BACKGROUND
profilins are small, ≈ 14-kda intracellular proteins which are crucial for actin microfilament dynamics .
their ubiquity, abundance, and necessity for life in higher organisms underscore their general importance .
despite their small size, their functions are amazingly diverse.
through binding to numerous protein ligands, profilins are components of complex protein networks .
interactions with components of the phosphatidylinositol cycle  <cit>  and the rac-rho pathway  <cit>  implicate them as a link through which the actin cytoskeleton communicates with the major signalling pathways of the cell.
accordingly, reducing the amount of profilin protein, e.g.
by gene deletion, often has severe or even fatal consequences on the viability of the afflicted organism.
profilins constitute a large and diverse protein family.
multiple isoforms exist in many species, being encoded by separate genes, or in some cases translated from mrna splice variants.
in animals and higher plants, isoforms may be expressed in a tissue-specific manner.
moreover, profilins are found at different subcellular locations .
enrichment at dynamic plasma membranes was confirmed for various cells types.
also, profilins were observed in association with internal membranes involved in vesicular transport  <cit> .
finally, profilins are constituents of the cell nucleus .
although the overall structure is conserved, sequence homologies between profilins from different species, and also between different isoforms from the same organism, are low .
it was, nevertheless, reported that the overall functional properties of different profilins are similar, and that one isoform can be interchanged with another one from quite a distant source  <cit> .
on the other hand, structural differences, which determine in vitro affinities for various ligands  <cit> , preferential protein complex formation in different cell types, and different subcellular locations may be important clues of divergent, possibly non-overlapping in vivo functions of different isoforms.
despite extensive studies, the significance of the multiple profilins, their tissue-specificity and distinct subcellular locations have remained enigmatic.
at least four different profilins were demonstrated to be present in the mammalian testis  <cit> , a complexity which was not observed in somatic tissues.
the mammalian testis may, thus, serve as a model to question whether profilin isoforms may fulfil distinct functions.
profilin- <dig>  is ubiquitous and essential for cell viability  <cit> ; its expression in all cell types of the testis, including spermatogenic cells, thus seems obvious.
in comparison, profilin- <dig>  is predominantly found in the nervous system and has acquired more specialized functions in regulating neuronal activity  <cit> ; it may represent a cell type-specific isoform also in the testis.
while pfn <dig> and pfn <dig> were both demonstrated in the somatic sertoli cells  <cit> , a third isoform, profilin- <dig> , is expressed solely in spermatids  <cit> .
most recently, profilin- <dig>  was characterized as a novel isoform.
it shows less than 30% amino acid identity with the other mammalian profilins; still, database searches produced significant alignments with the conserved profilin domain.
pfn <dig> is also highly expressed during spermatogenesis, but is distinct from pfn <dig> in its temporal expression pattern  <cit> .
based on sequence comparisons, it was speculated that the testis-expressed pfn <dig> and pfn <dig> might have altered binding capacities for actin and proline-rich ligands  <cit> .
however, such diverse characteristics remained to be experimentally shown.
in the present study, using yeast two-hybrid interaction assays and various biochemical methods, the binding capacities of pfn <dig> and pfn <dig> for proline-rich ligands, actin, and phosphoinositides were studied in comparison with pfn <dig> and pfn <dig>  subcellular locations in differentiating spermatids were studied by immunofluorescence.
three-dimensional structural models were also built to explain the functional properties of pfn <dig> and pfn <dig>
RESULTS
pfn <dig> and pfn <dig> have different in vitro affinities for protein ligands
actin monomers, poly-l-proline , and proline-rich proteins have been confirmed as in vitro ligands for each profilin isoform tested so far.
in addition, in vivo interactions of various profilins have been verified with actin , and proline-rich proteins, including the vasodilator-stimulated phosphoprotein  and the mammalian homologues of drosophila diaphanous , members of the formin gene family  <cit> .
in analogy with this, several databases of known and predicted protein-protein interactions suggested that the testis-expressed isoforms pfn <dig> and pfn <dig> would likewise associate with these ligands  <cit> .
we asked whether pfn <dig> and pfn <dig> indeed shared these affinities.
protein lysates containing pet-expressed  pfn <dig> and pfn <dig> proteins were employed in plp affinity chromatography.
pfn <dig> bound to the plp affinity column ; however, compared to pfn <dig> and pfn <dig>  the association was weaker, since pfn <dig> was eluted from the plp column at  <dig> to  <dig> m urea .
under the same experimental conditions, pfn <dig> failed to interact with plp , suggesting that different from the above predictions proline-rich protein ligands may not be targets for this isoform.
since plp affinity chromatography is routinely employed to purify native as well as recombinant profilins, the lack of polyproline interaction caused complications in the purification of untagged pfn <dig>
selective pair wise tests for yeast two-hybrid  interaction , assaying potential differences of pfn <dig> and pfn <dig> in their affinities for specific proline-rich proteins, i.e.
vasp and mdia <dig> , were performed as an independent method.
pfn2a, which is known to bind vasp and p140mdia  <cit> , was included as a positive control.
expression of the lacz reporter was used as a more quantitative indicator for the strength of protein-protein interactions.
mdia <dig>  but not vasp, was confirmed as an in vitro binding partner of pfn <dig> .
the interaction of mdia <dig> with pfn <dig> was weaker compared to pfn2a; parallel y2h assays employing pfn <dig>  in comparison, were negative , confirming the results of the plp affinity chromatography.
notably, our previous microarray analyses of human testis tissue  had shown that at least five mdia3-encoding mrnas variants, including the one employed in the y2h assays here, were highly expressed in the testis and were most abundant in tissue samples, which contained post-meiotic germ cells.
thus, mdia <dig> proteins may still represent in vivo interaction partners of pfn <dig>
binding of the testis-specific profilin isoforms to β-actin was similarly tested by a pair wise y2h interaction, confirming pfn <dig> as an actin-binding isoform .
parallel assays employing pfn <dig> were all negative , suggesting that neither proline-rich proteins nor actin were targets of this unusual isoform.
the failure of pfn <dig> to interact with actin contrasts with all other profilins studied thus far, most of which bind actin monomers with micromolar affinity.
the y2h method, however, is prone to false-negative discovery.
by attaching domains from a transcription factor to the bait and prey proteins, true interactions may be missed if fusions place these attachments at important interacting actin and/or profilin interfaces  <cit> .
to confirm the y2h results by two independent methods, co-immunoprecipitation of β-actin and actin polymerization assays were performed.
for co-immunoprecipitation pfn <dig>  pfn <dig>  and pfn <dig> were translated in vitro as n-terminal fusions to a c-myc-tag; a c-myc-antibody was employed to immunoprecipitate the profilins in the presence of ha-tagged β-actin.
while pfn <dig> and pfn <dig> effectively co-precipitated with β-actin as evidenced by their retention by protein a beads, pfn <dig> failed to associate with β-actin also under these experimental conditions .
in a control experiment, in the absence of profilins, β-actin was not precipitated with protein a beads .
we probed the influence of recombinantly expressed gst-fusions of pfn <dig> and pfn <dig> on salt-induced actin polymerization using the pyrenyl fluorescence assay  <cit> .
labelled skeletal muscle g-actin was incubated with or without the purified gst-fused profilins .
gst-pfn <dig>  which served as a control, showed a significant retardation of actin polymerization.
under the same experimental conditions, pfn <dig> also markedly reduced polymerization kinetics, albeit to a lesser extent.
pfn <dig>  on the other hand, did not influence the actin polymerization kinetics at all .
in summary, concerning pfn <dig> protein interaction partners, our results confirmed and extended the results by hu et al.
<cit> , suggesting that pfn <dig> showed major hallmarks of the well characterized profilins.
pfn <dig>  on the other hand, did not bind the tested protein ligands in vitro and thus may have in vivo functions different from regulating actin dynamics.
it should be noted that spermatids contain several actin-related proteins  <cit> .
recently, the actin-related protein arpm <dig> was identified as an interaction partner of pfn <dig>  <cit> .
the interaction of pfn <dig> and actin-related proteins expressed in spermatids deserves further evaluation.
pfn <dig> and pfn <dig> differentially interact with phospholipids
profilins interact with anionic phospholipids .
individual phospholipids show distinct subcellular distributions and perform distinct biological roles , with steady-state concentrations of ptdinsp <dig> predominating at plasma membranes, ptdinsp on endosomes, and ptdinsp on the golgi.
thus, through differential binding to these phospholipids in vivo, profilin isoforms may be selectively directed to functionally appropriate subcellular sites.
selective interaction with phosphoinositides was indeed the first functional difference reported between different profilin isoforms  <cit> .
moreover, it was shown that binding to ptdinsp <dig> may effectively compete for plp interaction of profilin  <dig>  <cit> .
more recently, neighbouring binding sites for these competing ligands have emerged which may explain the competing interactions  <cit> .
phospholipid interactions of gst-fusion proteins of pfn <dig>  pfn <dig>  and pfn <dig>  respectively, were assayed by protein-lipid overlay  <cit> .
each of the recombinant fusions strongly bound to ptdinsp, and to a lesser extent to its phosphorylated products and ptdinsp <dig> .
unlike gst-pfn <dig> , gst-pfn <dig> and gst-pfn <dig> both showed in vitro affinity towards ptdinsp, suggesting that in vivo, in addition to endosomes, pfn <dig> and pfn <dig> may be associated with the golgi apparatus.
for gst-pfn <dig>  a weak but reproducible binding to phosphatidic acid  was additionally observed .
pa is an abundant component  of most cellular membranes.
it is also a signalling lipid, since its regulated formation can constitute an important signal for many downstream responses, including actin polymerization in spermatozoa  <cit> .
in addition to this role, pa appears to regulate enzymes in phospholipase d pathways directly.
the list of pa-binding proteins is rapidly expanding ; however, pa-binding of profilins has not been previously reported.
the specificity of the observed interaction must be considered critically as the isoelectric point of pfn <dig> is basic ; still, gst-pfn <dig> did not bind pa under the same experimental conditions, despite of the highly basic character of pfn <dig> .
pfn <dig> and pfn <dig> localize in the acroplaxome-manchette complex in spermatids
cross-reactivity of the profilin antisera employed in immunostaining procedures was initially studied by western blot analysis using in vitro translated profilins .
antisera generated against pfn <dig> and pfn <dig> did not cross-react; weak cross-reactivity of the anti-pfn <dig> antiserum was observed only with in vitro-translated pfn <dig>  in protein extracts of human adult testis tissue , and also in rat testis samples collected during the first wave of rat spermatogenesis , anti-pfn <dig> and anti-pfn <dig> immunoreactive bands of ≈ 14-kda were seen only in those tissue samples which contained spermatids, confirming at the protein level the previously reported stage- and cell-type specific expression patterns of pfn <dig> and pfn <dig>  <cit> .
in comparison, anti-pfn <dig> and pfn <dig> immunoreactive bands were obvious in each testis sample analysed .
our previous studies suggested that pfn <dig> and pfn <dig> accumulated near the acrosome-acroplaxome-manchette complex of differentiating spermatids .
the acroplaxome is an f-actin/keratin 5-containing cytoskeletal plate, which anchors the acrosome to the spermatid nucleus  <cit> .
pro-acrosomal vesicles derived from the golgi apparatus  <cit>  are transported by f-actin- and microtubule-based molecular motors and dock and fuse along the acroplaxome .
the manchette is a transient structure developed subjacent to the marginal ring of the acroplaxome.
it consists of a perinuclear ring with inserted microtubules as well as associated f-actin.
the manchette participates in the transport of cargoes to the developing spermatid tail and in nucleo-cytoplasmic trafficking during spermatid head shaping  <cit> .
indirect immunofluorescence of spermatids isolated from spermatogenic stage-specific rat seminiferous tubules was used to determine the localization of pfn <dig> and pfn <dig> throughout spermiogenesis.
phalloidin-texas red was used to monitor the f-actin-containing acroplaxome; tubulin monoclonal antibody was used to determine the position of the manchette with respect to the acroplaxome.
pfn <dig> immunoreactivity was mainly observed in the acroplaxome of round spermatids and may be correlated with the presence of f-actin .
during the progression of spermiogenesis, pfn <dig> acroplaxome immunoreactivity gradually disappeared and became apparent in the developing manchette of spermatids step  <dig>  .
coinciding with the initiation of manchette disassembly in s <dig> spermatids, pfn <dig> immunoreactivity was seen in the cytoplasm subjacent to the disassembling manchette .
at this stage, the phalloidin-stained acroplaxome was devoid of pfn <dig> .
a similar immunoreactive staining pattern was observed with anti-pfn <dig> serum in rat  and also in human spermatids .
in agreement with the immunoblotting data, normal rabbit igg controls did not generate a fluorescence signal .
panel m in figure 6/ <dig> summarizes diagrammatically the localization of pfn <dig> and pfn <dig>  first in the acroplaxome and later in the manchette during spermatid head development.
these observations suggested that, despite their differing functional characteristics in vitro, the location sites of pfn <dig> and pfn <dig> proteins in spermatids were similar.
in vivo functions of the tissue- and cell type-restricted isoforms, however, remain obscure.
transgenic mouse models may help to clarify whether and how pfn <dig> and pfn <dig> impact on acrosome formation and/or spermatid head shaping.
structural models explain the observed biochemical properties of pfn <dig> and pfn4
in order to gain a deeper understanding of the observed functional differences, homology models were built for human pfn <dig> and pfn <dig>  and compared to the known structures of human pfn <dig> and pfn2a.
the sequence alignments of all known human profilins are shown in figure 7a, and those used for modelling in additional file  <dig>  both pfn <dig> and pfn <dig> are predicted to be folded like other profilins, with a central 7-stranded antiparallel beta-sheet covered on both faces by two alpha helices.
the pfn <dig> sequence, however, is ten amino acids shorter which is structurally reflected by three loops, i.e.
β1β <dig>  β4β <dig>  and β5β <dig>  being shorter in the pfn <dig> protein .
one of the loops, β5β <dig>  is also shorter in pfn <dig>
the poly-l-proline binding site
the plp binding site of profilins is formed by conserved aromatic amino acids located in the n- and c-terminal helices, such that the proline-rich peptide will bind to a groove between the two helices, interacting closely with the aromatic residues via hydrogen bonds and ch-pi interactions  <cit> .
for example, five conserved aromatic residues  and also asn <dig> are responsible for the plp interaction of pfn2a , and pfn <dig> is highly similar.
the two aromatic residues on the c-terminal helix  are not present in pfn <dig>  which could lead to an altered specificity and/or lower affinity towards proline-rich sequences, as also evidenced by our binding assay.
in pfn <dig>  of the abovementioned aromatic residues, only tyr <dig> is conserved .
thus, in this isoform, the aromatic surface of other profilins necessary for plp binding is not present at all, which is in line with its observed inability to bind proline-rich sequences.
while the overall sequence of pfn <dig> is more similar to the profilins of lower eukaryotes than to the other human/mammalian profilins  <cit> , pfn <dig> seems to be the only family member so far, in which the entire plp binding site is lacking, suggesting that it may have other binding partners instead.
the actin-binding site
in pfn <dig>  residues centrally involved in actin binding include phe <dig>  his <dig>  arg <dig>  arg <dig>  lys <dig>  lys <dig>  and tyr <dig> ; these are fully conserved in pfn2a.
in pfn <dig>  this site is conserved, with some rather conservative amino acid sequence differences, such as the replacement of phe <dig> and tyr <dig> by leu <dig> and his <dig>  respectively, compared to pfn2a.
these structural differences may have lowered or altered the affinity of pfn <dig> towards actin to some degree, when compared to pfn <dig> and pfn2a.
in the pfn <dig> sequences, in contrast, there is no conservation of the known actin-binding residues of other profilins, which is well in line with our experimental data described above.
the ptdinsp <dig> binding site
based on mutagenesis studies and the localisation of sulphate and phosphate ions in crystal structures, the binding site for ptdinsp <dig> has been approximately mapped in human pfn <dig>  <cit> .
it involves several conserved basic amino acid residues on the profilin surface, generating a large binding surface of positively charged residues.
in pfn2a, residues likely to contribute to ptdinsp <dig> binding include arginines  <dig>   <dig>   <dig>   <dig>  and  <dig>  and these binding determinants are largely conserved in pfn <dig>  interestingly, arg <dig> and arg <dig> are among the few residues, which are conserved throughout the known human profilin isoforms, including pfn <dig> .
in pfn <dig>  the putative common ptdinsp <dig> binding site is well conserved.
in pfn <dig>  many basic residues are present as well, but the putative phospholipid binding site seems to have shifted .
this structural difference may explain the distinct phospholipid binding characteristics of pfn <dig> .
CONCLUSIONS
we report functional differences between the testis-expressed profilins pfn <dig> and pfn <dig> as revealed by in vitro assays.
at the same time, structural homology models were built for human pfn <dig> and pfn <dig>  which explained their different functional characteristics.
by various methods, mdia <dig>  but not vasp, was identified as a novel in vitro binding partner of pfn <dig>  while pfn <dig> did not bind plp or proline-rich proteins.
so far, pfn <dig> seems to be the only profilin family member in which the entire plp binding site is lacking.
moreover, different from pfn <dig>  pfn <dig> did not interact with actin in three different in vitro assays deviating from all other profilins analysed thus far.
actually, the structure of pfn <dig> does not retain the actin-binding site of other profilins.
a much lower affinity for actin or actin-related proteins is thus conceivable, suggesting that pfn <dig> may perform in vivo functions, which are distinct from regulating actin dynamics.
pfn <dig> and pfn <dig> were both capable of in vitro phosphoinositide interaction, but differed in their selectivity towards specific phospholipids.
in pfn <dig>  the putative common ptdinsp <dig> binding site of profilins is conserved while it seems to have shifted in pfn <dig>  pfn <dig> and pfn <dig> coexist in the acroplaxome and the manchette of spermatids in a sequential developmental manner.
they vanish gradually from the acroplaxome when the manchette fully assembles and spermatid head shaping is in progress.
although the presence of pfn <dig> can be correlated with the presence of f-actin and actin-related proteins in the acroplaxome, and to a lesser extent in the manchette, the visualization of pfn <dig> in the acroplaxome-manchette complex is intriguing in light of our finding that it does not interact with actin in vitro.
an f-actin independent role of pfn <dig> may suggest novel functions related to the process of spermatid head shaping, and may have significant clinical implications in understanding idiopathic causes of male infertility associated with abnormal sperm head shaping.

 profilins are small, ≈ 14-kda intracellular proteins which are crucial for actin microfilament dynamics. their ubiquity, abundance, and necessity for life in higher organisms underscore their general importance. despite their small size, their functions are amazingly diverse. through binding to numerous protein ligands, profilins are components of complex protein networks. interactions with components of the phosphatidylinositol cycle and the rac-rho pathway implicate them as a link through which the actin cytoskeleton communicates with the major signalling pathways of the cell. accordingly, reducing the amount of profilin protein, e.g. by gene deletion, often has severe or even fatal consequences on the viability of the afflicted organism. profilins constitute a large and diverse protein family. multiple isoforms exist in many species, being encoded by separate genes, or in some cases translated from mrna splice variants. in animals and higher plants, isoforms may be expressed in a tissue-specific manner. moreover, profilins are found at different subcellular locations. enrichment at dynamic plasma membranes was confirmed for various cells types. also, profilins were observed in association with internal membranes involved in vesicular transport. finally, profilins are constituents of the cell nucleus. although the overall structure is conserved, sequence homologies between profilins from different species, and also between different isoforms from the same organism, are low. it was, nevertheless, reported that the overall functional properties of different profilins are similar, and that one isoform can be interchanged with another one from quite a distant source. on the other hand, structural differences, which determine in vitro affinities for various ligands, preferential protein complex formation in different cell types, and different subcellular locations may be important clues of divergent, possibly non-overlapping in vivo functions of different isoforms. despite extensive studies, the significance of the multiple profilins, their tissue-specificity and distinct subcellular locations have remained enigmatic. at least four different profilins were demonstrated to be present in the mammalian testis, a complexity which was not observed in somatic tissues. the mammalian testis may, thus, serve as a model to question whether profilin isoforms may fulfil distinct functions. profilin- is ubiquitous and essential for cell viability; its expression in all cell types of the testis, including spermatogenic cells, thus seems obvious. in comparison, profilin- is predominantly found in the nervous system and has acquired more specialized functions in regulating neuronal activity; it may represent a cell type-specific isoform also in the testis. while pfn and pfn were both demonstrated in the somatic sertoli cells, a third isoform, profilin-, is expressed solely in spermatids. most recently, profilin- was characterized as a novel isoform. it shows less than 30% amino acid identity with the other mammalian profilins; still, database searches produced significant alignments with the conserved profilin domain. pfn is also highly expressed during spermatogenesis, but is distinct from pfn in its temporal expression pattern. based on sequence comparisons, it was speculated that the testis-expressed pfn and pfn might have altered binding capacities for actin and proline-rich ligands. however, such diverse characteristics remained to be experimentally shown. in the present study, using yeast two-hybrid interaction assays and various biochemical methods, the binding capacities of pfn and pfn for proline-rich ligands, actin, and phosphoinositides were studied in comparison with pfn and pfn subcellular locations in differentiating spermatids were studied by immunofluorescence. three-dimensional structural models were also built to explain the functional properties of pfn and pfn  pfn and pfn have different in vitro affinities for protein ligands actin monomers, poly-l-proline, and proline-rich proteins have been confirmed as in vitro ligands for each profilin isoform tested so far. in addition, in vivo interactions of various profilins have been verified with actin, and proline-rich proteins, including the vasodilator-stimulated phosphoprotein and the mammalian homologues of drosophila diaphanous, members of the formin gene family. in analogy with this, several databases of known and predicted protein-protein interactions suggested that the testis-expressed isoforms pfn and pfn would likewise associate with these ligands. we asked whether pfn and pfn indeed shared these affinities. protein lysates containing pet-expressed pfn and pfn proteins were employed in plp affinity chromatography. pfn bound to the plp affinity column; however, compared to pfn and pfn the association was weaker, since pfn was eluted from the plp column at to m urea. under the same experimental conditions, pfn failed to interact with plp, suggesting that different from the above predictions proline-rich protein ligands may not be targets for this isoform. since plp affinity chromatography is routinely employed to purify native as well as recombinant profilins, the lack of polyproline interaction caused complications in the purification of untagged pfn selective pair wise tests for yeast two-hybrid interaction, assaying potential differences of pfn and pfn in their affinities for specific proline-rich proteins, i.e. vasp and mdia, were performed as an independent method. pfn2a, which is known to bind vasp and p140mdia, was included as a positive control. expression of the lacz reporter was used as a more quantitative indicator for the strength of protein-protein interactions. mdia but not vasp, was confirmed as an in vitro binding partner of pfn. the interaction of mdia with pfn was weaker compared to pfn2a; parallel y2h assays employing pfn in comparison, were negative, confirming the results of the plp affinity chromatography. notably, our previous microarray analyses of human testis tissue had shown that at least five mdia3-encoding mrnas variants, including the one employed in the y2h assays here, were highly expressed in the testis and were most abundant in tissue samples, which contained post-meiotic germ cells. thus, mdia proteins may still represent in vivo interaction partners of pfn binding of the testis-specific profilin isoforms to β-actin was similarly tested by a pair wise y2h interaction, confirming pfn as an actin-binding isoform. parallel assays employing pfn were all negative, suggesting that neither proline-rich proteins nor actin were targets of this unusual isoform. the failure of pfn to interact with actin contrasts with all other profilins studied thus far, most of which bind actin monomers with micromolar affinity. the y2h method, however, is prone to false-negative discovery. by attaching domains from a transcription factor to the bait and prey proteins, true interactions may be missed if fusions place these attachments at important interacting actin and/or profilin interfaces. to confirm the y2h results by two independent methods, co-immunoprecipitation of β-actin and actin polymerization assays were performed. for co-immunoprecipitation pfn pfn and pfn were translated in vitro as n-terminal fusions to a c-myc-tag; a c-myc-antibody was employed to immunoprecipitate the profilins in the presence of ha-tagged β-actin. while pfn and pfn effectively co-precipitated with β-actin as evidenced by their retention by protein a beads, pfn failed to associate with β-actin also under these experimental conditions. in a control experiment, in the absence of profilins, β-actin was not precipitated with protein a beads. we probed the influence of recombinantly expressed gst-fusions of pfn and pfn on salt-induced actin polymerization using the pyrenyl fluorescence assay. labelled skeletal muscle g-actin was incubated with or without the purified gst-fused profilins. gst-pfn which served as a control, showed a significant retardation of actin polymerization. under the same experimental conditions, pfn also markedly reduced polymerization kinetics, albeit to a lesser extent. pfn on the other hand, did not influence the actin polymerization kinetics at all. in summary, concerning pfn protein interaction partners, our results confirmed and extended the results by hu et al., suggesting that pfn showed major hallmarks of the well characterized profilins. pfn on the other hand, did not bind the tested protein ligands in vitro and thus may have in vivo functions different from regulating actin dynamics. it should be noted that spermatids contain several actin-related proteins. recently, the actin-related protein arpm was identified as an interaction partner of pfn. the interaction of pfn and actin-related proteins expressed in spermatids deserves further evaluation. pfn and pfn differentially interact with phospholipids profilins interact with anionic phospholipids. individual phospholipids show distinct subcellular distributions and perform distinct biological roles, with steady-state concentrations of ptdinsp predominating at plasma membranes, ptdinsp on endosomes, and ptdinsp on the golgi. thus, through differential binding to these phospholipids in vivo, profilin isoforms may be selectively directed to functionally appropriate subcellular sites. selective interaction with phosphoinositides was indeed the first functional difference reported between different profilin isoforms. moreover, it was shown that binding to ptdinsp may effectively compete for plp interaction of profilin. more recently, neighbouring binding sites for these competing ligands have emerged which may explain the competing interactions. phospholipid interactions of gst-fusion proteins of pfn pfn and pfn respectively, were assayed by protein-lipid overlay. each of the recombinant fusions strongly bound to ptdinsp, and to a lesser extent to its phosphorylated products and ptdinsp. unlike gst-pfn, gst-pfn and gst-pfn both showed in vitro affinity towards ptdinsp, suggesting that in vivo, in addition to endosomes, pfn and pfn may be associated with the golgi apparatus. for gst-pfn a weak but reproducible binding to phosphatidic acid was additionally observed. pa is an abundant component of most cellular membranes. it is also a signalling lipid, since its regulated formation can constitute an important signal for many downstream responses, including actin polymerization in spermatozoa. in addition to this role, pa appears to regulate enzymes in phospholipase d pathways directly. the list of pa-binding proteins is rapidly expanding; however, pa-binding of profilins has not been previously reported. the specificity of the observed interaction must be considered critically as the isoelectric point of pfn is basic; still, gst-pfn did not bind pa under the same experimental conditions, despite of the highly basic character of pfn. pfn and pfn localize in the acroplaxome-manchette complex in spermatids cross-reactivity of the profilin antisera employed in immunostaining procedures was initially studied by western blot analysis using in vitro translated profilins. antisera generated against pfn and pfn did not cross-react; weak cross-reactivity of the anti-pfn antiserum was observed only with in vitro-translated pfn in protein extracts of human adult testis tissue, and also in rat testis samples collected during the first wave of rat spermatogenesis, anti-pfn and anti-pfn immunoreactive bands of ≈ 14-kda were seen only in those tissue samples which contained spermatids, confirming at the protein level the previously reported stage- and cell-type specific expression patterns of pfn and pfn. in comparison, anti-pfn and pfn immunoreactive bands were obvious in each testis sample analysed. our previous studies suggested that pfn and pfn accumulated near the acrosome-acroplaxome-manchette complex of differentiating spermatids. the acroplaxome is an f-actin/keratin 5-containing cytoskeletal plate, which anchors the acrosome to the spermatid nucleus. pro-acrosomal vesicles derived from the golgi apparatus are transported by f-actin- and microtubule-based molecular motors and dock and fuse along the acroplaxome. the manchette is a transient structure developed subjacent to the marginal ring of the acroplaxome. it consists of a perinuclear ring with inserted microtubules as well as associated f-actin. the manchette participates in the transport of cargoes to the developing spermatid tail and in nucleo-cytoplasmic trafficking during spermatid head shaping. indirect immunofluorescence of spermatids isolated from spermatogenic stage-specific rat seminiferous tubules was used to determine the localization of pfn and pfn throughout spermiogenesis. phalloidin-texas red was used to monitor the f-actin-containing acroplaxome; tubulin monoclonal antibody was used to determine the position of the manchette with respect to the acroplaxome. pfn immunoreactivity was mainly observed in the acroplaxome of round spermatids and may be correlated with the presence of f-actin. during the progression of spermiogenesis, pfn acroplaxome immunoreactivity gradually disappeared and became apparent in the developing manchette of spermatids step. coinciding with the initiation of manchette disassembly in s spermatids, pfn immunoreactivity was seen in the cytoplasm subjacent to the disassembling manchette. at this stage, the phalloidin-stained acroplaxome was devoid of pfn. a similar immunoreactive staining pattern was observed with anti-pfn serum in rat and also in human spermatids. in agreement with the immunoblotting data, normal rabbit igg controls did not generate a fluorescence signal. panel m in figure 6/ summarizes diagrammatically the localization of pfn and pfn first in the acroplaxome and later in the manchette during spermatid head development. these observations suggested that, despite their differing functional characteristics in vitro, the location sites of pfn and pfn proteins in spermatids were similar. in vivo functions of the tissue- and cell type-restricted isoforms, however, remain obscure. transgenic mouse models may help to clarify whether and how pfn and pfn impact on acrosome formation and/or spermatid head shaping. structural models explain the observed biochemical properties of pfn and pfn4 in order to gain a deeper understanding of the observed functional differences, homology models were built for human pfn and pfn and compared to the known structures of human pfn and pfn2a. the sequence alignments of all known human profilins are shown in figure 7a, and those used for modelling in additional file both pfn and pfn are predicted to be folded like other profilins, with a central 7-stranded antiparallel beta-sheet covered on both faces by two alpha helices. the pfn sequence, however, is ten amino acids shorter which is structurally reflected by three loops, i.e. β1β β4β and β5β being shorter in the pfn protein. one of the loops, β5β is also shorter in pfn the poly-l-proline binding site the plp binding site of profilins is formed by conserved aromatic amino acids located in the n- and c-terminal helices, such that the proline-rich peptide will bind to a groove between the two helices, interacting closely with the aromatic residues via hydrogen bonds and ch-pi interactions. for example, five conserved aromatic residues and also asn are responsible for the plp interaction of pfn2a, and pfn is highly similar. the two aromatic residues on the c-terminal helix are not present in pfn which could lead to an altered specificity and/or lower affinity towards proline-rich sequences, as also evidenced by our binding assay. in pfn of the abovementioned aromatic residues, only tyr is conserved. thus, in this isoform, the aromatic surface of other profilins necessary for plp binding is not present at all, which is in line with its observed inability to bind proline-rich sequences. while the overall sequence of pfn is more similar to the profilins of lower eukaryotes than to the other human/mammalian profilins, pfn seems to be the only family member so far, in which the entire plp binding site is lacking, suggesting that it may have other binding partners instead. the actin-binding site in pfn residues centrally involved in actin binding include phe his arg arg lys lys and tyr; these are fully conserved in pfn2a. in pfn this site is conserved, with some rather conservative amino acid sequence differences, such as the replacement of phe and tyr by leu and his respectively, compared to pfn2a. these structural differences may have lowered or altered the affinity of pfn towards actin to some degree, when compared to pfn and pfn2a. in the pfn sequences, in contrast, there is no conservation of the known actin-binding residues of other profilins, which is well in line with our experimental data described above. the ptdinsp binding site based on mutagenesis studies and the localisation of sulphate and phosphate ions in crystal structures, the binding site for ptdinsp has been approximately mapped in human pfn. it involves several conserved basic amino acid residues on the profilin surface, generating a large binding surface of positively charged residues. in pfn2a, residues likely to contribute to ptdinsp binding include arginines and and these binding determinants are largely conserved in pfn interestingly, arg and arg are among the few residues, which are conserved throughout the known human profilin isoforms, including pfn. in pfn the putative common ptdinsp binding site is well conserved. in pfn many basic residues are present as well, but the putative phospholipid binding site seems to have shifted. this structural difference may explain the distinct phospholipid binding characteristics of pfn.  we report functional differences between the testis-expressed profilins pfn and pfn as revealed by in vitro assays. at the same time, structural homology models were built for human pfn and pfn which explained their different functional characteristics. by various methods, mdia but not vasp, was identified as a novel in vitro binding partner of pfn while pfn did not bind plp or proline-rich proteins. so far, pfn seems to be the only profilin family member in which the entire plp binding site is lacking. moreover, different from pfn pfn did not interact with actin in three different in vitro assays deviating from all other profilins analysed thus far. actually, the structure of pfn does not retain the actin-binding site of other profilins. a much lower affinity for actin or actin-related proteins is thus conceivable, suggesting that pfn may perform in vivo functions, which are distinct from regulating actin dynamics. pfn and pfn were both capable of in vitro phosphoinositide interaction, but differed in their selectivity towards specific phospholipids. in pfn the putative common ptdinsp binding site of profilins is conserved while it seems to have shifted in pfn pfn and pfn coexist in the acroplaxome and the manchette of spermatids in a sequential developmental manner. they vanish gradually from the acroplaxome when the manchette fully assembles and spermatid head shaping is in progress. although the presence of pfn can be correlated with the presence of f-actin and actin-related proteins in the acroplaxome, and to a lesser extent in the manchette, the visualization of pfn in the acroplaxome-manchette complex is intriguing in light of our finding that it does not interact with actin in vitro. an f-actin independent role of pfn may suggest novel functions related to the process of spermatid head shaping, and may have significant clinical implications in understanding idiopathic causes of male infertility associated with abnormal sperm head shaping. 
the testis-specific isoform pfn showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. the testis-specific isoforms profilin- and profilin- may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin- and profilin-. immunofluorescence microscopy of pfn and pfn revealed distinct subcellular locations in differentiating spermatids. pfn in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. β-actin, phosphoinositides, poly-l-proline and mdia but not vasp, were confirmed as in vitro interaction partners of pfn in parallel experiments, pfn bound to selected phosphoinositides but not to poly-l-proline, proline-rich proteins, or actin.
 multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. the testis-specific isoforms profilin- and profilin- may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin- and profilin-.  ligand interactions and spatial distributions of pfn and pfn were compared by biochemical, molecular and immunological methods; pfn and pfn were employed as controls. β-actin, phosphoinositides, poly-l-proline and mdia but not vasp, were confirmed as in vitro interaction partners of pfn in parallel experiments, pfn bound to selected phosphoinositides but not to poly-l-proline, proline-rich proteins, or actin. immunofluorescence microscopy of pfn and pfn revealed distinct subcellular locations in differentiating spermatids. both were associated first with the acroplaxome and later with the transient manchette. predicted 3d structures indicated that pfn has the actin-binding site conserved, but retains only approximately half of the common poly-l-proline binding site. pfn in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data.  the testis-specific isoform pfn showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. pfn on the other hand, did not interact with actin or polyproline in vitro. rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation. 