The Acrosome-Acroplaxome-Manchette Complex and the Shaping of the Spermatid Head*

Review Arch Histol Cytol, 67 (4): 271-284 (2004)

The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head*

Abraham L. Kierszenbaum and Laura L. Tres

Department of Cell Biology and Anatomical Sciences, The Sophie Davis School of Biomedical Education/ The City University of New York Medical School, New York NY 10031 USA

Summary. A combination of exogenous contractile forces stack of Sertoli cell F-actin-containing hoops applied to the generated by a stack of F-actin-containing hoops embracing elongating spermatid head. A tubulobulbar complex, formed the apical region of the elongating spermatid nucleus and an by cytoplasmic processes protruding from the elongating endogenous modulating mechanism dependent on the sper- spermatid head extending into the adjacent Sertoli cell, is matid-containing acrosome-acroplaxome-manchette com- located at the concave side of the spermatid head. The tubu- plex may play a cooperative role in the shaping of the sper- lobulbar complex might provide stabilizing conditions, matid head. In addition, the manchette is a key element in together with the actin-afadin-nectin-2/nectin-3 adhesion the transport of vesicles and macromolecules to the centro- unit, to enable sustained and balanced clutching exogenous some and developing spermatid tails as well as in nucleo- forces applied during the elongation of the spermatid head. cytoplasmic transport. The proposed model of spermatid head shaping is based on: 1) currently known structural and molecular components of the F-actin hoops, the main Introduction cytoskeletal element of the Sertoli cell ectoplasmic special- izations; 2) the molecular features of acrosome biogenesis; 3) One of the major causes of male infertility is the abnormal the assembly of a subacrosomal cytoskeletal plate called the development of the spermatid into a mature and motile acroplaxome; and 4) the spatial relationship of the acro- sperm. Globozoospermia (round-headed sperm) and coiled some-acroplaxome complex with the manchette, a transient sperm tails are two severe abnormalities that lead to infer- microtubular/actin-containing structure. During acrosome tility. Globozoospermia correlates with defective acrosome biogenesis, the acroplaxome becomes the nucleation site to biogenesis and tail defects that have a disruption in the which Golgi-derived proacrosomal vescicles tether and fuse. intramanchette (IMT, Kierszenbaum, 2002) and intrafla- The acroplaxome has at least two functions: it anchors the gellar transport systems (IFT, Rosenbaum and Witman, developing acrosome to the elongating spermatid head. It 2002) of molecules to the centrosome and the developing may also provide a mechanical scaffolding plate during the tail. Recent research has provided molecular data concern- shaping of the spermatid nucleus. The plate is stabilized by a ing the impact of acrosome biogenesis on spermatid nuclear marginal ring with junctional complex characteristics, shaping, in particular on the relationship of deficient acro- adjusting to exogenous clutching forces generated by the some development and round-headed sperm. Two exam- ples are the acrosome-deficient Hrb (Kang-Decker et al., 2001; Kierszenbaum et al., 2004) and the GOPC-deficient Received September 30, 2004 (Yao et al., 2002) mice mutants. Hrb is a protein associat- *Supported by USPHS grants HD36282 (ALK) and HD36477 ed with the cytosolic surface of proacrosomal transporting (LLT) vesicles in round spermatids, and a lack of Hrb protein pre- vents the vesicles from fusing to form the acrosome. The Address for correspondence: Dr. Abraham L. Kierszenbaum, GOPC protein is localized in the trans Golgi region of Department of Cell Biology and Anatomical Sciences, CUNY round spermatids, and a lack of this protein interferes with Medical School, 138th Street and Convent Avenue, Harris Hall the transport of vesicles from the Golgi apparatus to the Suite 306, New York NY 10031 USA acrosomal sac and fusion to form the acrosome. Spermatids Phone: +1-(212) 650-6811; Fax: +1-(212) 650-6812 in both mutants share a common feature: Golgi-derived E-mail: [email protected] proacrosomal vesicles attach to and align along the 272 A.L. Kierszenbaum and L.L. Tres:

acroplaxome, an actin-keratin-containing cytoskeletal plate the shaft of each tubulobulbar extension. It was proposed anchoring the developing acrosome to the nuclear envelope that the tubulobulbar-Sertoli cell ectoplasmic specialization (Kierszenbaum et al., 2003a). In the Hrb mutant, proacro- assembly may stabilize the spermatid heads undergoing somal vesicles fuse forming a flat sac, called pseudoacro- elongation (Russell, 1979). More recently, it has been pro- some, while the acroplaxome is deficient in the intermedi- posed that the tubulobulbar complex participates in both the ate filament protein called Sak57 (for spermatogenic disassembly of the ectoplasmic specializations and inter- cell/sperm-associated keratin with a molecular mass of 57 nalization of Sertoli cell-spermatid adhesion molecules kDa, Kierszenbaum et al., 1996), an ortholog of keratin 5 during spermiation (Guttman et al., 2004). (K5) (Kierszenbaum et al., 2003a). Nucleopodes, protru- From the foregoing account, it can be envisioned that an sions of the spermatid nucleus with a bulb-shaped ending exogenous contractile component, represented by Sertoli linked by a stalk to the main nuclear structure, develop at cell ectoplasmic specialization, and an endogenous compo- the Sak57/K5-deficient acroplaxome (Kierszenbaum et al., nent, represented by the acrosome-acroplaxome-manchette 2004). In the GOPC, proacrosomal vesicles are also seen complex, may interact to modulate the shaping of the sper- attached to the acroplaxome while an acrosome fails to matid head. The interaction of exogenous and endogenous develop (Yao et al., 2002). components may require a relatively stable head, a function When acrosome biogenesis is in progress, the transient presumably fulfilled by the tubulobulbar complex anchored microtubule-containing manchette develops caudally to the in the adjacent Sertoli cell. acrosome. A perinuclear ring, the insertion site of man- Details of the spermatid head shaping mechanism are not chette microtubules, assembles adjacent to the marginal fully understood but pieces of the puzzle are beginning to ring of the actoplaxome from which it is separated by a nar- emerge. In this review article, we focus on the mechanism row belt-like groove. A number of elongating spermatids of acrosome biogenesis and IMT involving the transport of from the mutant mouse azh (for abnormal sperm head- Golgi-derived vesicles containing the Rab27a/b receptor shape, Meistrich, 1993) display an abnormally constrict- and mobilized by the F-actin-dependent motor myosin Va ing manchette perinuclear ring as well as severe nuclear (Kierszenbaum et al., 2003b). These two events, together deformities at the acroplaxome site (Kierszenbaum et al., with the acroplaxome and the function of the ectoplasmic 2003a). In addition, spermatids and sperm from the azh specializations of the adjacent Sertoli cell, suggest a poten- mutant mouse have a lasso-like coiled tail with a high fre- tial mechanism of spermatid head shaping based on obser- quency of head dislocation and decapitation (Mochida et vations in mouse mutants displaying globozoospermia. It al., 1999; Kierszenbaum and Tres, 2002). A non-functional should be noted that in addition to vesicle transport, Hook-1 gene, predominantly expressed in azh spermatids, cytosol-derived protein cargos bound to protein rafts can be has been proposed as being responsible for the azh abnor- mobilized by molecular motors by IMT (see Kierszenbaum, mal spermatid and sperm phenotype (Mendoza-Lujambio et 2002). Therefore, both vesicle and protein cargo transport al., 2002). The presence of the Hook-1 protein, which is might take place simultaneously within the manchette and truncated in the azh mutant, appears to establish a link along the axoneme. between the membrane of vesicles and microtubules. Spermatids develop a tubulobulbar complex at the con- cave side of the elongating head, which projects into The Rab27a/b-MyRIP-myosin Va complex and matching infoldings of the adjacent Sertoli cells (Russell organelle transport and Clermont, 1976). Each tubular element of the tubu- lobulbar complex is cuffed by F-actin bundles; each bulbar Acrosome biogenesis consists of the transport of Golgi- element is associated to a piece of the Sertoli cell endoplas- derived vesicles containing acrosomal hydrolytic proteins mic reticulum (Guttman et al., 2004). The Sertoli cell region to the acrosomal sac, which is associated to one of the poles associated with the tubulobulbar complex can be regarded of the spermatid nucleus. The opposite pole of the nucleus as a confined modification of Sertoli cell ectoplasmic spe- harbors the centrosome, which gives rise to the axoneme, cializations, consisting of the plasma membrane, harboring one of the components of the developing spermatid tail. the afadin/nectin-2 complex (Mueller et al., 2003; Guttman The transport of proacrosomal vesicles requires micro- et al., 2004), F-actin bundles and associated cisternae of the tubule-based and actin-based motor proteins (kinesin/dynein endoplasmic reticulum as predominant elements. A clear and myosin Va, respectively). Vesicles transported by the distinction is the planar arrangement of Sertoli cell ecto- myosin Va motor require three elements: a vesicle receptor plasmic specializations at the convex side of the elongating (Rab27a/b), a molecular motor (myosin Va), and a molecu- spermatid head and with respect to the concave side, where lar motor recruiter (MyRIP; for myosin Va-Rab interacting the specializations adopt a folded arrangement in parallel to protein). Figure 1 illustrates the localization of myosin Va Spermatid head shaping 273

Fig. 1. Localization of myosin Va in the Golgi-acrosome-acroplaxome region and chromatoid body region and in the manchette of rat spermatids (A–G). The localization of Rab27a/b is shown in the Golgi and acrosome-acroplaxome region (H–K).

and Rab27a/b in the Golgi and acroplaxome region (in par- 2001). A mutation in Tg737, the gene encoding polaris, is ticular in the marginal ring) as well as myosin Va in the associated with immotile cilia and abortive sperm axoneme. manchette (Fig. 1E–G). In contrast, proteins not enclosed Therefore, a defective protein raft complex may hamper the in vesicles and synthesized in the cytosol can be bound to a timely delivery of molecules to the centrosome and protein raft and transported by molecular motors to the cen- developing spermatid tail. trosome and the developing sperm tail along microtubules Rab27a/b are members of a large family of Ras-related of the manchette (see Kierszenbaum, 2002 and references small GTPases. Rabs have distinct subcellular localizations therein). One of the components of the raft protein complex and are believed to regulate specific steps of intracellular is the polaris, found in the manchette (Taulman et al., trafficking. Rab27a is unique at present because it is the 274 A.L. Kierszenbaum and L.L. Tres:

Fig. 2. Gene expression determined by RT-PCR of the molecular motors myosin Va and Myosin VIIa, the vesicle receptors Rab27a and Rab 27b and the molecular motor linkers MyRIP and melanophilin in mouse testis. The numbers at the left indicate the molecular marker size in kilobases (kb). The base pair (bp) size of each transcript is indicated.

only known Rab associated with disease in humans (Seabra link between myosin tails and their cognate organelle. et al., 2002). Loss-of-function mutations in the Rab27a Genetic, morphologic, and biochemical analysis of three gene result in Griscelli syndrome, an autosomal disorder mouse coat color mutants, dilute, ashen, and leaden, gave characterized by partial cutaneous albinism and immunode- some clues regarding the mechanism regulating the binding ficiency due to the failure of cytotoxic T cells to release the of one class of myosin V to melanosomes, the pigment con- contents of their lytic granules. A natural mouse mutant, taining granules of skin melanocytes. The dilute locus in ashen (Rab27a¯), has a mutation in Rab27a and exhibits the mice encodes the murine ortholog of myosin Va. There are same phenotypic features of Griscelli syndrome patients two dilute mutants: dilute-lethal, with a severe neurological such as partial albinism and immunodeficiency (Wilson et condition leading to neonatal death due to loss of function, al., 2000). Rab27a is associated with melanosomes in pig- and dilute-viral, that exhibits only pigment dilution and mented cells of skin and retina and regulates melanosome expresses an alternative spliced form of myosin Va. transport via its interaction with actin-based cellular motors Myosin Va and myosin VIIa do not interact directly with such as myosin Va and myosin VIIa (Seabra et al., 2002; Rab27a but require linker proteins, melanophilin and Wu et al., 2002; Tolmachova et al., 2004). MyRIP, respectively, that bind GTP-bound Rab27a through Myosins share a common structure and are composed of a conserved N-terminal domain, and myosins through a three modules: the head domain comprising the motor medial domain (El-Amraoui et al., 2002; Fukuda et al., domain, which hydrolyzes ATP to power movement along 2002; Wu et al., 2002). Melanophilin and MyRIP are F-actin; the neck domain, which acts as a lever arm and products of the leaden gene. Recently, we reported the binds regulatory light chains such as calmodulin; and the expression of MyRIP (previously designated Slac2-c) in tail domain, which is highly divergent among the different mouse testis (Kierszenbaum et al., 2003b). MyRIP (or classes of myosin, serves to bind a specific cargo. Some Slac2-c, a homologue of Slac2-a, Kuroda et al., myosins are processive (i.e., can perform many consecutive 2002a, b) is widely expressed in most tissues and has the steps along F-actin without dissociating). Other myosins following characteristics: 1) it interacts specifically with can exert tensions between actin filaments, or actin Rab27a/Rab27b and myosin Va/myosin VIIa; 2) it displays filaments and membrane domains. an actin binding site at the C-terminal region; and 3) it is The divergent tail domain of myosins contains the neces- highly expressed in brain, lung, and testis (Fukuda and sary information for targeting cargos to specific cell com- Kuroda, 2002). Collectively, the ashen, dilute, and leaden partments. Until recently, little was known concerning the gene products may be responsible for the recruitment of Spermatid head shaping 275

melanosomes to actin filaments. The absence of any of motor kinesin variants mobilize the vesicles in an antero- these gene products leads to a striking clustering of grade direction. The model is based on recently reported melanosomes around the nucleus of melanocytes. However, data from our laboratory (Kierszenbaum et al., 2003a,b, in spermatids, the switch cytoskeletal track model antici- 2004) and on additional data demonstrating a coexisting pates the coexistence of kinesin and myosin Va/myosin kinesin/myosin V heteromotor complex on Golgi-derived VIIa motors in the same vesicle; one motor becomes inac- vesicles (Brown et al., 2004). tive when its companion motor binds to a filament. There- Rab27a is also required for secretion of lytic granules in fore, a molecular motor compensatory mechanism based on cytotoxic T cells (Menasche et al., 2000). In ashen mice, redundancy may ensure undisrupted vesicular transport and lytic granules polarize correctly but are unable to kill target a tug-of-war between motors does not occur. Figure 2 cells due to impaired secretion of the lytic granules (Stinch- shows that mouse testis expresses the molecular motors combe et al., 2001). Melanocytes and cytotoxic T cells are myosin Va and myosin VIIa, the molecular motor recruiters only a subset of cell types expressing Rab27a. The Rab27a MyRIP and melanophilin, and the vesicle receptors Rab27a protein is expressed in the hematopoietic lineage, spleen, and Rab 27b. Therefore, the possibility of a compensatory lung, eye, pancreas and the gastrointestinal tract (Seabra et vesicle microtubule-based transport mechanism in ashen al., 1995; Barral et al., 2002). In addition, recent studies spermatids can be readily explored. Figure 3 provides a suggest a role of Rab27a in exocytosis of insulin and chro- summary of the sorting patways of Golgi-derived proacro- maffin granules in endocrine cells (Fukuda et al., 2002; Yi somal and non-acrosomal vesicles mobilized along et al., 2002). Rab27a is present in cortical granules aligned manchette microtubules and manchette-associated F-actin under the zona pellucida of mature mouse follicles (Tolma- as well as the molecular components of microtubule- and chova et al., 2004; Seabra M, personal communication). F-actin-dependent vesicle transporting machinery. There The presence of immunoreractive Rab27a/b and myosin Va are two interesting aspects illustrated in the diagram to be during the early steps of acrosome biogenesis has been emphasized. First, vesicles may remain linked to micro- reported (Kierszenbaum et al., 2003b, 2004; see Fig. 1) and tubules in a “holding pattern” until microtubule- and F- myosin Va can be found bound to Golgi-derived vesicles actin-based motors recruit them for transport. A candidate copurified with manchettes (Kierszenbaum et al., 2003b). vesicle-microtubule linker is Hook1, a protein found in the Collectively, these data indicate that the Rab27a/b-myosin manchette and encoded by the Hook1 gene. Hook1 is defec- Va complex plays a role in immunity, pigmentation and tive in the azh mouse mutant (Mendoza-Lujambio et al., reproductive function as well as other physiological activi- 2002), which produces sperm with abnormal head shape, ties (Tolmachova et al., 2004) with specific characteristics head dislocation, and “lasso-like” coiled tails (Mochida et varying from one cell to another. al., 1999). It is likely that in addition to Hook1, other vesi- cle-microtubule protein linkers may be present in the manchette. Second, the chromatoid body, a mass associated The temporal development of the acrosome- to nuclei of developing spermatids, is surrounded by acroplaxome unit and the manchette myosin Va-decorated vesicles (see Fig. 1A–D). This observa- tion suggests a possible function of the vesicles in the dis- Golgi-derived proacrosomal vesicles initiate acrosome bio- posal of nuclear material generated during spermiogenesis. genesis by fusing into a single acrosomal sac bound to the A “switch cytoskeletal track model” has been proposed nuclear envelope. After acrosome biogenesis starts, the (Kierszenbaum et al., 2003b) to account for the coexistence microtubule-containing manchette assembles as the sper- in the manchette of F-actin with microtubules and the cor- matid nucleus initiates its elongation. The manchette disas- responding vesicle motors (myosin Va/myosin VIIa and sembles upon completion of nuclear elongation and con- kinesin/dynein, respectively) for the transport of cargos densation (reviewed by Clermont et al., 1993). The role of (Fig. 4). The model suggests that Golgi-derived vesicles, the manchette in spermatid nuclear shaping has been ana- required for acrosome biogenesis and spermatid tail lyzed for many years (Cole et al., 1988; reviewed by development, can switch from an F-actin to a microtubule Meistrich, 1993). Recent biochemical and structural evi- track and vice versa. The competition between molecular dences support an IMT mechanism for the delivery of mole- motors may be dictated by engaging effector molecules, cules to the centrosome and developing spermatid tail such as MyRIP and melanophilin, which upon binding to (Rivkin et al., 1997; Kierszenbaum et al., 2002; Kierszen- Rab27a/b, recruit myosin Va or myosin VIIa. If MyRIP, or baum, 2002). However, direct evidence of IMT of specific melanophilin, fail to bind to Rab27a/b on Golgi-derived cargos needs to be experimentally shown. vesicles, myosin Va and myosin VIIa will not be recruited. Because of the tight acrosome-nuclear relationship, In a default pathway, the already present microtubule-based we have looked into a nuclear-acrosome anchoring 276 A.L. Kierszenbaum and L.L. Tres:

Fig. 3. Diagrammatic representation of the two major intramanchette vesicle pathways and the components of the micro- tubule- and F-actin-based vesicle-motor complexes (diagram not to scale). 1: The acrosome biogenesis vesicle pathway includes transporting vesicles from the endoplasmic reticulum to the Golgi. The Golgi generates two types of vesicles: proacrosomal and non-acrosomal vesicles. Proacrosomal vesicles are transported to the acroplaxome by actin-based and microtubule-based molecular motors, where they fuse and organize the acrosome. 2: Non-acrosomal vesicles associate with microtubules of the manchette and are transported by microtubule-based molecular motors to the centrosome region and the developing spermatid tail (not shown). 3: Non-acrosomal vesicles can also associate with F-actin present in the manchette and transported by F-actin-based molecular motors. Non-acrosomal vesicles can remain in a “holding” pattern associated to manchette microtubules by vesicle-microtubule linkers until recruited by microtubule- or F-actin-based molecular motors for further intramanchette transport. The chromatoid body becomes surrounded by non-acrosomal vesicles probably mobi- lized by intramanchette transport.

mechanism. We have identified a cytoskeletal plate, the filaments connecting a plaque associated with the descend- acroplaxome, linking the developing acrosome to the ing edge of the inner acrosomal membrane and an oppo- spermatid nucleus (Kierszenbaum et al., 2003a). The site plaque bound to the spermatid nuclear envelope (Fig. acroplaxome consists of F-actin, Sak57, an ortholog of 5). The persistence in the acroplaxome marginal ring of keratin 5 [K5] (Kierszenbaum et al., 2003a), and myosin myosin Va, presumably associated to Rab27a/b via the Va (Kierszenbaum et al., 2003b). The marginal ring of the effector MyRIP, together with the report that another acroplaxome displays a desmosome-like structure consist- Rab27 effector, Munc13-14, is thought to regulate SNARE ing of a bundle of Sak57 [K5]-containing intermediate in vesicle fusion (Shirakawa et al., 2004), raises the interest- Spermatid head shaping 277

Fig. 4. A: Diagrammatic representation of the mechanism of microtubule based protein and vesicle intramanchette trans- port and F-actin-based vesicle intramanchette transport. MyRIP or melanophilin, recruiters of the molecular motors myosin Va or myosin VIIa, may be determinants for transporting vesicles allowing them to switch from a microtubule-actin track switch (indicated by the yellow arrow) and vice versa depending on the distance to be traveled and the delivery speed of the vesicle cargo (see text for additional details). The microtubule mantle of the manchette consists of tube-like assemblies formed by concentrically arranged microtubules connected to each other by inter-microtubule linkers. Adjacent tube-like formations may also be stabilized by linker. F-actin is depicted as distributed along the tube-like formations. B: Immuno- gold electron microscope localization ofβ-actin in the Sertoli cell ectoplasmic specialization, the spermatid acroplaxome, and along the microtubules of the manchette (denoted by circles). (Illustration from Kierszenbaum et al., 2003b)

ing possibility that Rabs may recruit hierarchically a set of reaction at fertilization. However, details of how this hierar- effectors and thereby coordinate proacrosomal vesicle chy could be achieved remain to be determined. transport with tethering to the F-actin component of the The lack of an acrosome in Hrb mutant spermatids does acroplaxome and fusion to form the acrosome. Therefore, it not prevent the development of the acroplaxome (Kierszen- is possible that the Rab27a/b-MyRIP-myosin Va complex baum et al., 2004). Yet, the acroplaxome in the Hrb mutant participates in both acrosome biogenesis and acrosome contains F-actin and myosin Va but is deficient in Sak57 278 A.L. Kierszenbaum and L.L. Tres:

Fig. 5. Diagrammatic representation of the acroplaxome plate and the marginal ring. The electron micro- graph illustrates the components of the acroplaxome marginal ring region. See Kierszenbaum et al. (2003a) for additional details.

[K5]. In wild-type and Hrb mutant spermatids, myosin Va acroplaxome location several nuclear protrusions, desig- is prominent in the Golgi region where proacrosome vesi- nated nucleopodes (Kierszenbaum et al., 2004). The cles are clustered (Fig. 1H–K). In the Hrb mutant, myosin acroplaxome appears to provide a focal point for myosin Va-bound proacrosome vesicles coalesce along the Va-driven proacrosome vesicles to congregate and form an acroplaxome and form a flat sac, the pseudoacrosome acrosome in wild-type spermatids or a pseudoacrosome in (Kierszenbaum et al., 2004). As spermiogenesis advances, Hrb mutant spermatids. round-shaped spermatid nuclei of the mutant display at the Spermatid head shaping 279

(Russell and Clermont, 1976; see Fig. 6). Although the pre- Intramanchette transport of cargos by actin- cise function of the tubulobulbar complex has not been and microtubule-based molecular motors experimentally determined, several possibilities have been considered. One of the possibilities is an attachment device In addition to the acroplaxome, F-actin is also present in between Sertoli cells and spermatids, which disappears the manchette (Mochida et al., 1998; 1999; Kierszenbaum upon spermiation (Russell and Clermont, 1976). Another et al., 2003b). The finding of discreteβ-actin immunore- possibility is that tubulobulbar complexes represent a activity along interconnected tube-like assemblies of dynamic modification of the preexisting Sertoli cell ecto- manchette microtubules (Fig. 4), together with myosin Va- plasmic specializations tailored to facilitate mechanical decorated vesicles associated with the manchette (Fig. events within the seminiferous epithelium during spermato- 1E–G), suggest that a dual microtubule- and actin-based genesis, but not necessarily restricted to spermiogenesis motor protein mechanism may operate to mobilize vesicle (Russell, 1979). It has also been proposed that tubulobul- cargos by IMT. Why might two transport systems be bar complexes are involved in a dual function: the dis- necessary in developing spermatids? Models have been assembly of Sertoli cell ectoplasmic specializations as well proposed to explain how the microtubule- and actin-based as in the uptake of cell adhesion molecules to enable the transport systems may interact with each other. One model release of mature spermatids into the seminiferous tubular postulates a fast long-range transport of cargos mediated by lumen (Guttman et al., 2004). microtubules, in contrast with an actin-based short-range The tubulobulbar complexes may also play a role in local transport (Goode et al., 2000). By contrast, another spermatid head shaping. The elongating spermatid head model assumes that different types of motors operate simul- displays a convex curvature in contact with Sertoli cell taneously and that the resulting motion of a vesicle cargo ectoplasmic specializations, and a concave curvature facing results from a balance of the forces acting upon the cargo the tubulobulbar complex (Fig. 6). The Sertoli cell surface (Gross et al., 2002). An alternate mechanism is that intra- facing the convex side of the spermatid head displays sig- manchette cargos may “switch” from a microtubule to an nificant characteristics: 1) It contains F-actin bundles stabi- actin track and vice versa by exchanging a microtubule- lized by the actin-bundling protein espin (Bartles et al., based motor (kinesin- or dynein-like) for an actin-based 1996). 2) F-actin is the binding site of the afadin, a protein motor (myosin Va/VIIa). Such a mechanism may involve that links the cytoplasmic domain of nectin-2, forming an Rab27a/b-specific motor recruiting proteins (such as actin-afadin-nectin-2 adhesion unit (Mueller et al., 2003 MyRIP/melanophilin) to dictate the final destination of the and references therein). 3) The extracellular domain of cargo. Essentially, Rab27a/b may determine whether vesi- nectin-2 (on Sertoli cell surfaces) and nectin-3 (on sper- cle membrane domains are assembled for vesicle fusion or matid cell surfaces) establish a heterophilic binding part- transport. Our finding that manchette-associated vesicles nership, thereby stabilizing adjacent Sertoli cell-spermatid decorated with anti-myosin Va can be fractionated (Kierszen- surfaces (Ozaki-Kuroda et al., 2002). 4) The nectin-2/ baum et al., 2003b), opens new experimental possibilities nectin-3 association appear critical to spermiogenesis as for determining how manchette microtubules (and demonstrated by a disrupted Sertoli-spermatid association, presumably F-actin) may regulate the mobility of vesicle abnormal spermatid head shape morphogenesis and infer- cargos. tility following inactivation of nectin-2 gene expression The microtubule-based motor kinesin KIFC1 and the (Bouchard et al., 2000; Ozaki-Kuroda et al., 2002; Mueller actin-based motor myosin Va have been proposed as par- et al., 2003). 5) Theα6β1 integrin complex has been iden- ticipating in the transport of Golgi-derived vesicles and tified at the Sertoli cell ectoplasmic specialization (Palombi acrosome biogenesis (Yang and Sperry, 2003; Kierszen- et al., 1992); yet, the binding partner on adjacent spermato- baum et al., 2003b, respectively). It remains to be deter- genic cells has not been determined. 6) The afadin-nectin-2 mined how these two motors interact with distinct complex undergoes a gradient redistribution during the cytoskeletal systems during acrosome biogenesis. elongation of the spermatid head; the concentration of the afadin-nectin-2 complex at the Sertoli cell convex curva- ture declines in correspondence to an increase in concentra- The tubulobulbar complex, a mechanical stabi- tion along the tubulobulbar complexes (Guttman et al., lizer during spermatid head shaping 2004). From these data, it can be concluded that a complex and dynamic cell-cell adhesion and mechanical mechanism Tubulobulbar complexes consist of several projections stabilizes the positioning of the spermatids within the apical from the spermatid plasma membrane and cytoplasm into domain of the seminiferous epithelium as head elongation corresponding invaginations of the adjacent Sertoli cell is in progress. 280 A.L. Kierszenbaum and L.L. Tres:

Fig. 6. Diagrammatic representation of the tubulobulbar complex arising from the concave face of the elongating spermatids. The convex face of the spermatid is associated with a planar distribution of the F-actin hoops present in the Sertoli cell ectoplasmic specialization. The Sertoli cell-spermatid head interface at the convex and concave sides is occupied by afadin-nectin adhesion complexes. See Guttman et al. (2004) for additional structural details.

the spatial arrangement of stacked F-actin-containing hoops The acrosome-acroplaxome-manchette com- embracing the apical one-third of the spermatid head (Fig. plex and sperm head shaping: a hypothesis 7). The hoops may be kept in place by the F-actin-linked afadin/nectin-2 and the nectin-2/nectin-2 adhesion com- Components of the developing spermatid, such as the plexes. How do hoops apply an exogenous constriction manchette and centrosomes, have been fractionated force? Conventional myosin motors, capable of interacting (manchette, Mochida et al., 1999; centrosomes, Kierszen- with F-actin and exerting contractile forces, have not been baum et al., 2002) and approaches are now being developed found along the Sertoli cell ectoplasmic specializations to fractionate the intact acroplaxome (our unpublished data) (Vogl and Soucy, 1985). However, unconventional for molecular and biochemical analysis. Yet, mouse myosins may be present. Dynein-type and kinesin-type mutants provide at present a convenient approach to deter- molecules have been found in association with micro- mine how the acrosome-acroplaxome-manchette complex tubules adjacent to the inner face of Sertoli cell endoplas- and Sertoli cell ectoplasmic specializations might con- mic reticulum components of the ectoplasmic specializa- tribute to the shaping of the sperm head. tions (Guttman et al., 2000). It has been proposed that Here, we present a model for spermatid head shaping molecular motors linked to microtubules adjacent to the based on structural data derived from normal rats and wild- endoplasmic reticulum at the ectoplasmic specialization site type and mutant mice. The model relies on two elements. could mobilize the F-actin-containing plaques in an up The first element, the exogenous clutch, is represented by direction (toward the seminiferous tubular lumen) and Spermatid head shaping 281

Fig. 7. Role of exogenous and endogenous clutching forces and aspects of intramanchette and intraflagellar transport oper- ating during the elongation of the spermatid head. A: The head of an elongating spermatid is enveloped by F-actin-contain- ing parallel hoops and associated proteins exerting an exogenous clutching effect on the spermatid nucleus by gradual reduction of the hoop length (indicated by facing arrows). The marginal ring of the acroplaxome and the adjacent perinu- clear ring of the manchette adjust their diameter in compliance to the elongation of the spermatid nucleus (endogenous clutch effect). A groove belt denotes the boundary between the acroplaxome marginal ring and the manchette perinuclear ring. B: In the elongated spermatid, the head is surrounded by F-actin hoops adjacent to endoplasmic reticulum cisternae on the Sertoli cell side. The shrinking marginal ring of the acroplaxome, anchored to the descending caudal edge of the acrosome and to the nuclear lamina as well as the narrowing perinuclear ring of the manchette, reach the caudal region of the nucleus. The manchette extends into the cytoplasm and is engaged in the intramanchette transport of vesicles and proteins to the centrosome and the developing tail. The manchette then disassembles. The progressive development of the axoneme and associated outer dense fibers and fibrous sheath (not shown) is facilitated by intraflagellar transport of the corresponding molecular building blocks.

down direction (toward the seminiferous tubular wall) to alternative to be explored is the depolymerization-poly- facilitate Sertoli cell displacement (Guttman et al., 2000). merization of F-actin, which could threadmill F-actin-con- However, a bidirectional up-down movement of F-actin taining hoops. Two regulatory molecules of F-actin assem- bundles is unlikely to generate constriction forces capable bly/disassembly are gelsolin and profilin. Gelsolin, a of participating in spermatid head shaping. An attractive protein that both caps and severs F-actin, is found in Sertoli 282 A.L. Kierszenbaum and L.L. Tres:

cell ectoplasmic specializations (Guttman et al., 2002). A is the finding in the acrosome-deficient Hrb mutant of a testis-specific isoform of profilin, a protein which blocks number of nucleopodes developed at the Sak57/K5-defec- the incorporation of G-actin into filamentous actin and also tive actoplaxome site (Kierszenbaum et al., 2004). Both the facilitates actin polymerization by facilitating the exchange marginal ring of the acroplaxome and the perinuclear ring of bound ADP for ATP in G-actin, has been reported (pro- of the manchette may act as endogenous constrictors in a filin-3, Braun et al., 2002). Given the existence of a number sleeve-like manner, steering the elongation of the spermatid of testis-specific actin-binding proteins (Kierszenbaum et nucleus as both rings descend from the caudal two-thirds al., 2003a and references therein), it is likely that an under- of the elongating spermatid head. The effective action of standing of the nucleation and disassembly of F-actin in the exogenous contractile forces requires the steady positioning Sertoli cell ectoplasmic specializations and the acro- of the spermatid head. In this regard, steady positioning plaxome can shed light on the mechanistic functions of may be provided by the tubulobulbar anchoring complex these two structures during spermatid head shaping. (Fig. 7) acting as a lever in concert with the interacting The second element in the hypothesis of spermatid head nectin-2/nectin-3 heterotypic complex andβ1 integrin. The shaping, the endogenous clutch, is the acrosome- various elements of the proposed model might provide a acroplaxome-manchette complex (Fig. 7). The acrosome is framework for further studies leading to the complete char- tightly bound to the acroplaxome plate, which in turn is acterization of the molecular components of the acroplax- anchored to the nuclear envelope of the elongating sper- ome and the manchette in a number of mutant mice where matid nucleus. The acroplaxome seems to play two impor- the development of the acrosome and the integrity of the tant roles. First, the acroplaxome is the site where proacro- acroplaxome and the manchette are defective. somal vesicles tether to the F-actin component after being mobilized by the Rab27a/b-myosin Va complex along F- actin tracks (Kierszenbaum et al., 2004) and microtubule tracks by kinesin molecular motors (Yang and Sperry, References 2003). Second, a specific feature of the acroplaxome is its marginal ring lodged in a shallow circumferential indenta- Barral DC, Ramalho JS, Anders R, Hume AN, Knapton HJ, tion of the spermatid nucleus. The indentation displays a Tolmachova T, Colinson LM, Goulding D, Authi KS, narrow dense plaque spanning across the indentation and Seabra MC: Functional redundancy of Rab27 proteins linked to the nuclear lamina. The subacroplaxome position and the pathogenesis of Griscelli syndrome. J Clin Invest of the nuclear plaque coincides with a distinct chromatin 110: 247-257 (2002). imprint denoted by propidium iodide staining (see Fig. 1D). Bartles JR, Wierda A, Zheng L: Identification and charac- On the opposite site, a bundle of Sak57/K5-containing terization of espin, an actin-binding protein localized to intermediate filaments is anchored to a dense acrosomal the F-actin-rich junctional plaques of Sertoli cell ecto- plaque (see Fig. 5). The marginal ring and associated dense plasmic specializations. J Cell Sci 109: 1229-1239 plaques are reminiscent of a junctional belt linking mem- (1996). branes of adjacent epithelial cells. 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