Rab27 and Rab3 sequentially regulate human sperm PNAS PLUS dense-core granule exocytosis

Matías A. Bustos, Ornella Lucchesi, María C. Ruete, Luis S. Mayorga, and Claudia N. Tomes1

Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina

Edited* by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved June 6, 2012 (received for review December 27, 2011) Two so-called “secretory Rabs,” Rab3 and Rab27, regulate late vesicle to the acceptor membrane (7–11), and participate in the steps during dense-core vesicle exocytosis in neuroendocrine cells. priming of synaptic vesicles to a readily releasable state (12). Sperm contain a single large dense-core granule that is released by Rab3 (A, B, C, and D) and Rab27 (A and B) constitute the regulated exocytosis (termed the acrosome reaction) during fertil- two main subfamilies directly implicated in regulated exo- ization or on exposure to inducers in vitro. Sperm exocytosis uses cytosis. These “secretory Rabs” localize to vesicles and secretory the same fusion machinery as neurons and neuroendocrine cells, granules in a variety of secretory cell types (1, 8, 13–15). They with an additional requirement for active Rab3. Here we show control the recruitment, tethering, and/or docking of secretory that Rab27 is also required for the acrosome reaction, as demon- vesicles to the plasma membrane through interaction with strated by the inability of inducers to elicit exocytosis when strep- effectors (16). The coexistence of Rab3 and Rab27 on the same tolysin O-permeabilized human sperm were loaded with inhibitory membrane in several secretory cells raises the questions of func- anti-Rab27 antibodies or the Rab27–GTP binding domain of the tional redundancy, and if and how their roles are coordinated to effector Slac2-b. The levels of GTP-bound Rab27 increased on ini- regulate exocytosis. A handful of laboratories have addressed tiation of exocytosis, as did the proportion of GTP-bound Rab3A. these issues by investigating the behavior of both Rabs in par- We have developed a fluorescence microscopy-based method for allel. In one study, for instance, in PC12 cells, both overex- detecting endogenous Rab3A-GTP and Rab27-GTP in the acroso- pressed and endogenous Rab3A and Rab27A localized to dense- mal region of human sperm. Challenge with an inducer increased core vesicles (17). Silencing each Rab individually reduced the the population of cells exhibiting GTP-bound Rabs in this subcel- number of vesicles docked at the plasma membrane and exo- CELL BIOLOGY lular domain. Interestingly, introducing recombinant Rab27A cytotic events. Both phenomena were further reduced by si- loaded with GTP-γ-S into sperm elicited a remarkable increase in lencing Rab3A and Rab27A simultaneously, which led to the the number of cells evincing GTP-bound Rab3A. In the converse conclusion that these small cooperate to regulate the condition, recombinant Rab3A did not modify the percentage of docking of dense-core vesicles to the plasma membrane (17). Rab27-GTP–containing cells. Furthermore, Rab27A-GTP recruited Another study conducted in the same neuroendocrine cell line a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings sug- and published a year later found that overexpressed Rab3A gest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense- cycles continuously between the cytosol and membranes, and core vesicle exocytosis. that this cycling is not coupled to exocytosis (18). Unlike Rab3A, Rab27A does not exchange between granules and cytosol (18). Rab cascade | GTP-binding protein | Rab pull down | calcium | In the pancreatic β-cell line MIN6, Rab27A localizes primarily cyclic AMP/Epac to membranes, whereas Rab3A distributes between membranes and cytosol. A study found that overexpression of WT or GTPase- volution has endowed secretory cells with sophisticated and deficient Rab27A enhanced K+ depolarization-induced insulin Ehighly regulated exocytosis mechanisms that are recruited by release by 50–60%, whereas overexpression of the equivalent the intracellular calcium fluctuations elicited by extracellular Rab3A proteins had no effect (19). The authors concluded that signals. These cells contain vesicles that store and release se- Rab27A and Rab3A have different roles in insulin secretion. creted products. The machinery of regulated exocytosis is highly Likewise, a kinetic study demonstrated that β-cells from − − conserved among conventional Golgi-derived granules in exo- Rab27A / (ashen) mice, but not from Rab3A null mice, exhibit crine and endocrine cells, lysosome-related organelles in hema- defects in refilling certain pools of insulin granules in response to topoietic cells and melanocytes, and synaptic or synaptic-like depolarization (20). Ashen islets exhibit decreased insulin se- vesicles in neurons and neuroendocrine cells (1–5). To reach the cretion from undocked granules (21). In the most recent study fusion competent state, vesicles must undergo a series of matu- analyzing Rab3 and Rab27 simultaneously, both proteomic and ration steps that include their tethering and docking to the quantitative Western blot analyses of subcellular fractions pre- plasma membrane and priming of the fusion machinery on both pared from rat brain homogenates demonstrated the marked membranes. One of the best-known functions of the Rab enrichment of three isoforms of Rab3 (A, B, and C), as well as GTPases is to govern the initial attachment, or tethering, of the Rab27B, on synaptic vesicles. Subpopulations of Rab3A mole- membranes that are fusing (6). Current knowledge suggests that cules also localize to brain cytosol and plasma membrane, Rabs function as molecular switches that alternate between whereas Rab27B is not found in the cytosol (15). In a recent a GTP and membrane-bound “on” state and a GDP-bound, cy- review, the authors hypothesized that the roles of Rab3A and tosolic, “off” state. During the activation phase of the Rab cycle, a GDP/GTP exchange factor (GEF) releases GDP bound in the nucleotide pocket. Rab immediately binds GTP, which has a much higher cytosolic concentration than GDP. Author contributions: M.A.B. and C.N.T. designed research; M.A.B., O.L., and M.C.R. per- During the inactivation phase, hydrolysis converts GTP to formed research; M.A.B., L.S.M., and C.N.T. analyzed data; and C.N.T. wrote the paper. GDP, a reaction catalyzed by Rab’s intrinsic GTPase activity The authors declare no conflict of interest. assisted by GTPase-activating proteins (GAPs). Guanine nucle- *This Direct Submission article had a prearranged editor. otide dissociation inhibitors recycle GDP-bound Rabs back to 1To whom correspondence should be addressed. E-mail: [email protected]. the cytosol. GTP-bound Rabs recruit other proteins called This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. effectors, forming protein complexes that tether the transport 1073/pnas.1121173109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1121173109 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 Rab27B in the synaptic vesicle cycle are complementary, and antibody and the anti-Rab3A antibody and confirmed a lack of that these isoforms act in successive steps (22). cross-reactivity between their target proteins. In brief, we elec- The acrosome is a dense-core secretory vesicle that overlies trophoresed recombinant GST-Rab3A and GST-Rab27A and the nucleus of the mature spermatozoon. In response to physi- probed them on Western blots. The anti-Rab3A antibody detec- ological or pharmacologic stimuli, the acrosome undergoes a ted Rab3A, but not Rab27A, and vice-versa; an anti-GST anti- special type of calcium-dependent exocytosis termed the acro- body recognized both (Fig. S1A). some reaction (AR), which is a prerequisite for fertilization (23). Probing extracts of whole human sperm (Fig. 1A) and mouse We and others have shown that the AR relies on the same highly testis (Fig. 1C) with the anti-Rab27 antibody on Western blots conserved molecules and goes through the same stages as exo- revealed a single protein band with apparent molecular mass cytosis in neuronal, endocrine, and all other types of cells studied of ∼28 kDa corresponding to Rab27. We inferred that Rab27 is to date (reviewed in refs 24–26), so much so that some authors geranylgeranylated in sperm because it partitioned entirely into “ refer to the anterior region of the acrosomal cap as the acro- the detergent phase after Triton X-114 phase separation (Fig. ” somal synapse (27). Rab3A is present in the acrosomal region 1A). To determine its subcellular localization, we fractionated of human (28), rat, and mouse sperm (29); in the latter it is human sperm into membrane and cytosolic fractions and probed predominantly membrane-bound and disappears from cells that them on Western blots. Rab27 was predominantly membrane- have undergone the AR (30). Rab3A is required for the bound, with a negligible amount detected in the cytosol (Fig. AR triggered by calcium (31, 32), cAMP (33), and sphingosine 1B). All membrane-bound Rab27 partitioned into the detergent 1-phosphate (34) in human sperm. Treatment with AR inducers fraction after Triton X-114 phase separation (Fig. 1C). increases the levels of GTP-bound Rab3A (35). Here we combine biochemical with functional and micros- Rab27 Is Required for Human Sperm AR. Streptolysin O (SLO) copy-based methods to show that, like Rab3A, Rab27 is present permeabilization is a widespread methodology used in analysis in, localizes to the acrosomal region of, and is required for of dense-core granule exocytosis that we and others have applied exocytosis in human sperm. Whereas Rab27 is predominantly to studying the AR (28, 36–39). This method permits the in- membrane-bound, Rab3A binding to membranes increases when troduction of exogenous factors—which are not otherwise able sperm are challenged with AR inducers. Both Rabs exchange to cross membranes—into the cytosol through pores in the GDP for GTP in response to exocytosis initiators. By means of plasma membrane generated by SLO. Given the important role a protocol developed in our laboratory, we are able to show that of Rab3 in the AR and its coexistence with Rab27 in human Rab3A-GTP and Rab27-GTP localize to the acrosomal region of sperm, we predicted that Rab27 also would be required for the sperm head. More importantly, we present direct evidence sperm exocytosis. We tested this hypothesis and characterized that active Rab27 increases the exchange of GDP for GTP on ’ Rab3A. The molecular mechanism for this activation appears to Rab27 s site of action during human sperm AR by means of a plasma membrane permeabilization technique. To do so, we be through a Rab-GEF cascade, given that active Rab27 recruits fi a Rab3 GEF from human sperm extracts. Therefore, we propose rst introduced anti-Rab27 antibodies into SLO-permeabilized that Rab27 and Rab3A work sequentially during sperm dense- human sperm and monitored their effects on the calcium-trig- – core granule exocytosis, with Rab27 acting first. gered AR. The antibodies inhibited the AR in a dose response fashion (Fig. 2A). To determine whether active Rab27 is required Results for the AR, we conducted similar experiments, but substituting Presence and Subcellular Localization of Rab27 in Human Sperm. the anti-Rab27 antibodies with the Rab27-GTP binding domain Rabs from the Rab3 and Rab27 families usually coexist in se- of the effector Slac2-b. This protein blocked calcium-triggered cretory cells. A few years ago, we and others reported that exocytosis in a concentration-dependent manner (Fig. 2B). Fi- Rab3A is present in mammalian sperm and exhibits a role in nally, we tested the inhibitory effect of the Rab27 blockers when the AR, as discussed in the Introduction. In the present study, initiating the signaling pathway leading to the AR with a different we investigated the presence of Rab27 in human sperm using exocytosis inducer. When we challenged sperm with the Epac- a polyclonal antibody raised against purified recombinant full- selective cAMP analogue 8-(p-chlorophenylthio)-2′-O-methyl- length rat Rab27A. According to the manufacturer, this antibody adenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) recognizes Rab27A and B, but does not cross-react with other instead of calcium, we observed that the anti-Rab27 antibodies Rab proteins. Nevertheless, we specificity controls for this and Slac2-b blocked exocytosis (Fig. 2C, black bars). Taken

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Fig. 1. Rab27 is membrane-associated in human sperm. (A and B) Whole sperm homogenates were partitioned in Triton X-114 (A) or subjected to subcellular fractionation (B) as described in SI Materials and Methods. Proteins from unfractionated (wc extract) sperm extract, from the aqueous (ap) and detergent phases (dp), and from the particulate (membranes) and soluble (cytosol) fractions were electrophoresed on 10% Tris-tricine-SDS-polyacrylamide gels, 3 transferred to nitrocellulose, and immunoblotted with the anti-Rab27 antibody. Mr standards (× 10 ) are indicated on the left. Data are from experiments representative of three repetitions. Quantification (expressed as percent Rab27 in each fraction, carried out with ImageJ) is depicted to the right of each immunoblot as mean ± SEM from all replicates. (C) Human sperm samples were subjected to subcellular fractionation into membranes and cytosol, followed by Triton X-114 partition. Proteins from the aqueous and detergent phases were precipitated with organic solvents, resolved on 10% Tris-tricine-SDS- 3 polyacrylamide gels, transferred to nitrocellulose, and probed with the anti-Rab27 antibody. Mr standards (× 10 ) are shown on the left. Data are from an experiment representative of two repetitions.

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Fig. 2. Active Rab27 is required for human sperm AR. (A and B) SLO-permeabilized human sperm were treated for 15 min at 37 °C with increasing con- CELL BIOLOGY centrations of the anti-Rab27 antibody (A) or the Rab27-GTP binding domain of Slac2-b (B). The AR was then initiated by adding 0.5 mM CaCl2 and incubating for 15 min at 37 °C. In B, ▪ indicates that Slac2-b alone has no effect on exocytosis. (C) Permeabilized spermatozoa were incubated with 7 nM anti-Rab27 or

140 nM Slac2-b for 15 min at 37 °C. Acrosomal exocytosis was then initiated by adding 0.5 mM CaCl2 (calcium) or 50 μM 8-pCPT-2′-O-Me-cAMP (8pCPT) and another 15-min incubation at 37 °C. (D) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 10 min at 37 °C to chelate intra-acrosomal

calcium. The AR was then initiated by adding 0.5 mM CaCl2. After another 15-min incubation at 37 °C to allow exocytosis to proceed up to the intra-acrosomal calcium-sensitive step, sperm were treated for 15 min at 37 °C with 7 nM anti-Rab27 antibodies or 140 nM Slac2-b. All of these procedures were carried outin the dark. UV flash photolysis of the chelator was induced at the end of the incubation period (hν), and the samples were incubated for 5 min (NP→calcium→inhibitor→hν, black bars). Controls (gray bars) included background AR in the absence of any stimulation (control), AR stimulated by 0.5 mM

CaCl2 (calcium), the inhibitory effect of NP-EGTA-AM in the dark (NP→calcium→dark) and recovery on illumination (NP→calcium→hν), and the effect of the inhibitors when present throughout the experiment (NP→inhibitor→calcium→hν). Exocytosis was evaluated by FITC-PSA binding, and the data were nor- malized as described in Materials and Methods. Plotted data represent the mean ± SEM of at least three independent experiments.

together, these data indicate that active Rab27 is necessary for the calcium from the intracellular store (32). To assess when Rab27 human sperm AR. is required with respect to intracellular calcium release, we Because Rab27 exhibits homology with members of the Rab3 conducted identical experiments, combining the anti-Rab27 subfamily (40% similarity at the amino acid level) (19, 40), and antibodies and recombinant Slac2-b with the photolabile calcium because we derive strong conclusions from experiments con- chelator O-nitrophenyl EGTA-acetoxymethyl ester (NP-EGTA- ducted with antibodies, we performed specificity controls for the AM). In the SLO-permeabilized human sperm model, NP- effect of the anti-Rab3A and anti-Rab27 antibodies on the AR. EGTA-AM crosses the plasma and outer acrosomal membranes, We had previously shown that the inhibitory effect of anti- accumulates inside the acrosome, and prevents the AR triggered Rab3A antibodies in human sperm exocytosis is abolished when by all inducers by sequestering intra-acrosomal calcium for as the antibodies are preblocked with recombinant Rab3A (33). long as the system is kept in the dark. UV photolysis of NP- Here we tested the effect of anti-Rab27 antibodies on the AR EGTA-AM rapidly replenishes the acrosomal calcium pool, re- after blocking their active sites by preincubation with recombi- suming exocytosis (36, 39, 41). In NP-EGTA-AM–loaded per- nant Rab27A before adding them to permeabilized sperm. Un- meabilized sperm, anti-Rab27 and Slac2-b blocked exocytosis der these conditions, anti-Rab27 was no longer able to prevent when added before, but not after, initiation of the AR by calcium the AR elicited by calcium (Fig. S2A, black bar), suggesting (Fig. 2D). These data indicate that Rab27 acts in exocytosis that its inhibitory effect is due to binding to endogenous Rab27. before the start of intra-acrosomal calcium efflux. In contrast, preincubation of the anti-Rab27 antibody with recombinant Rab3A did not abolish the inhibitory effect (Fig. Effects of AR Inducers on Rab3A and Rab27 Targeting to Membranes S2C, black bar). In the reverse condition, recombinant Rab27A and Activation Status. Having established that Rab3A and Rab27 was unable to prevent the exocytotic block caused by the anti- are present in human sperm and play a role in the AR, we then Rab3A antibody (Fig. S2B). These results confirm the specificity asked whether these Rabs are activated during the onset of of the anti-Rab antibodies in sperm cells, in agreement with what exocytosis. We first investigated whether challenging sperm to we observed with recombinant proteins (Fig. S1A). undergo the AR correlates with the subcellular localization of The acrosome behaves as an internal store of releasable cal- the secretory Rabs. To this end, we incubated sperm with 2- cium (41–44); efflux from this reservoir through inositol 1,4,5 aminoethoxy-diphenylborate (2-APB), an IP3-sensitive calcium triphosphate (IP3)-sensitive channels is required for the AR channel blocker, to prevent acrosomal loss due to exocytosis. We initiated by all inducers (32, 33, 41, 45). We had previously then added the exocytotic inducers calcium ionophore A23187 or demonstrated that Rab3A acts in the AR before the release of 8-pCPT-2′-O-Me-cAMP, fractionated the cells, and assessed the

Bustos et al. PNAS Early Edition | 3of10 Downloaded by guest on October 1, 2021 levels of membrane-bound Rabs by Western blot analysis. We associated with membranes and significantly bound to GTP in observed no differences in the amount of membrane-bound resting human sperm. Initiation of the AR elicits further acti- Rab27 from resting cells and from sperm treated with A23187 or vation of this small GTPase. 8-pCPT-2′-O-Me-cAMP (Fig. 3A). In contrast, treatment with We previously reported that sperm contain a GEF activity either AR inducer increased the levels of membrane-bound that exchanges GDP for GTP on Rab3A when challenged with ∼ A Rab3A by 50% compared with untreated cells (Fig. 3 ). These calcium or 8-pCPT-2′-O-Me-cAMP (35, 47). We conducted those results indicate that targeting of Rab3A, but not (or to a lesser earlier experiments in SLO-permeabilized human sperm, but extent) of Rab27, to membranes appears linked to the initiation because we performed the Rab27 pull-down experiments repor- of exocytosis. We next applied a pull-down strategy to examine the effects of ted in this paper with nonpermeabilized sperm, we wished to AR inducers on the GTP status of endogenous Rab27 in sperm. verify the Rab3A data under the same conditions. To proceed, we fi E. coli We first expressed Slac2-b fused to GST in Escherichia coli and rst expressed in the Rab3-binding domain (RBD) of conducted far-Western blot analyses to verify that our recom- Rab3-interacting molecule (RIM), an effector that specifically binant Slac2-b preparations were active. In brief, we ran GST binds the GTP-bound form of Rab3, fused to GST. RIM-RBD and Slac2-b on SDS gels, then transferred the proteins to pulled-down GTP-γ-S–loaded recombinant Rab3A; GDP-loaded nitrocellulose and incubated with GTP-γ-S– or GDP-bound Rab3A was pulled down to a substantially lesser extent (Fig. S1C). Rab27A, before continuing as for standard Western blot analy- These results indicate that our RIM-RBD preparation was able to sis, probing with anti-Rab27 and anti-GST antibodies. The far- discriminate between active and inactive Rab3A; thus, we used it γ Western blots showed much higher binding of Rab27A-GTP- -S to verify the capacity of sperm to activate Rab3A during the AR. B than Rab27A-GDP to Slac2-b; neither bound GST (Fig. S1 ). Given the difficulty of detecting endogenous Rab3A in sperm, we These results indicate that our Slac2-b preparation was able to introduced a membrane-permeant version of Rab3A into the cells discriminate between active and inactive Rab27. We used Slac2- ′ b in pull-down assays as described previously (46) to determine before performing the challenge with 8-pCPT-2 -O-Me-cAMP. the levels of Rab27-GTP in sperm. The threshold level of en- We found that the levels of GTP-bound Rab3A increased by D dogenous Rab27-GTP in unstimulated sperm was high; never- approximately twofold in response to the AR trigger (Fig. 3 ). theless, A23187 elicited an ∼1.5-fold increase in active Rab27 Taken together, our data indicate that the onset of the AR cor- (Fig. 3C). Taken together, these results indicate that Rab27 is relates with the activation of both Rab3A and Rab27.

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Fig. 3. Initiation of the AR activates Rab27 and Rab3A. (A) Capacitated human sperm were incubated without (control) or with the AR inducers A23187 or 8pCPT, washed, and subjected to subcellular fractionation as described in SI Materials and Methods. Rab27 (Top) and Rab3A (Middle) were detected in the particulate (membrane) fractions by Western blot analysis using the rabbit polyclonal antibodies as probes. Anti-α- was used as a loading control 3 (Bottom). Mr standards (× 10 ) are indicated on the left. (B) Quantification of 12 experiments (n = 3 for each panel) identical to those in A, with mean ± SEM from all replicates. Data were evaluated using the t-test for paired comparisons for means. NS indicates that statistical differences between control and treated samples were nonsignificant (P > 0.05). **P < 0.01. (C and D) Pull-down assays using GST-Slac2b-Sepharose (C) and GST-RIM-RBD-Sepharose (D). The levels of GTP-bound Rab27 (C) and Rab3A (D) were determined by Western blot analysis probed with the rabbit polyclonal anti-Rab27 and mouse monoclonal 3 anti-Rab3A antibodies. Anti–α-tubulin was used as an input control. Mr standards (× 10 ) are indicated on the left. Shown are representative blots, with quantifications depicted as mean ± SEM from all replicates (n = 2 for Rab27; n = 4 for Rab3A) to the right of each blot. Data were evaluated using the t-test for paired comparisons for means. **P < 0.01.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1121173109 Bustos et al. Downloaded by guest on October 1, 2021 Detecting the GTP Status of Endogenous Rab3A and Rab27 by To rule out the possibility that calcium might influence GST- PNAS PLUS Fluorescence Microscopy. We were interested in determining the Slac2-b/RIM binding to the acrosomal region per se (in a Rab/ percentage of the sperm population that exhibit activated Rab3 GTP-independent manner), we conducted an additional control and Rab27 in response to AR inducers, and in identifying where experiment in which cells were offered calcium but no GTP. The in the cell these active Rabs are localized. Unfortunately, pull- numbers of cells decorated with the Slac2-b cassette were in- down assays are not suitable for addressing these issues (in ad- distinguishable regardless of whether we loaded cells with GDP dition to being lengthy to perform and only semiquantitative). or with GDP/calcium (Fig. 4C, black bars). Likewise, the active Thus, we designed a protocol based on the principles underlining Rab3 probe labeled the same number of cells in the acrosomal far-Western blot analysis, but with indirect immunofluorescence region in both conditions (Fig. 4D, black bars). The percentage as the readout. We reasoned that if the interaction between of acrosome-reacted sperm was low in all conditions (Fig. 4 C Rab27 and Slac2-b survived the harsh conditions of SDS/PAGE and D, gray bars). Taken together, our data suggest that sperm and Western blot analysis (Fig. S1B), it also might be expected to contain Rab27-GEF and Rab3-GEF activities that are stimu- survive the comparatively milder conditions applied in indirect lated in response to the AR inducer calcium. immunofluorescence protocols. This was indeed the case. We To determine whether the two secretory Rabs act sequentially first validated the assay to demonstrate that the probes can dis- to achieve the AR, we ran experiments similar to those described tinguish between GDP-bound and GTP-bound Rabs in this above but replacing calcium with exogenous, persistently acti- protocol. To do so, we incubated SLO-permeabilized sperm with vated Rabs. The number of cells decorated with Slac2-b in the EDTA to chelate endogenous Mg2+ cations and allow for the acrosomal region was similar in cells treated with GDP and cells release of endogenous guanosine nucleotides. We then loaded treated with recombinant Rab3A-GTP-γ-S plus GTP (Fig. 4A). γ the cells with GDP-β-S or GTP-γ-S to determine the minimum In contrast, the addition of recombinant Rab27A-GTP- -S to and maximum levels of active Rabs detectable by this assay. We SLO-permeabilized sperm doubled the number of cells dis- B smeared and fixed sperm on slides, overlaid with GST-RIM- playing Rab3-GTP in the acrosomal region (Fig. 4 ). These — RBD or GST-Slac2-b, washed the slides, and proceeded as for results indicate that active Rab27 stimulates/recruits directly or — standard immunostaining, using anti-GST antibodies to detect indirectly a Rab3-GEF in human sperm. More importantly, the activity probes. The anti-GST antibodies immunodecorated they suggest that these Rabs act sequentially, rather than in the acrosomal region in 70% of the cells loaded with GTP-γ-S a loop, during the AR. and overlaid with RIM-RBD, whereas only 29% of sperm trea- Having established that Rab27 somehow activates Rab3 in ted with GDP-β-S gave a detectable signal (Fig. S3). Likewise, human sperm, we reasoned that endogenous Rab27 must act CELL BIOLOGY Slac2-b bound the acrosomal region in 23% of GTP-γ-S–treated upstream of Rab3 during the AR cascade put into motion by sperm, but in only 6% of the GDP-β-S–treated sperm (Fig. S3). calcium. To test this hypothesis, we needed to set up functional In the control condition, overlaying with GST and probing with assays in which anti-Rab antibodies halted the AR reversibly. Coincubation of anti-Rab antibodies with the corresponding the anti-GST antibodies did not accomplish immunostaining Rabs abolished the inhibitory effect of the former (33) (Fig. S2). (Fig. S3). Thus, we validated our protocol by demonstrating that To assess the reversibility of the antibody block, we investigated RIM-RBD and Slac2-b can distinguish between GDP-bound and whether recombinant Rabs could rescue exocytosis when added GTP-bound Rab3A and Rab27. Our results indicate that GTP- at the end of the sequence of additions, and found that in fact bound Rab3A and Rab27 localize to the acrosomal region in they did (Fig. 4 E and F; anti-Rab → calcium → Rab). We be- human sperm, as expected for proteins with a role in exocytosis lieve that this rescue stems from reversal of the antibodies’ effect and consistent with indirect immunofluorescence data for anti- Lower rather than from reconstitution of the systems by functional Rab3A (28) and anti-Rab27 (Fig. S3, ) antibodies. recombinant Rabs, because they are neither prenylated nor loaded with GTP and thus should be inactive by themselves (Fig. Rab27 and Rab3A Act Sequentially During Human Sperm AR. The 4 E and F). In addition, inert Rab27A (heat-inactivated) as well coexistence of more than one Rab GTPase in the same mem- as the Rab3A mutant V55E, which does not bind the effectors brane raises the question of how their functions are coordinated. Rabphilin or RIM (50, 51) and is inactive in PC12 cell secretion Different models have been proposed to address this question (52), rescued the corresponding antibody block (Fig. S2 D and E, based on data from yeast and mammalian endomembranes black bars). (for recent reviews, see refs. 7, 11, 48, 49). How Rab3 and Rab27 When added after the AR inducer calcium in the presence of cooperate to achieve dense-core granule exocytosis is a question anti-Rab27 antibodies (rescued at the end of the incubation by that has not yet been addressed in any model system. We in- recombinant Rab27A), the Rab3-sequestering cassette RIM- vestigated this issue in the AR by applying our recently de- fl RBD, which does not bind Rab27 (53), prevented the AR (Fig. veloped immuno uorescence assay. First, we pretreated the cells 4E, black bar). These results indicate that RIM blocks the AR with 2-APB to prevent loss of the acrosomal granule due to by sequestering Rab3-GTP at a stage downstream of Rab27. In a exocytosis; thus, the percentage of acrosome-reacted sperm was parallel experiment, we loaded sperm with an anti-Rab3A anti- A B low under all conditions (Fig. 4 and , gray bars). Second, we body, challenged with calcium, and offered Slac2-b, which does loaded endogenous sperm GTPases with GDP and stabilized 2+ not bind Rab3 (54), before rescuing the antibody block with the bound nucleotide with high Mg concentrations. Third, recombinant Rab3A. Under these conditions, Slac2-b failed to when indicated, we bathed the cells in a buffer containing GTP. inhibit exocytosis (Fig. 4F, black bar). These findings suggest that We found the same percentage of cells depicting Rab27-GTP by the time we added Slac2-b, the system had already traversed whether we treated with GDP alone or with GDP followed by the step catalyzed by Rab27-GTP. Given that this was achieved GTP; in contrast, when we added calcium to the GTP-containing in the presence of anti-Rab3 antibodies, we conclude that the medium, the number of cells harboring Rab27-GTP in the ac- action of Rab27 in the AR occurs upstream of that of Rab3. rosomal region increased by more than fourfold (Fig. 4A). These results suggest that sperm do not exchange GDP for GTP on Rab27 Recruits a Rab3 GEF Activity. What molecular mechanism Rab27 spontaneously; in other words, sperm Rab27-GEF is not drives the Rab27/Rab3 cascade? As mentioned earlier, some constitutively active. Likewise, the percentage of cells exhibiting Rabs activate later-acting Rabs through GEF cascades. To test Rab3-GTP in the acrosomal region more than doubled when whether a similar cascade might be involved in regulated dense- cells were exposed to GTP/calcium compared with GDP or GTP core granule exocytosis, we loaded human sperm extracts on alone (Fig. 4B). GST-Rab27A-GTP-γ-S immobilized on glutathione-Sepharose

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anti-Rab27 calcium RIM Rab27A anti-Rab3A calcium Slac2-b Rab3A

020406080 100 020406080 100 120 acrosome exocytosis index acrosome exocytosis index

Fig. 4. Rab27 and Rab3A play sequential roles during exocytosis; active Rab27 increases the level of Rab3A-GTP. (A–D) SLO-permeabilized human sperm treated as described in Materials and Methods were overlaid with GST-Slac2-b to detect active Rab27 (A and C) and with GST-RIM-RBD to detect active Rab3 (B and D) and then triple-stained with the anti-GST antibody to visualize the activity probes, with FITC-PSA to confirm that the AR was effectively prevented by 2-APB (PSA, gray bars), and with Hoechst 33342 to visualize all cells in each field (not shown). The percentage of cells immunodecorated in the acrosomal region by the anti-GST antibodies is represented by the black bars. The Tukey–Kramer post hoc test was used for pairwise comparisons. (A and B)The

population of active Rabs increased on incubation with GTP plus CaCl2 compared with controls incubated with GDP (***P < 0.001; cell counts: 170 cells probed with Slac2-b and 484 cells probed with RIM-RBD). Note that by itself, GTP (cell counts: 220 cells probed with Slac2-b and 405 cells probed with RIM-RBD) was unable to increase the number of cells depicting active Rabs compared with those treated with GDP (NS, statistically nonsignificant; cell counts: 219 cells probed with Slac2-b and 406 cells probed with RIM-RBD). Recombinant Rab27A augmented the number of cells depicting endogenous GTP-Rab3A in the

acrosomal region (B, NS with respect to GTP/CaCl2; 396 cells counted; ***P < 0.001 compared with GDP). The converse was not true; recombinant Rab3A did not influence the number of cells with active Rab27 in the head; (A, NS compared with controls loaded with GDP; 180 cells counted). The data represent mean ± SEM of at least three independent experiments. (C and D) Control experiments in which the cells were maintained in GDP instead of in GTP when calcium was added. Shown are quantifications (mean ± SEM, three independent experiments) of GTP-bound Rab27 (C; we counted 201 cells loaded with GDP and 192 cells loaded with GDP/calcium) and GTP-bound Rab3 (D; we counted 206 cells loaded with GDP and 205 cells loaded with GDP/calcium). (E) SLO-permeabilized spermatozoa were loaded with 7 nM anti-Rab27 antibodies for 8 min at 37 °C to block the signaling pathway in which Rab27 is required. The AR was

subsequently initiated by adding 0.5 mM CaCl2. After an 8-min incubation at 37 °C to allow exocytosis to proceed to the Rab27-sensitive step, sperm were treated with 67 nM GST-RIM-RBD and incubated for an additional 8 min at 37 °C. Finally, we added 14 nM recombinant GST-Rab27A (unprenylated and not loaded with nucleotides) to rescue the anti-Rab27 antibody block and incubated as before (black bar). (F) Same as E, except that the reversible pair was 70 nM anti-Rab3A mouse monoclonal antibody rescued by 140 nM GST-Rab3A and that 140 nM GST-Slac2-b substituted for GST-RIM-RBD. We ran several controls in

parallel (gray bars), including background AR in the absence of any stimulation (control), AR stimulated by 0.5 mM CaCl2 (calcium), lack of effect of 140/14 nM recombinant Rab3A/27A alone (Rab3A/27A), inhibition by 70/7 nM anti-Rab3A/27 antibodies (anti-Rab3A/27→calcium) and by 67 nM GST-RIM-RBD or 140 nM GST-Slac2-b (RIM/Slac2-b→calcium), rescue of the anti-Rab3A/27 antibodies by their cognate Rab3A/27A proteins (anti-Rab3A/27→calcium→Rab3A/27), and the inhibitory effect of the cassettes when present throughout the experiment (anti-Rab3A/27→Slac2-b/RIM→calcium→Rab3A/27). Sperm were fixed, and AR was measured by FITC-PSA binding as described in Materials and Methods. The data represent mean ± SEM of at least three independent experiments.

beads. After washing, we incubated the beads with recombinant tivity, and that stimulated by Rab3A GAP is only 12% of that of His6-Rab3A-GDP in the presence of GTP (a schematic of the WT Rab3A (55). His6-Rab3A Q81L-GTP was pulled down at protocol is provided in Fig. 5A). To maximize the probability the end of the assay by GST-RIM-RBD (Fig. 5B, Top). The GDP of finding detectable levels of active Rab3A, we conducted for GTP exchange on recombinant Rab3A was catalyzed by the these experiments with Rab3A Q81L, which has a 3.2-fold higher Rab3 GEF activity present in human sperm extracts; the koff (GDP) value and an eightfold lower koff (GTP) value than WT. was retained on the Rab27A column. In contrast to the high In addition, this mutant has undetectably low basal GTPase ac- levels of Rab3A activated by the GEF immobilized by Rab27,

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1121173109 Bustos et al. Downloaded by guest on October 1, 2021 AB greatest homology (41–44%) to members of the Rab3 subfamily PNAS PLUS (40). Rab27A and Rab27B share 66% identity at the nucleotide level, and 71% identity and 81% similarity at the amino acid sequence level. Rab27A is expressed in many mouse and human tissues and tumor cell lines, whereas Rab27B has a more limited expression pattern. Rab27B is present in human and mouse testis (19, 40, 56). Probing of whole human sperm extracts with the anti-Rab27 antibody on Western blot analysis revealed a single protein band corresponding to Rab27 (Fig. 1). Because Rab27A and Rab27B have very similar electrophoretic mobility, and be- cause the anti-Rab27 antibody that we used is supposed to rec- ognize both isoforms, we cannot distinguish which of the two is present in sperm. By introducing specific anti-Rab antibodies that sequester the endogenous proteins or recombinant cassettes that selectively bind the active forms into SLO-permeabilized human sperm, we found that both Rab3A (32, 35) (Fig. 4 and Fig. S2) and Rab27 (Figs. 2 and 4 and Fig. S2) are necessary for regulated exocytosis. In short, our findings reinforce the idea that Rab3A plays a positive role in sperm secretion and point to a similar positive role for Rab27. Do these secretory Rabs cycle constantly, as described for Rab families that drive constitutive membrane traffic? We mined the literature for data on the coupling of subcellular localization and activation status of secretory Rabs to exocytosis stimuli in an attempt to unveil a unifying picture, but failed to find such Fig. 5. Rab27 recruits a Rab3 GEF. (A) Schematic of the protocol. Beads a synthesis. We trust that the results reported here using the AR represent glutathione-Sepharose; blue spheres, geranylgeranylated GST- as a model system provide insight into these issues, given that we

Rab27A-GTP-γ-S; orange pentagons, Rab3 GEF isolated from human sperm compared both Rab3 and Rab27 targeting to membranes and CELL BIOLOGY extracts; red sphere, His6-Rab3A-GDP; green sphere, His6-Rab3A-GTP. (B)GST their activation status in resting and stimulated human sperm. and GST-Rab27A-GTP-γ-S were immobilized on glutathione-Sepharose (i; Our findings can be summarized as follows: (i) The levels of anti-GST blot shows equal amounts of immobilized proteins), incubated with membrane-associated Rab27 were similar in resting and stimu- –α human sperm extracts (ii; anti -tubulin blot of the unbound fraction shows lated sperm (Fig. 3A); (ii) in contrast to Rab27, the levels of equal loading), and washed as described in Materials and Methods. The GEF activity from human sperm retained by immobilized Rab27 was assessed membrane-bound Rab3A in untreated cells were lower than those in sperm treated with AR inducers (Fig. 3A); and (iii) by incubating the beads with His6-Rab3A-GDP. After centrifugation, His6- Rab3A-GTP was pulled down from the supernatants with the GST-RIM-RBD challenging sperm to undergo exocytosis increased the pro- cassette (iii; anti-GST blot shows equal amounts of immobilized cassette) and portion of GTP-bound Rabs (Fig. 3 C and D). In other words, probed with the anti-Rab3A monoclonal antibody. Mr standards (× 103)are inducing the AR activated both Rabs and targeted Rab3 to indicated on the left. Shown is an experiment representative of two repetitions. sperm membranes, whereas Rab27 was mainly membrane-bound under all conditions. Our findings pose the fundamental but as- yet unanswered question of how Rab27, which is already pre- little active Rab3A was detected in the negative control (immo- dominantly attached to membranes and in large proportion bilized GST). These background levels were likely due to spon- bound to GTP in resting sperm, relays extracellular signals to taneous (nonenzymatic) exchange, given that we used a mutant the intracellular fusion cascade to achieve exocytosis only when fi that is GTPase-de cient and does not release GTP easily. These cells are stimulated. Like all exocytosis, that of the acrosome is results support the idea that Rab27 and Rab3 represent a Rab a multistage process comprising the opening of ion (mainly cal- cascade involved in regulated exocytosis. Because Rab27 re- cium) channels, activation of multiple signaling pathways, or- cruits a Rab3 GEF, our findings establish an example of a Rab- chestration of numerous protein–protein interactions to put the GEF cascade. fusion machinery in place, and other steps. Morphologically, this process relies on the swelling of the dense-core granule and Discussion dramatic remodeling of the acrosomal membrane that lead to A central challenge in the field of reproductive biology is to the latter contacting the plasma membrane at the points of understand how sperm regulate their secretion machinery to eventual fusion. It would appear that the mere presence of active release their acrosomal contents only when they encounter the and membrane-bound endogenous Rab27 is not sufficient to egg. At the most reductionist level, the first step in addressing accomplish exocytosis by itself. this challenge is to delineate the cellular and molecular processes How are the actions of Rab3A and Rab27 coordinated to by which sperm mount an exocytotic response at the appropriate accomplish the AR? Are they part of a single time. Our laboratory has contributed to the understanding of the pathway or separate pathways? In an attempt to answer these molecular mechanisms that drive human sperm exocytosis by questions, we introduced the Rab-GTP–binding domains of RIM identifying and characterizing the role and point of action of and Slac2-b into SLO-permeabilized sperm, and found that these a number of components of the fusion machinery (reviewed in Rabs act sequentially to accomplish the secretion of the sperm refs. 24 and 25). Here we focus on the role of two secretory Rabs, dense-core granule, with Rab27 regulating the earliest step (Fig. Rab3A and Rab27, in the release of the sperm single dense-core 4 E and F). It has been suggested that Rab3 and Rab27 exhibit secretory granule. Over the last several years, we have published opposite effects (Rab3 inhibitory and Rab27 stimulatory) on several papers that demonstrate the positive role of Rab3A in insulin exocytosis, based on results from overexpression of con- sperm exocytosis (32, 33); in this paper, we describe the presence stitutively active mutants of these two GTPases (see ref. 57 for of Rab27 in sperm and its role in the AR. Rab3A and Rab27A a review). However, a study conducted in PC12 cells demon- are phylogenetically similar (54); Rab27A and Rab27B have only strated the need for caution when interpreting data derived from moderate homology to other human Rab sequences, with the overexpression experiments; transfection with high levels of

Bustos et al. PNAS Early Edition | 7of10 Downloaded by guest on October 1, 2021 Rab3A or Rab27A plasmids was found to substantially inhibit permeabilized in ice-cold methanol for 20 s. An alternative fixation protocol evoked exocytosis, an effect not observed when transfecting with (described in Indirect Immunofluorescence) produced identical results. Ac- rosomal status was evaluated by staining with FITC-coupled Pisum sativum smaller amounts of DNA (18). Thus, we stress the importance of μ our findings, which were obtained by scrutinizing the roles of the agglutinin (FITC-PSA, 25 g/mL in PBS) for 40 min at room temperature, followed by a 20-min wash in water (59). We scored at least 200 cells per endogenous Rabs and working with primary cultures instead of condition using an upright Nikon Optiphot II microscope equipped with immortalized cell lines. epifluorescence optics. Basal (“control,” no stimulation) and positive (“cal- fl We have developed an indirect immuno uorescence assay to cium,” 0.5 mM CaCl2, corresponding to 10 μM free calcium estimated by determine the subcellular localization of GTP-bound Rabs. We MAXCHELATOR, a series of programs for determining the free metal con- are excited about the potential applications of this assay. It is centration in the presence of chelators; available at http://maxchelator. important to bear in mind that localization of active Rabs is stanford.edu/) controls were included in all experiments. Acrosomal exo- otherwise impossible to ascertain, because pull-down assays re- cytosis indices were calculated by subtracting the number of spontaneously ∼ port the activation status of these proteins, but not the location reacted spermatozoa (basal control without stimulation, ranging from 8% to 20% before normalization) from all values and expressing the results as in the cell where they are activated or how many cells respond to a percentage of the AR observed in the positive control (ranging from ∼18% the stimuli. The only attempt to address the issue of subcellular to 40% before normalization; assigned 100% of responsive cells for nor- localization of active Rabs reported in the literature deals with malization). Our analysis included only results derived from experiments the overexpression of GTPase-deficient mutants, which entails that produced similar responses and in which the difference between basal the inherent problem of overexpression itself, compounded by the and calcium-stimulated conditions was of at least 8–10 percentage points. inability of these mutants to cycle as the endogenous small G Samples with more than 20% of spontaneously reacted sperm were ex- proteins do. Using our assay, we were able to detect the presence cluded from our analysis. Data were evaluated using the Tukey–Kramer post hoc test for pairwise comparisons. Differences were considered significant at of active Rabs in the acrosomal region of human sperm, a local- < ization expected of proteins relevant for exocytosis. We found an the P 0.05 level.

increasing percentage of cells expressing acrosomal Rab3-GTP 5 Indirect Immunofluorescence. Between 2 and 3.5 × 10 capacitated sperm and Rab27-GTP after challenging sperm with calcium to initiate – A B fi were attached to poly-L-lysine coated (stock 0.1%, diluted 1:20 in water; the AR (Fig. 4 and ). These ndings were expected, because Sigma-Aldrich), 12-mm round coverslips by incubating for 30 min at room they lend support to our model indicating that the onset of the temperature in a moisturized chamber. The cells were then fixed in 2% (wt/ AR relies on the activation of Rab proteins, and also demonstrate vol) paraformaldehyde in PBS for 15 min at room temperature. The fixative the robustness of our assay in detecting this activation. We are was neutralized by overnight incubation at 4 °C in PBS containing 100 mM currently designing experiments aimed at determining whether glycine. Next, the cells were permeabilized with 0.1% Triton X-100 in PBS for calcium stimulates Rab-GEFs and/or inhibits Rab-GAPs. Cur- 10 min at room temperature and washed three times with PBS containing fi rently, we lack information on the molecular identities of these 0.1% polyvinylpyrrolidone (PBS/PVP; average MW = 40,000). Nonspeci c staining was blocked by incubation in 5% (wt/vol) BSA in PBS/PVP for 1 h at proteins in sperm, except for Rab3-GAPs 1 and 2, which were 37 °C. Anti-Rab27 antibodies were diluted at 4 μg/mL in 3% (wt/vol) BSA in detected in a murine haploid germ cell proteome (58). Finally, PBS/PVP, added to the coverslips, and incubated for 1 h at 37 °C in a mois- the assay described in this paper allowed us to obtain one of the turized chamber. After two washes for 10 min with 2% (wt/vol) saponin in most relevant findings that we report here: an increasing number PBS, Cy3-conjugated donkey anti-rabbit IgG (2.5 μg/mL in 1% BSA in PBS/ of cells depicting endogenous GTP-bound Rab3 in the acrosomal PVP) was added, and the mixture was incubated for 1 h at room tempera- region in response to the introduction of recombinant ger- ture while protected from light. Coverslips were washed six times for 6 min anylgeranylated and persistently active Rab27A into SLO-per- with PBS/PVP. Cells were subsequently stained for acrosomal contents as meabilized human sperm (Fig. 4B). These results reinforce the described earlier, but without air-drying, mounted with 1% propyl-gallate/ 50% (vol/vol) glycerol in PBS containing 2 μM Hoechst 33342, and stored at idea that Rab27 acts upstream of Rab3A during the exocytotic −20 °C in the dark until examination with an Eclipse TE2000 Nikon micro- cascade, leading to acrosomal release. The literature contains scope equipped with a Plan Apo 40×/1.40 oil objective and a Hamamatsu several examples of how small GTPases form networks to co- digital C4742-95 camera operated with MetaMorph 6.1 software (Universal ordinate transport pathways, such as GAP cascades, Rab GEF Imaging). Background was subtracted, and brightness/contrast were ad- cascades, and positive feedback loops (48). We show here that the justed to render an all-or-nothing labeling pattern using ImageJ freeware. molecular mechanism that governs the activation of Rab3 by Rab27 acts through a Rab GEF cascade, in which active Rab27A In Vitro Geranylgeranylation of Recombinant Rab3A and Rab27A. Recombinant recruits, directly or indirectly, a Rab3A GEF activity (Fig. 5B). His6-R-Rab3A and His6-Rab27A (Figs. 3D and 4 A and B) were prenylated in a solid-phase assay developed in our laboratory before being eluted from Ni- This report directly demonstrates the existence of sequential roles – μ for these exocytotic Rabs, establishing an example of a Rab GEF NTA-agarose beads. In brief, 10 20 M Rabs immobilized on Ni-NTA-agarose were incubated in a geranylgeranylation mixture comprising 80 μM ger- cascade in regulated secretion. anylgeranyl pyrophosphate, 200 μM GDP, 1 mM DTT, 3 mM MgCl2, 1 mg/mL BSA, 1 mg/mL bovine brain cytosol as a source of Rab geranylgeranyl Materials and Methods and Rab escort protein, a protease inhibitor mixture (Sigma- Human Sperm Sample Preparation. Human semen samples were obtained Aldrich P2714), and 20 mM Hepes-K (pH 7) for 2 h at 37 °C. After this in- from normal healthy donors. Semen was allowed to liquefy for 30–60 min at cubation, we added four volumes of the same washing buffer (300 mM NaCl, 37 °C. After a swim-up protocol to isolate highly motile cells, sperm con- 10 mM imidazole, 20 mM TrisHCl; pH 7.4) used during the purification of × 6 centrations were adjusted to 7 10 /mL before incubation for at least 2 h His6-fused proteins and incubated the mixture for 30 min at 4 °C. (This step under capacitating conditions [Human Tubal Fluid media (as formulated by favors the recapture of prenylated Rabs dissociated from the resin.) Beads fi Irvine Scienti c) supplemented with 0.5% BSA (HTF medium) HTF, 37 °C, 5% were washed three times with His6-washing buffer and geranylgeranylated (vol/vol) CO2/95% (vol/vol) air]. Sperm were washed twice with PBS and Rabs eluted with His6-elution buffer (300 mM NaCl, 400 mM imidazole, 20 resuspended in cold PBS containing 2.1 U/mL SLO for 15 min at 4 °C. Cells mM TrisHCl; pH 7.4). GST-Rab27A was isoprenylated following a similar were washed once with PBS resuspended in ice-cold sucrose buffer (250 mM protocol except that it was immobilized on glutathione-Sepharose, and the sucrose, 0.5 mM EGTA, 20 mM Hepes-K; pH 7) containing 2 mM DTT. For AR buffers were those used in the purification of GST-fusion proteins (Fig. 5). assays, inhibitors and stimulants were added sequentially, with incubation Geranylgeranylation was confirmed by Triton X-114 partitioning. When in- for 8–15 min at 37 °C after each addition. When indicated, SLO-per- dicated, various versions of Rab3A and Rab27A were loaded for 1 h at 37 °C meabilized sperm was preloaded with photosensitive NP-EGTA-AM before with 0.125 mM of the appropriate guanosine nucleotide in a buffer con-

incubation in the presence of inhibitors and/or calcium, with all procedures taining 5 mM MgCl2, 0.03% Igepal or Nonidet-P40, 1 mM DTT, 25 mM EDTA, performed in the dark. Photolysis was induced after the last incubation by and 50 mM Hepes/KOH (pH 7.2). Rabs were separated from free nucleotides two exposures (1 min each) to a UV transilluminator (FBTIV-614; Fisher by gel filtration in Sephadex G25 minicolumns equilibrated in PBS containing

Scientific), and exocytosis resumed by mixing, and incubation for 5 min at 1% BSA added 1 mM MgCl2, and stored at −20 °C until use. Recombinant 37 °C. Sperm were spotted on Teflon-printed slides, air-dried, and fixed/ protein concentrations were determined by the Bradford method (Bio-Rad)

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1121173109 Bustos et al. Downloaded by guest on October 1, 2021 using BSA as a standard in 96-well microplates, and quantified on a BioRad by SDS/PAGE, and cellular Rab27-GTP and Rab3A-GTP levels were analyzed PNAS PLUS 3550 Microplate Reader or from the intensities of the bands in Coomassie by immunoblotting as described below. blue-stained SDS/PAGE gels. Recruitment of Human Sperm Rab3 GEF Activity by Immobilized Rab27. Human Detection of Nucleotide-Binding Status of Endogenous Rab3 and Rab27. sperm extracts were prepared as described in SI Materials and Methods, but Capacitated, SLO-permeabilized sperm were incubated with 100 μM 2-APB with Triton X-100 substituted for Triton X-114. Nonspecific binding sites on for 10 min at 37 °C. Then the endogenous nucleotides were released by glutathione-Sepharose beads were blocked by incubating three times with incubating sperm in 5 mM EDTA (which increases the off rate of bound two volumes each of 0.1% BSA in 20 mM Hepes (pH 7.4). Then 2.5 μg of GST γ nucleotides; ref. 60)/1 mM MgCl2 for 10 min at 37 °C. Cells were loaded with or geranylgeranylated GST-Rab27A-GTP- -S was incubated for 1 h at 4 °C 40 μMGDP-β-S or GTP-γ-S for 10 min at 37 °C, and bound nucleotides were with constant rotation with 20 μL of glutathione-Sepharose beads in binding

stabilized with 15 mM MgCl2 for 5 min at 37 °C (Fig. S3). A similar protocol buffer [20 mM Hepes (pH 7.4), 150 mM KAcO, 15 mM MgCl2, 0.05% Tween was applied for the experiments depicted in Fig. 4 A and B and Fig. 3 C and 20, 5 μM GTP, and protease inhibitors] in a final volume of 200 μL. Beads D, except that cells were loaded with 40 μM GDP before the addition of high containing immobilized recombinant proteins were washed twice with 200 μ MgCl2 concentrations. After this step, aliquots were incubated with or without L of binding buffer and incubated for 30 min at 4 °C under constant ro- × 6 200 μM GTP with and without 0.5 mM CaCl2 or 300 nM geranylgeranylated tation with 0.5 mL of sperm extract (50 10 cells). Unbound sperm proteins His6-Rab3A or His6-Rab27A loaded with GTP-γ-S. After a 15-min incubation at were precipitated with CCl3H-CH3OH-H2O and dissolved in sample buffer 37 °C, cell suspensions were fixed in 2% (wt/vol) paraformaldehyde, neutral- (anti–α-tubulin Western blot; Fig. 5B). The beads containing the immobilized ized with 100 mM glycine, attached to poly-L-lysine–coated coverslips, and recombinant proteins plus any sperm proteins retained by them were used stored overnight at 4 °C in a moisturized chamber. Sperm membranes were as the enzyme source in the subsequent Rab3A GEF assay. In brief, beads permeabilized in 0.1% Triton X-100 in PBS for 10 min at room temperature, were washed twice with 0.5 mL of binding buffer and incubated for 10 min cells were washed three times for 6 min each with PBS/PVP, and nonspecific at 30 °C with 8 nM geranylgeranylated His6-Rab3A-GDP in 0.5 mL of binding reactivity was blocked in 5% (wt/vol) BSA in PBS/PVP for 1 h at 37 °C. Slides buffer to promote the exchange of GDP for GTP. After centrifugation at were overlaid with 140 nM GST, GST-RIM-RBD, or GST-Slac2-b in 3% (wt/vol) 3,000 × g for 2 min at 4 °C, His6-Rab3A-GTP was pulled down from the BSA in PBS/PVP for 1 h at 37 °C. After three 6-min washes in PBS/PVP, the supernatants with the GST-RIM-RBD cassette as described above. protocol proceeded as described in Indirect Immunofluorescence,exceptwith anti-GST antibodies (31.5 μg/mL; 210 nM) substituted for anti-Rab27. The SDS/PAGE and Western Blot Analysis. Proteins were separated on Tris-tricine- presence of immunostaining in the acrosomal region was scored by manually SDS gels (61) or Tris-glycine-SDS gels (62) and transferred to 0.22-μm nitro- counting between 100 and 200 cells either directly in the fluorescence mi- cellulose membranes (Hybond; GE Healthcare). Nonspecific reactivity was croscope view or in digital images from at least 10 fields. Data were evaluated blocked by incubation for 1 h at room temperature with 5% (wt/vol) skim using the Tukey–Kramer post hoc test for pairwise comparisons. milk dissolved in washing buffer [PBS (pH 7.6) and 0.2% Tween 20]. Blots were incubated with the anti-Rab27 (0.2 μg/mL), anti-Rab3A rabbit poly- CELL BIOLOGY GST Pull-Down Assay. Capacitated sperm (20–50 × 106 cells) were treated for clonal (0.1 μg/mL; to detect endogenous Rab3A), anti-Rab3A mouse mono- 10 min at 37 °C with 100 μM 2-APB loaded (Fig. 3D) or not (Fig. 3C) with 10 clonal (0.2 μg/mL; to detect recombinant Rab3A), or anti–α-tubulin (0.2 μg/

nM (0.25 ng) His6-R-Rab3A, and finally challenged with 10 μM A23187 (Fig. mL) antibodies in blocking solution for 1 h at room temperature. Anti-GST 3C)or50μM 8pCPT (Fig. 3D) for 15 min at 37 °C. Cells were lysed in GST pull- antibodies were diluted to 40 ng/mL in washing buffer and incubated as for

down buffer [200 mM NaCl, 2.5 mM MgCl2, 1% (vol/vol) Igepal, 10% (vol/vol) the other primary antibodies. HRP-conjugated goat anti-mouse IgG or goat glycerol, 1 mM PMSF plus the protease inhibitor mixture, and 50 mM TrisHCl; anti-rabbit IgG were used as secondary antibodies (0.1 μg/mL in washing pH 7.4] by sonication on ice (two times for 15 s each). Proteins were allowed buffer) with 1-h incubations. Excess first and second antibodies were re- to diffuse into the lysis buffer for 15 min at 4 °C. These whole-cell detergent moved by washing five times for 10 min each in washing buffer. Detection extracts were clarified by centrifugation at 14,000 × g for 5 min and was performed with a Millipore chemiluminescence system and a Fujifilm used immediately. LAS-4000 Luminescent Image Analyzer. Quantification of signal intensities Glutathione-Sepharose beads were washed twice with GST pull-down was performed with ImageJ. buffer and incubated with bacterial lysates containing GST-Slac2-b or GST- RIM-RBD for 1 h at 4 °C under constant rocking. Beads were washed twice ACKNOWLEDGMENTS. We thank M. Furlán, N. Domizio, and A. Medero for with PBS and once with GST pull-down buffer and used immediately. Twenty excellent technical assistance; Drs. C. López, D. Munafó, R. Regazzi, R. Shirakawa, and P. Stahl for plasmids; Dr. S. Patterson for critical reading of the manu- μL of glutathione-Sepharose containing 5–10 μg of the appropriate fusion script; and S. Laurito and Dr. F. Rodriguez for helpful suggestions. This work protein was added to sperm lysates in a total volume of 1 mL, and incubated was supported by grants from the Argentinian Consejo Nacional de Investi- by rotation at 4 °C for 30 min. The resin was recovered by centrifugation at gaciones Científicas y Técnicas, Agencia Nacional de Promoción Cientifica y 4 °C (2 min at 950 × g) and washed three times with ice-cold GST pull-down Tecnológica, and Secretaría de Ciencia y Técnica-Universidad Nacional de buffer. The resin-bound fractions were boiled in sample buffer and resolved Cuyo (to C.N.T.).

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