long as the expressed fusion protein contained one of these two motifs (Fig. 1). When a GFP fusion protein containing the T2a motif but not the T2b motif (amino acids 327–398) was expressed, multiple foci in the perinuclear region were observed (Fig. 6A). A protein containing only the T2b motif (amino acids 398–451) also was present in numerous foci, but these structures localized not only in the cell body but also in the extensions of the cells (Fig. 6C). To confirm that the T2a and T2b motifs were responsible for conferring the punctate appearance to AKAP4, specific amino acids in each region were mutated (Fig. 6E). Because these regions generally are hydrophobic and contain a core domain of L–K–R–(V/L)–(L/V), we modified this string to include two -helix-breaking proline residues. The resulting GFP fusion proteins containing the mutated AKAP4 fragments showed diffuse cytoplasmic localizations (Fig. 6, B and D).
|
AKAP4 Interacts with Microtubular Cytoskeleton
As discussed above, the punctate localization pattern shown by AKAP4 persisted as long as the expressed fusion protein contained either the T2a or T2b domains (Fig. 1). Because these domains are homologous to the T1 and T2 domains of rat AKAP5 that mediate its colocalization with the cortical actin cytoskeleton of HEK293 cells [5], we investigated whether AKAP4 could localize with the actin cytoskeleton. When cells were transfected with the construct AKAP4 (amino acids 180–569) plus GFP and stained with phalloidin, AKAP4 and F-actin did not colocalize (Fig. 7A). Furthermore, the disruption of the actin cytoskeleton with cytochalasin did not alter the fibrous appearance of AKAP4-GFP fusion protein, suggesting that no direct interaction occurred between AKAP4 and the actin cytoskeleton (data not shown).
|
The proximity of the longitudinal columns of the FS and microtubular doublets 3 and 8 of the axoneme suggested to us that AKAP4 interacts with the microtubular cytoskeleton. When NIH3T3 cells were transfected with AKAP4 (amino acids 180–569) plus GFP and stained for tubulin, regions of overlapping fluorescence were observed, suggesting that AKAP4 and tubulin colocalized (Fig. 7, B–D). To test the association of AKAP4 with microtubules further, transfected cells were treated with nocodazole to disrupt the microtubular network. Under these conditions, AKAP4 (amino acids 180–569) plus GFP was dispersed to multiple foci throughout the cytoplasm (Fig. 8). After removing the nocodazole by washing the cells to allow re-establishment of the microtubular cytoskeleton, the fibrous appearance of AKAP4 reappeared (Fig. 8).
|
DISCUSSION
The study of flagellar biogenesis is complicated by the lack of appropriate spermatogenic cell lines for in vitro studies. Thus, it is a challenge to study how the proteins of the sperm tail are synthesized and then assembled during spermatogenesis. Furthermore, the developing flagellum lacks ribosomes, requiring that proteins designated for this structure must be synthesized in the cell body and then transported to the site of assembly in the tail. To circumvent the lack of spermatogenic cell lines, somatic cell lines have been used as surrogates for studying the protein targeting and assembly of sperm-specific structures. Although the FS would not be expected to be assembled in somatic cells, we and others have used this approach to identify domains within the germ cell-specific isoforms of hexokinase (an FS component) that are responsible for its subcellular compartmentalization [17, 18].
Several functional domains are contained within AKAP4. Previously, we demonstrated that the majority of pro-AKAP4 is processed to the mature form by the proteolytic removal of the 179-amino-acid pro domain at the N-terminal end of the protein [6]. Subsequently, binding sites for both the RI and RII subunits of PKA were identified [14, 15]. In our initial identification and characterization of AKAP4 (then called p82), we noted that two regions in the protein resemble the T2 domain of AKAP5 [6]. In the present study, we have determined that both the T2-like regions and the pro domain contain information that confers specific localization patterns to AKAP4. In contrast, neither the RI- nor RII-binding domains appeared to have a role in the intracellular distribution of the protein.
The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in the AKAP4-GFP fusion protein having a diffuse cytoplasmic localization. This result suggests that the pro domain keeps the precursor unassembled so that it can be transported to the tail before being assembled into an insoluble cytoskeletal structure. The extractability experiments in somatic cells (Fig. 4) more closely approximates the situation in sperm rather than the situation in spermatids [13]. In this regard, we previously showed that pro-AKAP4 is extractable and not processed to AKAP4 in the cell body of spermatids before being transported to the developing flagellum. The precursor first moves to the tail and then is processed to the mature AKAP4, which is now insoluble [13]. Given these results, another possible (although not mutually exclusive) function of the pro region could be to act as a "zip code" for delivering AKAP4 to the tail.
Fusion proteins appeared in a punctate pattern with a number of foci when the AKAP4 region containing the T2a and T2b domains, either singly or in combination, were expressed. Computer analysis of the T2a and T2b domains showed that both have a low probability of exposure on the surface, most likely resulting from their hydrophobic nature. In addition, both domains are predicted to be relatively inflexible. Disruption of the rigid nature of these structures by mutations of the core amino acids in the T2a and T2b regions to residues containing kink-inducing proline residues (Fig. 6) destroyed the ability of the fusion proteins to assemble into punctate structures. As noted previously, the amino acid sequences of T2a and T2b are similar to the T2 domain of AKAP5. Both the T1 and T2 domains of AKAP5 are critical for targeting the protein to the actin-rich cortical cytoskeleton of HEK293 cells [5]. Deletion of either the T1 or the T2 domain or multiple amino acid substitutions within a particular domain leads to AKAP5 being localized throughout the cytoplasm and not at the cortex, indicating that an intact T domain is essential for the selective anchoring of AKAP5 in the cortical actin cytoskeleton. The difference in location of these two proteins, despite the presence of a T2 domain in each, was surprising and should be addressed in future studies to determine whether minor amino acid substitutions in the T2 region dictate the cytoskeletal specificity or if other motifs are important in targeting each AKAP to discrete cytoskeletal locations.
The findings that AKAP4 did not interact with actin are evocative of results reported for another FS protein, TSGA10, which contains an ERM domain found in the AKAPs ezrin, radizin, and moesin [19, 20]. When a TSGA10-GFP fusion protein is transiently expressed in mouse fibroblasts, the fusion protein also does not colocalize with actin, but it is observed in thick, short filaments. The formation of these filaments is mediated by the ERM domain, because elimination of this domain in a mutated fusion protein abolished the filament production by TSGA10 and resulted in the local accumulation of TSGA10 in several cytoplasmic foci, reminiscent of the punctate patterns shown in Figures 3C and 5C. Similar to AKAP4, TSGA10 was processed during mouse spermatogenesis; however, it was not determined whether the processed protein can form filaments.
We have shown that a truncated form of AKAP4 (amino acids 180–569) interacts with the microtubular cytoskeleton but not with actin. Among the microtubular structures in germ cells is the manchette, a transient structure that develops during spermiogenesis [21]. It is assembled shortly after the axoneme organizes, and proteins destined for distinct regions of the sperm, such as the flagellum, are transiently associated with this structure. Kierszenbaum and Tres [22] have formulated a model in which the manchette is positioned in the developing spermatid to be a temporary storage site for structural and signaling proteins, some of which ultimately are targeted to the flagellum. Given the association of AKAP4 (amino acids 180–569) with microtubules, it is possible that the unassembled pro-AKAP4 transiently associates with other proteins that expose the microtubular binding region (amino acids 180–569) to enable interaction with manchette before or as part of its transport to the developing flagellum.
In the principal piece of the sperm tail, the longitudinal columns of the FS replace the two ODFs associated with the outer microtubular doublets 3 and 8 of the axoneme. When demembranated sperm flagella are prepared and perfused with Mg-ATP to induce microtubular sliding, the ODFs and those doublets associated with the ODFs are extruded [23]. In addition, the FS slides proximally toward the midpiece. Our finding that an AKAP4-GFP fusion protein colocalizes with the microtubular cytoskeleton in transfected somatic cells suggests that AKAP4 may mediate the association of the FS with the axonemal microtubules in sperm.
The assembly of the FS in the principal piece of the flagellum is a complex and critical process for the production of mature sperm. The dual localization of AKAP4 in the ribs and columns of the FS indicates that its assembly is coordinated to form such a highly detailed structure [13]. The diffuse localization of pro-AKAP4 in transfected somatic cells (present study) and the Triton X-100 solubility of this protein in developing spermatids [13] indicate that the pro domain may keep the protein unassembled until AKAP4 is incorporated into the FS. In transfected cells, the elimination of the pro domain results in a punctate intracellular distribution of the mature AKAP4 protein, a finding that we propose is related to the mechanism of FS assembly into an SDS-insoluble structure. Further analysis of the AKAP4 domains and specific protein-protein interactions will provide a better understanding of the differential assembly of ribs and columns leading to the formation of a functional FS.
ACKNOWLEDGMENTS
We thank Lisa Haig-Ladewig for her expert technical assistance and the anonymous reviewers for their insights and suggested experiments.
FOOTNOTES
1 Supported by NIH HD06274 to S.B.M. and G.L.G. 
2 Correspondence: Stuart B. Moss, Center for Research on Reproduction and Women's Health, Rm. 1312 BRB II/III, 421 Curie Blvd., University of Pennsylvania Medical Center, Philadelphia, PA 19104-6142. FAX: 215 573 7627; smoss{at}mail.med.upenn.edu
3 Current address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.
4 Current address: Neotropix, Inc., 351 Phoenixville Pike, Malvern, PA 19355.
5 These authors contributed equally to this work.
6 These authors were coprincipal investigators of this work.
Received: 16 January 2006.
First decision: 2 February 2006.
Accepted: 8 May 2006.
REFERENCES
regulatory subunits on sperm fibrous sheath protein FSC1 [in process citation]. J Biol Chem 1998 273:34384-34390
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
