The Multi‐PDZ domain protein MUPP1 as a lipid raft‐associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

Abstract

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca 2þ chelator which allows a controlled release of acrosomal Ca 2þ. The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.

CaMKIIα interacts with multi-PDZ domain protein MUPP1 in spermatozoa and prevents spontaneous acrosomal exocytosis

“…Fig. 2C shows that in cortex-(left panel) and in sperm-derived preparations (right panel), total CaMKII␣ as well as the raft marker protein caveolin-1 (Anderson, 1998) co-migrated with the Triton-X-100-insoluble pellet, indicating colocalization with MUPP1 in detergent-resistant membrane fractions (Ackermann et al, 2008). A similar lipid raft targeting was also observed for the sperm-derived Thr286-P CaMKII␣ (data not shown).…”

“…In these cells, MUPP1 is involved in recruiting molecules that control the initial tethering and/or docking of the acrosomal vesicle at specific sites of the plasma membrane (Ackermann et al, 2008). In vitro, MUPP1 interacts with distinct isoforms of the neuronal Ca 2+ -calmodulin kinase II (CaMKII) (Krapivinsky et al, 2004), a holoenzyme of several protein subunits (␣, ␤, ␥ and ␦), encoded by four closely related genes (Hudmon and Schulman, 2002b).…”

“…In vitro, MUPP1 interacts with distinct isoforms of the neuronal Ca 2+ -calmodulin kinase II (CaMKII) (Krapivinsky et al, 2004), a holoenzyme of several protein subunits (␣, ␤, ␥ and ␦), encoded by four closely related genes (Hudmon and Schulman, 2002b). In analogy to the fact that the CaMKII is functionally active in recruiting synaptic vesicles to the active zone of the presynaptic nerve terminal LealOrtiz et al, 2008), the MUPP1-controlled recruitment of the acrosomal vesicle to the plasma membrane (Ackermann et al, 2008) might be mediated by MUPP1-associated CaMKII (Krapivinsky et al, 2004). However, it is also conceivable that a CaMKII-catalyzed phosphorylation reaction is responsible for 'freezing' the acrosomal vesicle in an intermediate pre-assembled fusion state (Wang, 2008), thereby preventing accidental spontaneous acrosomal secretion.…”

“…Since MUPP1, which binds CaMKII␣ in in vitro translated systems (Krapivinsky et al, 2004), is also concentrated in Triton-X-100-insoluble plasma membrane microdomains derived from brain and sperm preparations (Ackermann et al, 2008;Krapivinsky et al, 2004), we asked whether both proteins were colocalized to detergent-insoluble membrane clusters in sperm cells. Therefore, we extracted rat brain cortex tissue and freshly isolated epididymal rat spermatozoa with Triton X-100.…”

“…A biotinylated antimouse IgG as well as fluorescein avidin were purchased from Vector Laboratories (Burlingame, CA); Alexa-Fluor-546-conjugated goat anti-rabbit antibody was from Invitrogen (Karlsruhe, Germany). Anti-MUPP1 antibody was generated against a protein region between PDZ domains 5 and 6 of MUPP1 (Ackermann et al, 2008). The KN-CaMKII inhibitors 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93), as well as its inactive analogue 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine phosphate (KN92) (Marley and Thomson, 1996), and 1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl piperazine (KN62) (Tokumitsu et al, 1990), which competitively interact with the calmodulinbinding site of CaMKII (Sumi et al, 1991) were from Calbiochem-Novabiochem (Bad Soden, Germany); likewise, the non-cell-permeable non-phosphorylatable CaMKII-peptide-substrate inhibitor autocamtide-2 inhibitory peptide II (AIPII) was derived from the autoinhibitory region of CaMKII (Ishida et al, 1995;Ishida et al, 1998) and its cell-permeable analogue fused to the antennapedia transport peptide sequence (Passafaro et al, 1999), as well as the CaM antagonist W7 [8 (N-86-aminohexyl)-5-chloro-1-naphthalenesulfonamide] (Hidaka et al, 1981), the phosphatase inhibitor okadaic acid (Cohen et al, 1990) and a protease inhibitor cocktail III.…”