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Synergistic Activation of ENaC by Three Membrane-bound Channel-activating Serine Proteases (mCAP1, mCAP2, and mCAP3) and Serum- and Glucocorticoid-regulated Kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (INa) by a GPI-anchored serine protease (mouse channel–activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase INa 6–10-fold in the Xenopus oocyte expression system. INa and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I125-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases INa 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcPo). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances INa by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcPo. Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in INa (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on INa was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.

I N T R O D U C T I O N
Sodium balance, extracellular volume, and blood pressure are maintained by the tight control of the activity of the epithelial sodium channel (ENaC)* (Verrey et al., 2000). We have recently identified a membrane-bound serine protease that acts as channel-activating protease, namely CAP1 (Vallet et al., 1997Vuagniaux et al., 2000). This regulation defines a novel extracellular signaling pathway, which appears to be highly conserved throughout evolution from Xenopus (xCAP1) (Vallet et al., 1997) to mouse (Vuagniaux et al., 2000), rat (Adachi et al., 2001), and human (Yu et al., 1995). CAP1 activates ENaC through its extracellular serine protease activity, as evidenced by patch-clamp experiments (Chraibi et al., 1998). The activation of ENaC by CAP1 can be mimicked by external addition of trypsin, showing that both CAP1 and trypsin act via the same pathway. Aprotinin, an inhibitor of serine proteases, can block this effect (Vallet et al., 1997;Vuagniaux et al., 2000). In epithelial cell lines, like the Xenopus kidney cell line (A6), ENaC appears to be constitutively activated through the presence of endogenously expressed serine proteases, since basal transepithelial Na ϩ transport cannot be further activated by addition of trypsin (Vallet et al., 1997). However, the base line Na ϩ transport can be inhibited by the addition of aprotinin on the apical side of the cell and this inhibition can be reversed by the addition of trypsin (Vallet et al., 1997). In Xenopus kidney cells, up to 90% of the amiloride-sensitive electrogenic Na ϩ transport can be blocked by aprotinin (50 M) (Vallet et al., 1997), whereas the mouse mpkCCD cl4 cell line (derived from the cortical collecting duct; Bens et al., 1999) appears to be only 50% sensitive to aprotinin (Vuagniaux et al., 2000). These findings suggest that ENaC activation is achieved by either a constitutive, serine protease-independent mechanism or, alternatively, that the activation depends on more than one serine protease with different sensitivity to Edith Hummle and Bernard C. Rossier contributed equally to this work. aprotinin and that acts in combination within the same cell. The mechanism by which serine proteases like CAP1 activates ENaC is not yet understood. CAP1 leads to a very substantial increase in the open probability of the Na ϩ channel, whereas the number of channels at the cell surface is either unchanged (Vallet et al., 1997) or even diminished (Vuagniaux et al., 2000).
The activity of ENaC is tightly controlled by hormones, including aldosterone and vasopressin (for review see Verrey et al., 2000). Components of the aldosterone-dependent signaling pathway have been identified recently. Aldosterone rapidly induced the expression of Sgk1 kinase (serum-and glucocorticoidregulated kinase), a member of the PKB-AKT family of serine-threonine kinases (Webster et al., 1993). When coexpressed in Xenopus laevis oocytes, Sgk1 stimulates ENaC activity by 2-3-fold Naray-Fejes-Toth et al., 1999). In Xenopus oocytes, Sgk1 increases cell surface expression of ENaC without changing its open probability (Alvarez de La Rosa et al., 1999;Loffing et al., 2001). Recently, it has been demonstrated that the phosphorylation of Nedd4-2, an ubiquitin protein ligase, by Sgk1 may regulate epithelial sodium channel cell surface expression in the Xenopus oocyte system (Debonneville et al., 2001). It was further demonstrated that the phosphorylation of Nedd4-2 decreases its affinity for ENaC, thereby diminishing ENaC endocytosis and/or degradation (Debonneville et al., 2001;Snyder et al., 2002). These data provided evidence for the missing link between aldosterone binding to its receptor, transcription activation of an aldosterone-induced protein (Sgk1) and the molecular mechanism leading to an increased cell surface expression of ENaC.
The aims of the present study were twofold: (a) to test the interaction between two regulatory pathways that can activate ENaC through extracellular signaling via CAPs and intracellular signaling via Sgk1, and (b) to identify further putative membrane-bound serine proteases able to activate ENaC; in particular, to search for an aprotininresistant protease that could account for the pharmacological data observed in the mouse CCD cell line.
We demonstrate here the existence of three channel-activating proteases, namely mouse channel-activating protease, mCAP1, mCAP2, and mCAP3 within the same kidney cell. Whereas mCAP2 is inhibited by aprotinin as well as mCAP1, mCAP3 is not significantly inhibited by this serine protease inhibitor. Each of these membrane-bound serine proteases increases ENaC-mediated I Na and potentiates the effect mediated by Sgk1.

Electrophysiological Experiments in Xenopus Oocytes
For functional expression studies, cDNA for mCAP1, mCAP2, mCAP3, and mouse Sgk1 (provided by O. Staub, Institute of Pharmacology and Toxicology, Lausanne, Switzerland) were subcloned into pSDS expression vector and in vitro transcribed, as described previously (Canessa et al., 1994). Purified cRNA were injected into stage V/VI Xenopus oocytes. Routinely, oocytes were injected with 0.33 ng of each cRNA coding for the rat ␣ -, ␤ -and ␥ -ENaC subunits in the presence or absence of varying concentrations of mCAP1, mCAP2, and mCAP3 and incubated in a modified Barth (low sodium) solution, as described (Firsov et al., 1996). Oocytes were incubated overnight in modified Barth saline solution in the presence or absence of 100 g/ml aprotinin. This protocol insures that intracellular sodium is low at the beginning of the measurement and independent of the level of ENaC expression at the cell surface. Electrophysiological measurements were performed 1-2 d after cRNA injection using the two-electrode voltage-clamp technique and one bath electrode, allowing corrections for serial resistance. The oocytes were exposed to 2 g/ml trypsin during 2-3 min and amiloride-sensitive Na ϩ current (I Na ) was measured in the presence of 120 mM of Na ϩ in frog Ringer's solution with 5 M amiloride at a holding potential of Ϫ 100 mV.

Measurement of N and I Na within the Same Oocyte
Cell surface expression has been performed as described (Firsov et al., 1996). Briefly, 1 ng of tagged rat ␣-, ␤-, and ␥-ENaC subunits was expressed in oocytes either alone or with 4 ng (mCAP1), 8 ng (mCAP2) or 2 ng (mCAP3) of each CAP and/ or 4 ng Sgk1 cRNA. The density of the channel was then calculated by binding of iodinated anti-FLAG monoclonal antibody (M2Ab*) (Sigma-Aldrich). The binding assay was performed at 4ЊC, and I Na was measured at room temperature as described. To deduce the whole cell P o (wcP o ), we have used a macroscopic approach, as described (Firsov et al., 1997) in which the starting point is the equation, derived from Ohm's law: (1) where (a) I Na is the macroscopic amiloride-sensitive current that measures the total activity of all channels expressed in a cell. (b) g Na is the single channel conductance measured by patch clamp in independent experiments. In the presence of sodium it is 5 and 8 pS in the presence of Li (Canessa et al., 1994). (c) (E Ϫ E Na ) is assessed by the measurement of membrane potential and the reversal potential for sodium. For practical purposes, E Ϫ E Na can be set constant by clamping the membrane at Ϫ100 mV or higher, allowing the measurement of changes in P o in function of the various experimental conditions. (d) N is the total number of channel molecules (active and inactive) expressed at the cell surface. The binding assay permits to quantitate the total number of channel molecules at the cell surface, independently of their function. Having determined with good accuracy I Na , g Na , E Ϫ E Na , and N, one can deduce a whole-cell P o (wcP o ) that reflects the activity of all channels expressed in an intact whole cell. The main advantage of this macroscopic approach is that it takes into account all channels expressed at the cell surface, whatever their activity. Our method is therefore distinct from a microscopic approach that is the classical measurement of P o in membrane patches (mpP o ). This is performed by determining the overall N и P o of all the active channels and the number of the channels N present in the patch membrane.

Identification of Two Membrane-bound Serine Proteases
in the mpkCCD cl4 Cell Line mCAP2. By using degenerated oligos to the previously isolated serine protease from Xenopus xCAP1 and RACE technique, we identified mCAP2, which encodes a 435-amino acid protein. Analysis of the amino acid sequence and the hydrophobicity plot reveals a type II-oriented membrane-bound serine protease with a predicted transmembrane segment (V30-151) and an extracellular COOH terminus containing a serine protease catalytic domain with the classical catalytic triad at H243, D288, and S385 and with a potential proteolytic activation cleavage site at R202V203 ( Fig. 1 A). Two cysteines at position C194 and C308 are predicted to connect the prodomain to the main serine protease domain via a disulfide bond at the extracellular surface of the cell (Hooper et al., 2001). The extracellular domain has two potential N-glycosylation sites (N128 and N176) that are used when mCAP2 is expressed in the Xenopus oocyte system (unpublished data). The catalytic domain is preceded by a low density lipoprotein receptor class A domain (LDLR, F60-V91) and a group A scavenger receptor domain (SRCR, V102-G142) ( Fig. 1 A). The serine protease domain shares homology with mouse trypsinogen precursor (49%), xCAP1 (45%), mCAP1 (43%), and 80% with a human orthologue hTMPRSS4 (Wallrapp et al., 2000) (Fig. 1 B).
mCAP3. By analysis of the transcriptome of an mpkCCD cl4 cell line library by SAGE (Robert-Nicoud et al., 2001) and EST database analysis, we identified a serine protease we termed mCAP3. mCAP3 is identical to the recently cloned mouse epithin . At the time we started this project, epithin cDNA sequence had not been published. To underline the functional relevance of this serine protease, we name the third serine protease throughout our paper mouse channel-activating protein three (mCAP3). Analysis of the primary sequence revealed the presence of a transmembrane domain with predicted type II orientation ( Fig. 1 A). COOH terminus contained the serine protease domain with the catalytic triad (H656, D711, and S805) and a potential activation cleavage site at R605V606. The catalytic domain was preceded by two complement factor IRurchin embryonic growth factor-bone orphogenetic proteins (CUBs) (C214-F331 and C340-Y444) and four LDLR (C453-R487, C488-S524, C525-N563 and C567-L603) domains ( Fig. 1 A)  . mCAP3 shares 47% homology with the mouse trysino-gen, 49% with xCAP1, 40% with mCAP2 and 83% with the human orthologue hMT-SP1 (Takeuchi et al., 1999) (or Matriptase;Lin et al., 1999) (Fig. 1 B).
Primary sequence comparison of the catalytic domain of mCAP1, mCAP2, mCAP3, and trypsin revealed the presence of an aspartate (mCAP1, D232; mCAP2, D379; mCAP3, D799) at the S1 site (Schechter and Berger, 1967) and 2 glycines at the entry of the S1 pocket (mCAP1, G259 and G269; mCAP2, G405 and G415; and mCAP3, G827 and G837) which direct the specificity of serine proteases Craik, 1995, 1997) (Fig. 1 C). This suggests that all three serine proteases are specific for the positively charged P1 amino acids. The presence of a proline residue (mCAP1, P268; mCAP2, P414; and mCAP3, P836) instead of a tyrosine residue at the Na ϩ -induced allosteric regulation of catalytic activity site in serine proteases suggests that the catalytic activity of three serine proteases is not sensitive to extracellular Na ϩ concentration variations (Dang and Di Cera, 1996) (Fig. 1 C).
Northern blot analysis showed that mCAP1, mCAP2, mCAP3, and ␣ENaC subunit are expressed, as expected, in mpkCCD c14 cell line. Whereas mCAP3 expression was expressed in the cell line and the kidney at a level similar to that of mCAP1, mCAP2 expression was low and not detectable in the whole kidney (Fig.  2). The three membrane-bound proteases and the ␣ENaC subunit were also expressed in epithelia known to express an amiloride-sensitive sodium transport (lung and colon). A similar pattern of expression was also found in small intestine and stomach. In these tissues, the role of ENaC and/or CAPs remains to be established. Figure 1. mCAP1, 2, and 3 are membrane-bound serine proteases. (A) Structural properties of the three mCAPs (mCAP1, mCAP2, and mCAP3). The amino acid sequence of each protein was scanned using ProfileScan algorithm to confirm the presence of domains indicated. Numbers delineate the location of each domain. (B) A representative phylogenetic tree of the GPI-anchored mCAP1 and type II transmembrane mCAP2 and mCAP3 serine protease family proteins. Multiple alignment was generated by the clustal W program (mCAP1, AAG1705; hProstasin, Q16651; xCAP1, AAB969054; mCAP2, AY043240; hTMPRSS4-1, CAC60389; hTMPRSS4-2; MT-SP2, Q9NRS4; mTMPRSS2; epitheliasin, AAF97867; hTMPRSS2, AAC51784; mTMPRSS3, CAC83350; hTMPRSS3, P57727; mCAP3; epithin, AAD02230; hMT-SP1, AAF00109; xMT-SP1, BAB08218). (C) Amino acid alignment of the serine protease catalytic domain of mCAP1, mCAP2, and mCAP3 with mouse trypsinogen precursor (1-246), was performed using Pileup program (Genetic Computer Group). Gray and black boxes indicate similar and identical residues, respectively. The catalytic triad (H, D, and S, asterisks), the amino acids implicated in the P1 substrate specificity (closed circle), and the position for Na ϩ sensibility (open circle) are indicated.

ENaC Activation by mCAP1, mCAP2, and mCAP3 in Xenopus Oocytes
In the Xenopus oocytes, ENaC activity is recorded as an amiloride-sensitive Na ϩ current (I Na ). As shown in Fig. 3 A  (lanes 1-4), mCAP1 increased ENaC activity 5-10-fold. Increasing amounts of cRNA coding for mCAP1 were tested and a maximal effect was observed with 4 ng mCAP1. mCAP2 and mCAP3 (Fig. 3, lanes 5-8 and 9-12) were also able to increase ENaC activity by 4-8-fold, the maximal effect for mCAP2 was observed with 8 ng of injected cRNA and for mCAP3 with only 2 ng cRNA. At higher concentrations of mCAP3 (4-12 ng), ENaC activation was inhibited, (P Ͻ 0.01) presumably by competition for the translational machinery. Consequently, in all further experiments we used 4 ng (mCAP1), 8 ng (mCAP2), and 2 ng (mCAP3) that induce maximal activation of ENaC.
Trypsin can activate the I Na (Fig. 3 B, lane 2 vs. 1). The activity of ENaC can be partially blocked by incubating the oocytes in the presence of the serine protease inhibitor aprotinin (lane 3), suggesting that ENaC has been weakly activated by endogenous serine proteases. This block can be overcome by further addition of trypsin in the medium (lane 4). Activation of ENaC in the presence of mCAP1 (lane 5) cannot be further increased by trypsin (lane 6), whereas preincubation in the presence of aprotinin inhibits ENaC activity to base line level (lane 7), which can then be restored by trypsin (lane 8). In the same assay, one can observe that mCAP2 similarly activates ENaC (lanes 9-12). mCAP3 was also able to fully activate ENaC (lane 13), to a level that was not further increased by trypsin (lane 14). Unlike mCAP1 or mCAP2, this effect was aprotinin resistant (lane 15) and could not be further increased by perfusion of trypsin (lane 16). These data indicate that the mCAP3 aprotinin binding site, unlike that of mCAP1 and mCAP2, has a low affinity for the inhibitor. mCAP3 is therefore a good candidate for being a serine protease responsible for the aprotinin-resistant component observed in mpkCCD c14 cells.

Functional Interaction between CAPs and Sgk1 in the Xenopus Oocyte System
To test whether mouse CAPs and Sgk1 interact functionally, oocytes injected with rENaC FLAG-tagged subunits (rENaCf) were coexpressed either individually or with each protease (mCAP), or with Sgk1 or both together. If the two activation pathways are fully independent of each other but have a final common effector (ENaC), a true potentiation should be observed, i.e., any synergistic effect should be more than additive. In Table I (mCAP1-Skg1), Table II (mCAP2-Sgk1), Table  III (mCAP3 ϩ Sgk1), the data are presented in absolute values, allowing to quantify the synergism observed.

mCAP1 and Sgk1
When rENaCf was coexpressed with mCAP1 (Table I), N did not change (P Ͻ 0.9) (column a) whereas I Na (column b) increased 11-fold (P Ͻ 0.001) compared with rENaCf expressed alone. When rENaCf was coexpressed with Sgk1, we observed a 1.6-fold in N (P Ͻ 0.01) and a 2.6-fold increase in I Na (P Ͻ 0.001). When Sgk1 and mCAP1 were coexpressed, I Na increased 9.1fold (P Ͻ 0.001) and N by only 0.3-fold (P Ͻ 0.05) when compared with oocytes injected with Sgk1 alone. If the effect of the two pathways was just additive, their combined effect would not exceed the sum of the two cRNA given alone. For I Na it should not exceed 13 A per oocyte, but we do observe 24 A/oocyte. Thus, the data indicate a strong and highly significant (P Ͻ 0.001) potentiation between mCAP1 and Sgk1 for the I Na effect, whereas the synergism for the N effect was small (though significant) and less than additive. To verify that mCAP1 was expressed for its maximal effect, we tested the effect of external trypsin. As shown in Table I, trypsin (column d) was able to increase baseline I Na only in ENaC or ENaC ϩ Sgk1-injected oocytes. By contrast, trypsin did not elicit any further increase in I Na when mCAP1 was expressed. These data indicate that mCAP1 has already fully activated ENaC expressed at the surface of the oocyte. I Na /N (column c) is a parameter from which one can deduce the average open probability in a whole cell wcP o (see materials and methods and discussion). As shown in Table I, mCAP1 increased I Na /N by 13-fold (P Ͻ 0.001), Sgk1 alone had a minimal effect (1.5-fold; P Ͻ 0.05), mCAP1 and Sgk1 lead to an 11-fold increase in I Na /N (P Ͻ 0.001), not significantly different from stimulation observed with CAP1 alone. Upon activation by trypsin (column e), I Na /N increased 5.9-fold in controls (P Ͻ 0.001) and 4.7-fold in Sgk1 injected oocytes (P Ͻ 0.001). No further significant increase in I Na /N was observed in mCAP1 or mCAP1 ϩ Sgk1-coinjected oocytes.

mCAP2 and Sgk1
When rENaCf was coexpressed with mCAP2 (Table II), N did not change (P ϭ 0.6) (column a), whereas I Na (column b) increased 8.4-fold (P Ͻ 0.001) compared with rENaCf expressed alone. When rENaCf was coexpressed with Sgk1, we observed a 1.6-fold increase in N (P Ͻ 0.01) and a 2.4-fold increase in I Na (P Ͻ 0.001). When Sgk1 and CAP2 were coexpressed, I Na increased 7.6-fold (P Ͻ 0.001) and N by 1.4-fold (P Ͻ 0.06) when compared with oocytes injected with Sgk1 alone. If the effect of the two pathways was just additive, their combined effect would not exceed the sum of the two cRNA given alone. For I Na it should not exceed 5.4 A/ oocyte, but we do observe 9.1 A/oocyte. Thus, the data indicate a strong and highly significant (P Ͻ 0.001) potentiation between mCAP2 and Sgk1 for the I Na effect, whereas the synergism for the N effect did not reach the level of significance (P Ͻ 0.06) and was less than additive. To verify that mCAP2 was expressed maximally, we tested the effect of external trypsin. As shown in Table II, trypsin (column d) was able to increase baseline I Na only in ENaC or ENaC ϩ Sgk1injected oocytes. By contrast, trypsin did not elicit any further increase in I Na when mCAP2 was expressed. These data indicate that mCAP2 has already fully activated ENaC expressed at the surface of the oocyte. As shown in Table II (column d), mCAP2 increased I Na /N by ninefold (P Ͻ 0.001), Sgk1 alone had a small effect (2.4-fold; P Ͻ 0.05), and mCAP2 and Sgk1 led to a 10fold increase in I Na /N (P Ͻ 0.001), not significantly different from the stimulation observed with mCAP2 alone (P Ͻ 0.76). Upon activation by trypsin (column e), I Na /N increased 7.3-fold in controls (P Ͻ 0.001) and 4.4-fold in Sgk1-injected oocytes (P Ͻ 0.001). No further significant increase in I Na /N was observed in mCAP2 or mCAP2 ϩ Sgk1-coinjected oocytes.

mCAP3 and Sgk1
When rENaCf was coexpressed with mCAP3 (Table III), N did not change (P Ͻ 0.7) (column a), whereas I Na (column b) increased 6.8-fold (P Ͻ 0.001) compared with rENaCf expressed alone. When rENaCf was coexpressed with Sgk1, we observed a 1.8-fold increase in N (P Ͻ 0.001) and a 3.6-fold increase in I Na (P Ͻ 0.001). When Sgk1 and mCAP3 were coexpressed, I Na increased 7.3-fold (P Ͻ 0.001) and N by only 0.14-fold (P ϭ 0.4) compared with oocytes injected with Sgk1 alone. If the effect of the two pathways was just additive, their combined effect would not exceed the sum of the two cRNAs given alone. For I Na , it should not exceed 5.2 A/ oocyte, but we do observe 13.2 A/oocyte. Thus, the data indicate a strong and highly significant (P Ͻ 0.001) potentiation between mCAP3 and Sgk1 for the I Na effect, whereas the synergism for the N effect was significant but small and less than additive. To verify that mCAP3 was expressed maximally, we tested the effect of external trypsin. As shown in Table III, trypsin (column d) was able to increase baseline I Na only in ENaC or ENaC ϩ Sgk1-injected oocytes. By contrast, trypsin did not elicit any further increase in I Na when mCAP3 was expressed alone or with Sgk1. These data indicate mCAP3 has already fully activated ENaC expressed at the surface of the oocyte. As shown in Table III, mCAP3 increased I Na /N by 9.4-fold (P Ͻ 0.001), Sgk1 alone had a small effect (twofold (P Ͻ 0.01), and mCAP3 and Sgk1 led to 7.6-fold increase in I Na /N (P Ͻ 0.001), not significantly different from stimulation observed with CAP3 alone (P Ͻ 0.14). Upon activation by trypsin (column e), I Na /N increased 6.7-fold in controls (P Ͻ 0.001) and 5.3-fold in Sgk1-injected oocytes (P Ͻ 0.001). No further significant increase in I Na /N was observed in mCAP3 or mCAP3 ϩ Sgk1-coinjected oocytes. In summary, qualitatively, the effects of mCAP1, mCAP2, or mCAP3 are remarkably similar. Since base-T A B L E I I I Na and N were measured in the same oocyte as described in materials and methods. 15-34 oocytes from a minimum of 5 different batches were measured for each experimental condition. line I Na varies by a factor of two from experiment to experiment (see column b, ENaC-injected oocytes of each table), comparisons between CAPs are also shown in Fig. 4 and summarized as relative changes normalized to ENaC and/or water-injected oocytes. It is evident that, when corrected for the different baseline values, the effects of each CAP are also quantitatively very similar.

Membrane-bound Serine Proteases: a Novel Signaling Cascade in Epithelial Cells?
In this study, we describe the identification of three distinct mammalian membrane-bound serine proteases that are present in the same cell type. mCAP1 is a GPIanchored protein, whereas mCAP2 and mCAP3 belong to the type 2 transmembrane serine proteases (TTSPs) family (Hooper et al., 2001). A few members of membrane-bound serine proteases have been so far biochemically characterized or their cell surface localiza-tion has been demonstrated (Hooper et al., 2001). Based on a conserved disulfide bond linking the proand the catalytic domains, one can predict that TTSPs are active in their membrane-bound form. Interestingly, some TTSPs have a high degree of autocatalytic activity and the targeting of the TTSPs to the apical membrane of an epithelial cell involves its proteolytic domain (Zheng et al., 1999;Hooper et al., 2001). Recently, a recombinant Kunitz-type serine protease inhibitor, which is similar to aprotinin, was tested in human bronchial epithelial cells and shown to efficiently block ENaC-mediated Na ϩ transport in the lung (Bridges et al., 2001). Homology screening of a human airway epithelial cDNA library identified human prostasin (the orthologue of mCAP1) and TMPRSS2, a homologue of mCAP2 or the human TMPRSS4 (Donaldson et al., 2002). These authors demonstrated that prostasin increased ENaC activity, whereas TMPRSS2 had a strong antagonist effect in the Xenopus oocyte system. In our screen, we were not able to identify epitheliasin, the mouse homologue of TMPRSS2, in the mpkCCD c14 cell line. In our study, mCAP2 clearly has an agonist effect on ENaC activation, which is very similar to that of mCAP1 or prostasin. It is not clear, at present time, what the structural difference is between the two proteases that might explain this striking functional difference. Our present working hypothesis is that mCAP1-3 are coexpressed at the apical membrane of, for example, kidney cells, and exert their effect either together on channel activation or by a catalytic cascade similar to the serine protease cascade of the blood clotting system. For xCAP1, we have reported recently the importance of the GPI anchor for the activation of ENaC at the plasma membrane (Vallet et al., 2002), but the role of the transmembrane domains of mCAP2 or mCAP3 in ENaC activation has not yet been studied. How and in which sequence each of the serine proteases may activate ENaC is not known.

Control of Cell Surface Expression of ENaC and its Open Probability by Two Distinct and Synergistic-signaling Pathways Extends the Dynamic Range of Channel Regulation
The Xenopus oocyte expression system allows to quantify cell surface expression of epithelial sodium channels and to determine the channel open probability in a whole cell (wcP o ) (Firsov et al., 1996(Firsov et al., , 1997) (see materials and methods). According to the single channel measurement after trypsin activation reported by Chraibi et al. (1998), one can make the assumption that gNa remains unchanged under our experimental conditions. Under a voltage clamp set constant at Ϫ100 mV, one can now get a fairly good estimate of the wcP o and how it is affected by our experimental maneuvers. Under low sodium incubation, we reported a I Na /N of 4.82 mA/fmole (Firsov et al., 1996). We can calculate a wcP o of around 0.018, assuming a heterotetrameric channel . In the present study, we injected three times less ENaC cRNA, leading to a lower cell surface expression of ENaC (N varies from 0.06-0.08 fmole/oocyte [present study] vs. 0.5 fmole/ oocyte ]) but with a relatively high current (I Na , 0.5-1 A/oocyte with a I Na /N varying between 9 and 14 A/fmole (See Table I-III). From these data, one can calculate a wcP o of 0.05. For ENaC, the estimation of P o in membrane patches (mpP o ) has been reported to be extremely variable (Palmer and Frindt, 1988;Palmer and Frindt, 1996). In native membranes from salt-repleted rat, no channel activity was detectable. It is likely that this observation is due to the lack of sensitivity of the patch method to detect channels with very low P o or very few channels with higher P o . In native membrane from salt-depleted animals with high plasma aldosterone levels, mpP o ranged from 0.05 to 0.9 with an average value of 0.5 (Pacha et al., 1993). Due to the wide distribution of spontaneous P o and the slow transitions between open and closed states, the quantitation of the number of active channels in the membrane patch is difficult, leading to overestimation of P o . This overestimation could be experimentally documented by direct comparisons of the two methods for a mutant channel of the ␤ subunit (G37S) leading to a 50% decrease in Po (Firsov et al., 1997). The alternative wcP o method used in the present study is therefore a useful quantitative approach to assess the effect of CAP or Sgk1 on P o in an intact cell.

mCAP1, mCAP2, and mCAP3 Increase wcP o without Changing N
Upon CAP stimulation, it is evident that the wcP o of ENaC can reach very high values (up to 0.65 for mCAP1), which cannot be further increased by external trypsin. None of the three CAPs tested changed the cell surface expression of ENaC. It would appear that this signaling pathway is unique in controlling the gating of the channel from an external site, which is trypsin sensitive (Chraibi et al., 1998). The molecular mechanisms remain elusive. It has been suggested that the ␥ subunit was a substrate for CAP (Masilamani et al., 1999) but, so far, no biochemical experimental evidence has supported this hypothesis. A second important issue is to know whether CAPs are regulated by hormones or other factors. In our experimental systems, we have not been able to demonstrate any effect of aldosterone on the mRNA abundance of either of the three CAPs expressed in mpkCCD cells (unpublished data). It is, however, interesting to note a recent article reporting that aldosterone increased in vitro the expression of mCAP1/prostasin mRNA and protein in a kidney cell line (M1) and in vivo in adrenalectomized rats (Narikiyo et al., 2002).

Dual Effect of Sgk1 on N and wcP o
Our data clearly indicate that Sgk1 increases N significantly in three large and independent series of experiments (Table I-III and Fig. 4), confirming previous reports showing an over twofold increased expression of ␣␤␥-FLAG ENaC measured by 125 I-protein G binding to the anti-FLAG monoclonal antibody (Alvarez de La Rosa et al., 1999), with no evidence of an effect on P o measured in membrane patches. More recently, it has been shown that Sgk1 induced a proportional increase in I Na and in N (about threefold) in the Xenopus oocyte system, suggesting that the effect is entirely mediated by a change in ENaC cell-surface expression (Loffing et al., 2001) that may occur in vivo only in a restricted part of the aldosterone-sensitive distal nephron (proximal ASDN). Our data differ somewhat from these two studies in the sense that we do observe a small but highly significant and highly reproducible effect of Sgk1 on I Na /N (1.52, P Ͻ 0.001, Table I; 2.4, P Ͻ 0.001 Table II; and 2.0, P Ͻ 0.001) that is consistently higher than that observed on N (Table III), suggesting that Sgk1 has an additional effect on wcP o . The reasons for that differ- ence are not clear, but our experimental protocol differs significantly from those of the two previous studies in the sense that we have measured I Na and N in the same individual oocyte, allowing a better quantitation of any effect on N and/or P o . On the other hand, values of mpP o reported in one study were 0.83 for ENaCinjected oocytes and 0.87 for ENaC ϩ Sgk1-injected oocytes (Alvarez de La Rosa et al., 1999). As discussed above, such high baseline values may not reflect the physiological regulation of ENaC in a kidney cell and the technique may underestimate the number of silent or weakly active channels in the membrane. Interestingly, we reported previously that the Liddle mutation has also a dual effect on N and P o (Firsov et al., 1996), very similar to the effect of Sgk1 reported here. Since Sgk1 is proposed to mediate its effects on ENaC through the phosphorylation of Nedd4-2 (Debonneville et al., 2001), which binds to the PPXY motif of ENaC, which is precisely the consensus for the Liddle mutation, it would make sense that the Sgk1-Nedd4-2-ENaC-signaling cascade involves a dual mechanism of ENaC activation.

CAP and Sgk1: Synergism of Potentiation
This paper demonstrates a synergism between the two signaling pathways in the oocyte expression system (Figs. 4 and 5). mCAP1, mCAP2, or mCAP3 have no effect on cell surface expression of ENaC, but increase wcP o by 7-10-fold. On the other hand, Sgk1 increases the number of channels expressed at the cell surface by twofold, without changing wcP o . When the two signaling pathways are activated together, a 20-30-fold increase is observed. The synergism is therefore more than additive and is a true potentiation. The possible mechanisms for this synergism has not yet been studied. It will be interesting to examine whether mCAPs can bind directly to ENaC by protein-protein interaction through their extracellular (mCAP1, mCAP2, or mCAP3) and/or through their cytoplasmic domains (mCAP2 or mCAP3). Another possible mechanism would imply a protein-protein interaction between Sgk1 and the intracellular COOH termini of mCAP2 or mCAP3 as a potential target for phosphorylation. It remains to demonstrate that the synergism we describe here operates in vivo in the principal cell of CCD in response to aldosterone. Gene inactivation of Sgk1 (Wulff et al., 2001) and mCAP1 (Rubera et al., 2002), mCAP2, and mCAP3 will help to dissect the respective role of the two signaling pathways. Despite large variations in salt intake, the kidney is able to maintain sodium balance and the extracellular volume within narrow margins, an important factor in the control of blood pressure. The ASDN plays a key role in adjusting sodium reabsorption to diet intake that may vary from 1 g NaCl/d to 40 g/d. The fine control of ENaC through the control of its cell surface expression and its open probability by aldosterone acting, on one hand, on an intracellular signaling cascade and, on the other hand, on a serine protease that controls gating from the extracellular compartment, should provide the large range required for rapid and long term regulation of sodium transport in ASDN.


extfieldsofstudy: Biology Medicine

text:
Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation.

Introduction
Studies in the yeast Saccharomyces cerevisiae have provided insight into the mechanism and control of eukaryotic DNA replication. Yeast possess specific, well-defined origins of DNA replication onto which complexes of replication factors assemble. Generally, yeast origins are 200 bp or less and consist of an 11-bp A-T-rich autonomously replicating sequence (ARS) consensus sequence, as well as the B1 and B2 elements. The pre-replication complex (pre-RC)* assembles onto these regions during the G1 phase of the cell cycle, resulting in origins that are competent to initiate DNA replication and serving as a molecular beacon to recruit the replication fork machinery (for reviews see Bell and Dutta, 2002).
A combination of approaches in S. cerevisiae has identified components of the pre-RC and the replication fork machinery (for reviews see Dutta and Bell, 1997;Bell and Dutta, 2002). The six-member origin recognition complex (ORC) was identified as a pre-RC component by its ability to bind to yeast replication origins (Bell and Stillman, 1992). ORC binds to the ARS consequence sequence and B1 elements, and then recruits the pre-RC factors cell division cycle (Cdc)6/ Cdc18 and double-parked protein (DUP)/Cdt1. In turn, DUP/Cdt1 and Cdc6/Cdc18 load the hexameric minichromosome maintenance proteins (MCM)2-7 complex onto pre-RCs. MCM2-7 are necessary for initiation, but are also required for elongation and travel with replication forks (Aparicio et al., 1997;Labib et al., 2000). Furthermore, MCM4, -6, and -7 have helicase activity in vitro, suggesting that they function as the replicative helicase (Ishimi, 1997).
Once MCM2-7 are loaded, additional replication factors are recruited to origins and replication initiates. Cdc45 and Mcm10 are two other factors necessary for both initiation and elongation that travel with replication forks (Merchant et al., 1997;Aparicio et al., 1999;Tercero et al., 2000;Wohlschlegel et al., 2002). CDK and Cdc7-Dbf4 kinase activity are required for initiation, with MCM2-7 and Cdc45 as potential targets Zou and Stillman, 2000). Replication fork components must also be recruited for origin firing. These include the single-stranded DNA The online version of this article contains supplemental material. binding protein RPA, Pol ␣ /primase, the clamp loader replication factor (RF)C; the sliding clamp proliferating cell nuclear antigen (PCNA), DPB11, and the replicative polymerases Pol ␦ / (for reviews see Waga and Stillman, 1998;Bell and Dutta, 2002).
Although the pre-RC and replication fork components are structurally conserved in metazoans (Donaldson and Blow, 1999), analysis of replication initiation and elongation is limited by the lack of model replicons. Using cells and extracts from humans, Xenopus , or Drosophila pre-RCs can assemble on model templates and DNA replication can initiate in vitro, giving results consistent with the yeast paradigm of pre-RC and replication fork composition and activity (Chesnokov et al., 1999;Mendez and Stillman, 2000;Blow, 2001). However, obstacles such as multiple potential initiation sites and complex cis-regulatory sequences have hindered the progress of in vivo replication initiation studies (for reviews see DePamphilis, 1999;. In addition, a lack of genetic assays has made it difficult to study the precise localization and properties of the trans-factors necessary for replication. Thus, the available models in vertebrates have yielded information about either cis-elements or trans-factors necessary for replication, but a single system has not provided information about both. In contrast, amplification in the Dipteran flies Drosophila melanogaster and Sciara coprophila has provided the framework to study DNA replication in which the cis-regulatory sequences are well defined and trans-acting replication factors can be examined (Calvi and Spradling, 1999;. In Sciara , the replication start site within an amplified salivary puff origin, ori II/9A , is understood at the single nucleotide level and displays similarities to the yeast ARS. Furthermore, Drosophila ORC has been shown to bind to an 80bp region adjacent to this replication start site . In Drosophila , amplification of the chorion gene clusters provides another powerful system for the study of metazoan DNA replication. The ovarian follicle cells, somatic cells that surround the developing oocyte, synthesize and secrete the chorion, or eggshell. In response to developmental signals at stages 9 and 10 of egg chamber development, the follicle cells end genomic DNA replication and begin to amplify several clusters of genes throughout the genome, including two clusters of chorion genes (Calvi et al., 1998).
Amplification of the chorion clusters occurs via a bidirectional replication mechanism, in which initiation occurs repeatedly from defined origins and forks progress outward to ف 50 kb on either side of the origins (Spradling, 1981;Spradling and Mahowald, 1981;Osheim et al., 1988;Delidakis and Kafatos, 1989;Heck and Spradling, 1990). By stage 13 of egg chamber development, a gradient of copy number results, with the origins and chorion genes located at the central, maximally amplified region. Quantitative Southern blots detect a maximum copy level of 16-20-fold for the X chromosome chorion cluster, and 60-100-fold for the third chromosome chorion cluster (Spradling, 1981;Delidakis and Kafatos, 1989). P-element-mediated transformation of DNA fragments from the third chromosome cluster defined the cis-regulatory element, amplification control element ( ACE ) on third chromosome ( ACE3 ), which is required for high levels of amplification and sufficient for low levels of amplification (de Cicco and Spradling, 1984;Carminati et al., 1992). Two-dimensional gel analysis demonstrated that repeated firings occur from a preferred origin, ori ␤ , ف 1.5 kb downstream of ACE3 (Delidakis and Kafatos, 1989;Heck and Spradling, 1990). Further transformation experiments showed that ACE3 interacts with ori ␤ (Lu et al., 2001).
Genetic studies took advantage of female-sterile mutations to demonstrate an essential role for known replication factors in chorion amplification. Females mutant for orc2 , dbf4-like , mcm6 , and dup/cdt1 lay eggs with thin or otherwise abnormal eggshells due to defects in chorion amplification (Underwood et al., 1990;Landis et al., 1997;Landis and Tower, 1999;Whittaker et al., 2000;Schwed et al., 2002).
In addition to genetic approaches, the process of chorion amplification can be visualized directly. Bromodeoxyuridine (BrdU) incorporation at amplicons can be detected throughout the amplification process, from stages 10B to 13 (Calvi et al., 1998). The replication proteins ORC2, ORC1, ORC5, DUP/Cdt1, and CDC45 localize specifically to amplicons during chorion amplification in follicle cells (Asano and Wharton, 1999;Austin et al., 1999;Royzman et al., 1999;Loebel et al., 2000;Whittaker et al., 2000). In this study, we use a cell biology approach coupled with quantitative realtime PCR to decipher the dynamics of DNA replication at the chorion loci in a developmental context. We find that all initiation at chorion origins occurs during one part of amplification, whereas in subsequent stages, only the existing replication forks elongate. We also observe that the localization pattern of several replication factors during amplification correlates with the roles of these proteins in initiation or elongation.

Localization patterns of ORC2 and BrdU throughout chorion amplification
We performed high-resolution deconvolution microscopy to analyze the pattern of ORC localization with respect to BrdU incorporation at the third chromosome chorion locus throughout amplification. Previous observations showed that ORC2 localizes to amplified regions for only a portion of amplification, from egg chamber stages 10A to 11 (Royzman et al., 1999). In contrast, BrdU incorporation begins in stage 10B and persists until stage 13 of egg chamber development (Calvi et al., 1998;Royzman et al., 1999;Calvi and Spradling, 2001). These differences in localization patterns suggest that DNA replication continues in the absence of ORC2 at chorion loci; that is elongation exclusively may occur during stages 12 and 13.
When BrdU incorporation became detectable early in stage 10B follicle cell nuclei, ORC2 localized to the X and third chromosome chorion clusters and was coincident with BrdU (unpublished data). As stage 10B continued, ORC2 no longer localized to the X chromosome cluster, but persisted at the third chromosome cluster, coincident with BrdU ( Fig. 1, A and B; Video 1, available at http:// www.jcb.org/cgi/content/full/jcb.200207046/DC1). At this time, ORC2 was present at origin sequences, as the ORC2 signal colocalized with that of a FISH probe spanning ACE3 and ori ␤ on the third chromosome ( Fig. 1, C-E). Addi-tionally, chromatin immunoprecipitation experiments have shown that in vivo, ORC is bound in the vicinity of ACE3 and ori ␤ in amplifying stage-10 follicle cells . Furthermore, our observations are consistent with previous results obtained by Calvi, localizing the same FISH probe relative to BrdU incorporation (Calvi et al., 1998;Calvi and Spradling, 2001). It should also be noted that even though the follicle cells are polyploid (16C), the fact that there is a single BrdU spot (or set of double bars, see below) for each amplicon demonstrates that all the chromosome copies must be tightly aligned as polytene chromosomes (Calvi and Spradling, 2001). These data demonstrate that ORC2 is at chorion origins when they fire and begin to incorporate BrdU.
As chorion amplification proceeded, deconvolution microscopy revealed that the pattern of BrdU incorporation diverged from that of ORC2. In stage-11 egg chambers, the BrdU staining pattern resolved into a coffee bean-like structure, with bands of BrdU incorporation flanking ORC2 present at the origins (Fig. 1, F and G). Furthermore, and consistent with the results of Royzman et al. (1999), during stage 11, ORC2 dissipated from the origins and a higher level of diffuse nuclear and cytoplasmic ORC2 staining was observed. Although ORC2 staining was undetectable at chorion loci after stage 11, BrdU incorporation continued, and during stages 12 and 13, the BrdU pattern resolved into a double bar structure (see Figs. 3 E and 4 E,BrdU and Fig. 5, C and D, lack of ORC2). Similar results were observed for ORC1 (unpublished data).
Deconvolution microscopy enabled us to measure the dimensions of the fluorescent signals at the third chromosome amplicon from stages 10B to stage 13. We examined the gap from the inside of one BrdU (or DUP, see below) signal to the inside of the second BrdU signal, the length of the bars, and the depth of each of the bars (Fig. 1 I). Based on the onionskin or reinitiation model of chorion amplification (Botchan et al., 1979;Osheim et al., 1988) (Fig 1, H and I), the gap should represent: the extent of replication fork progression; the length and number of origin firings; and the depth and complexity of the onion skin as replication forks progress outward and are arranged in three dimensions. The dimensions during stages 10B, 11, and 13 are summarized in Table I. The length of the bars remained constant after stage 11 (stage 11, 1760 nm; stage 13, 1740 nm), suggesting that the maximum number of origin firings occurred by stage 11. The depth measurement increased dramatically throughout the later stages of amplification, from 400 nm in stage 10B to 1040 nm in stage 13. The gap measurement increased from 300 nm in stage 10B to 740 nm in stage 13. The gap measurement can be used to calculate the distance in kilobases the forks have progressed at a particular stage, with the conversion factor of 100 nm ف 10 kb. This conversion factor was calculated based on data by Calvi and Spradling (2001), in which the distance of two FISH probes 46 kb apart and flanking ACE3 was measured to be ف 480 nm, giving the conversion factor of 480 nm ف 46 kb, or ف 100 nm ف 10 kb. Thus, in stage 10B, replication forks have traveled a total distance of 30 kb (an average of 15 kb on either side of ACE3 ), and by stage 13 they have moved out across a 74-kb total region (an average of 37 kb on each side).
Considering the lack of ORC at chorion loci after stage 11, the essential role ORC plays in initiation, and the microscopy measurements, we propose that amplification can be separated into two phases. The first phase of amplification occurs during stages 10B and 11, is ORC dependent, and involves initiation coupled with elongation ( Fig. 1 H). After this discrete period of initiation, ORC is lost from chorion origins and only the existing replication forks progress outward, in an elongation-only phase, to give the double bar structure seen in stages 12 and 13 ( Fig. 1 I).

Quantitative realtime PCR measurement of DNA copy number along the third chromosome chorion amplicon
The immunofluorescence studies suggested that if the relative DNA copy number along the amplified regions were measured, a maximum copy number at origin sequences would be detected by stage 11. Furthermore, as replication forks progress outward by stages 12 and 13, we would expect to see a sequential increase in copy number of the loci proximal and distal to origins. To test this model, we used realtime PCR to quantify copy number in 5-kb intervals along the third chromosome chorion locus during each stage of egg chamber development (see Materials and methods).
Quantitation of fold amplification in each of the stages allowed us to measure both inititiation and elongation events. In stage 1-8 (preamplification) egg chambers, no amplification was observed (Fig. 2 A). Stage 10B egg chambers, in which chorion amplification has begun, showed an increase in copy number at and around ACE3 , (from 25 to -15 kb) with a maximum of 15-fold amplification at 0 kb ( Fig. 2 B). Loci proximal and distal to ACE3 , from 25 to 35 kb and -20 to -40 kb, also showed some amplification during stage 10B (two-to fourfold). This suggests that a subset of forks had replicated the entire amplicon. By stage 11, 30-fold amplification was observed at ACE3 , as further rounds of initiation occurred. We did not observe integral doublings of copy number at ACE3 between stages 10B and 11, probably because pools of egg chambers were used, and the result obtained represents the average of the pool. An increase in copy number from ف 5 to Ϫ 20 kb also was detected in stage 11 (Fig. 2 C).
Strikingly, in stage 12 and 13 reactions (Figs. 2, D and E, respectively), no further increase in copy number was detected at ACE3 , with 29-and 27-fold amplification, respectively. This indicates that no further initiation occurred. In contrast, at loci proximal and distal to ACE3 , an increase in copy number was detected as the existing replication forks progressed outward to approximately -40 and 35 kb. For example, at 35 kb, ninfold chorion amplification was detected in stage 13, and 7.5-fold chorion amplification was detected in stage 12, as compared with four and twofold in stages 11 and 10B, respectively. We observed only half the maximum level of amplification detected by Spradling's original quantitative Southern blots ( ف 30-vs. 64-fold; Spradling, 1981), probably because of the increased sensitivity of fluorescent PCR detection and the uniformity of the intervals used to measure amplification here.
When data from all stages are compared ( Fig. 2 F), it is clear that the final rounds of initiation occur between stages 10B and 11 and the copy number of flanking regions increases throughout subsequent stages. The results in Fig. 2 were obtained using the 3R nonamplified control to determine fold amplification, and similar results were observed using the ry control (unpublished data).
Localization patterns of PCNA and MCM2-7 during chorion amplification Both lines of data described above indicate that initiation and elongation occur simultaneously during one phase of chorion amplification, whereas only elongation occurs dur- ing a separate developmental phase. As an additional test of this hypothesis, we studied the localization patterns of replication factors known to travel with the replication forks, PCNA, and MCM2-7. We observed a compelling pattern of PCNA localization in follicle cell nuclei. PCNA was nuclear throughout stages 1-9 (unpublished results), but by stage 10B, foci of PCNA staining were detected above faint nuclear staining (Fig. 3, A and C). As chorion amplification proceeded, PCNA remained localized and resolved into the double bar structure (Fig. 3, D and F). To ensure that PCNA was localized to chorion regions, colabeling with BrdU was performed, and PCNA was Figure 2. Quantitative realtime PCR performed on staged egg chamber DNA confirms the timing of initiation and elongation. DNA from egg chambers before chorion amplification, stages 1-8, and during amplification, stages 10B, 11, 12, and 13 was used in quantitative realtime PCR reactions. Primer sets used for chorion loci spanned the third chromosome 50 kb on either side of ACE3 (denoted as 0 distance), in 5-kb intervals, and control primer sets (nonamplified) were to an intergenic region on chromosome arm 3R. The Y axis represents fold amplification, measured as the ratio of the chorion locus to the 3R locus and errors are the standard deviation of the sample. The X axis represents distance along the chorion locus in kilobases, with the major origin, ori␤ located between 0 and 5 kb. (A) In stage 1-8 egg chambers, no chorion amplification has occurred and the ratio of chorion to control loci is centered at .1ف Note that the scale in A is different from the scale in B-F. (B) By stage 10B, chorion gene amplification has initiated and there is an increase in fold amplification over 53ف kb total. (C) By stage 11, additional initiation has occurred at the origins, as fold amplification increases to .03ف (D) During stage 12, no further increases in copy number are detected at origins, but an increase in fold amplification both proximal and distal to origins is detected. (E) By stage 13, replication forks have progressed out further, as an increase in fold amplification is detected out to 53ف and -40 kb. No further initiation events occurred. The stage 13 reactions were performed on two separate samples of stage 13 DNA and similar results were observed in both trials (unpublished results). (F) A composite graph of A-E showing fold amplification at the third chromosome chorion locus throughout egg chamber development.
shown to colocalize with BrdU (Fig. 3, A-C and D-F). These data support the idea that the double bar structure arises from fronts of bidirectional replication fork movement.
Previously, polyclonal antibodies raised against MCM2, -4, and -5 O'Farrell, 1997, 1998; showed nuclear staining with no localization to chorion foci (Royzman et al., 1999). This was true even when egg chambers were treated with a high salt, high detergent buffer in an attempt to remove nonchromatin-bound MCMs from the nucleus (Schwed et al., 2002). We reexamined the localization of the MCM2-7 complex during amplification using a monoclonal antibody that recognizes an epitope present in all six Drosophila MCM subunits (Jayson Bowers, Anthony Schwacha, and Stephen Bell, personal communication), thereby enhancing the sensitivity of detection. Additionally, egg chambers were washed with a high salt, high detergent buffer to remove nonchromatin-bound MCM proteins from the nuclei.
Under these conditions, we saw that MCM2-7 localized throughout amplification. MCM2-7 localization first became visible as foci in stage 10B (unpublished data; Fig. 3, G and I) and progressed to the double bar structure by stages 12 and 13 (Fig. 3, J and L). To confirm that MCM2-7 were localized to the chorion regions, we costained with PCNA and observed colocalization throughout all stages of amplification (Fig. 3, G-I and J-L). Thus, MCMs are present at chorion amplicons during initiation and persist throughout amplification, presumably moving with the replication forks. The correlation of MCM2-7, PCNA, and BrdU staining patterns supports our model for chorion amplification.  (Calvi et al., 1998), and the X chromosome cluster (arrow) has already resolved into the double bar structure by this stage. In addition to being at the chorion loci, PCNA is diffusely present throughout the nucleus during this stage. (D-F) A single follicle cell nucleus from a stage 13 egg chamber shows this pattern of PCNA and BrdU staining, which is characteristic of replication fork movement. The 2 smaller foci of staining in this image may be the X chromosome amplicon. (G-I and J-L) MCM2-7 are in red and PCNA is in green. (G-I) MCM2-7 and PCNA colocalize in stage 11 follicle cell nuclei (arrows represent third chromosome clusters in two nuclei). (J-L) MCM2-7 staining, like PCNA, persists throughout chorion amplification and resolves into the double bar structure by stage 12. One stage 12 nucleus is shown. Bars, 1 m.

The localization pattern of DUP/Cdt1 during chorion amplification
We then characterized the properties of the pre-RC component, DUP/Cdt1 (Maiorano et al., 2000;Nishitani et al., 2000;Whittaker et al., 2000;Devault et al., 2002;Tanaka and Diffley, 2002). DUP/Cdt1 requires ORC2 to localize to chorion origins (Whittaker et al., 2000) and DUP/Cdt1 homologues in yeast and Xenopus have been shown to interact with Cdc6/18 to load MCM2-7 onto origins (Maiorano et al., 2000;Nishitani et al., 2000;Tada et al., 2001;Devault et al., 2002;Tanaka and Diffley, 2002). In Xenopus extracts, fission yeast, and budding yeast, Cdt1 is dispensable after initiation (Maiorano et al., 2000;Nishitani et al., 2000;Devault et al., 2002;Tanaka and Diffley, 2002). Furthermore, Cdt1 appears to be lost from chromatin or the nucleus at the onset of S phase (Maiorano et al., 2000;Tanaka and Diffley, 2002). These data suggest that Cdt1 is not necessary after performing its role in pre-RC formation. In contrast, the initial description of DUP/Cdt1 staining during amplification showed that DUP/Cdt1 localized to chorion loci throughout amplification, and was present during stage 13 in the double bar structure (Whittaker et al., 2000). Therefore, we examined the localization pattern of DUP/Cdt1 during amplification in relation to BrdU and ORC2, using confocal and deconvolution microscopy, to investigate whether DUP/Cdt1 could be traveling with replication forks.
DUP/Cdt1 colocalized with BrdU throughout amplification. In stage 10B, DUP/Cdt1 staining was detected as foci ( Fig. 4 A) that overlapped completely with BrdU staining (Fig. 4, B and C). By stage 13, DUP/Cdt1 staining resolved into the double bar structure (Fig. 4 D) and was coincident with BrdU (Fig. 4, E and F). The fact that DUP/ Cdt1 remained localized to chorion regions throughout the elongation phase suggests that DUP/Cdt1 travels with the replication forks.
We precisely localized DUP/Cdt1 with respect to ORC2 by deconvolution microscopy, and in contrast to the colocalization of DUP/Cdt1 and BrdU, the ORC2 and DUP/Cdt1 staining patterns diverged as amplification proceeded. In early stage 10B, ORC2 and DUP/Cdt1 staining overlapped (unpublished data), similar to the results with ORC2 and BrdU costaining (Fig. 1, A and B). However, by late stage 10B and stage 11, DUP/Cdt1 staining became fainter at the origins and resolved into a coffee bean-like structure (Fig. 5, A and B; Video 2, available at http://www.jcb.org/cgi/content/ full/jcb.200207046/DC1). This change in the DUP/Cdt1 localization pattern occurred while ORC2 remained bound to origins. By stage 13, DUP/Cdt1 was detected in the double bar structure, with no evidence of ORC2 staining at origins (Fig. 5, C and D). Similar results were seen for DUP/ Cdt1 and ORC1 (unpublished data). The pattern of DUP/ Cdt1 localization in relation to BrdU and the fact that DUP/Cdt1 clears from origin sequences while ORC2 remains bound strongly indicate that DUP/Cdt1 travels with elongating replication forks.

DUP/Cdt1 is necessary to localize MCM2-7 during amplification
Given the unexpected presence of DUP/Cdt1 during elongation, we wanted to know if DUP/Cdt1 functioned in this system to load MCM2-7 during initiation. To test this, we studied the localization pattern of MCM2-7 in the dup PA77 female-sterile mutant ovaries. These mutants have thin eggshells and decreased and delayed BrdU incorporation during amplification (Underwood et al., 1990;Whittaker et al., 2000).
In dup PA77 homozygous mutant ovaries, we did not detect the localization of MCM2-7 to chorion loci at any stage of amplification (Fig. 6). Furthermore, MCM2-7 appeared to cluster at the nuclear envelope, where it colocalized with nuclear lamins. These data indicate that DUP/Cdt1 is necessary to localize MCM2-7 to origins during chorion amplification, the same as the role of DUP/Cdt1 orthologues. The clustering of MCM2-7 at the nuclear periphery suggests that DUP/Cdt1 may be necessary for the nuclear transport of MCM2-7, consistent with the findings in S. cerevisiae that Cdt1 and MCM2-7 display an interdependence for nuclear trafficking (Tanaka and Diffley, 2002).

Discussion
We demonstrated by three independent lines of evidence that initiation and the bulk of elongation at a chorion amplicon occur during two separate developmental periods. First, deconvolution microscopy shows that ORC and BrdU ini-tially colocalize at origins and then diverge, as ORC is lost in stage 11 and BrdU resolves into a double bar structure. Second, elongation factors PCNA and MCM2-7 follow the same pattern as BrdU, resolving from foci early in amplification to a double bar structure by stage 12 to 13. Third, quantitative realtime PCR shows a peak increase in DNA copy number at the origins by stage 11, with increases in flanking sequences becoming substantial in stages 12 and 13. Thus initiation ends by stage 11, and during stages 12 and 13 only the existing forks progress outward. Furthermore, these observations led to the unanticipated conclusion that DUP/Cdt1 travels with replication forks.
Our realtime PCR and immunofluorescence data are remarkably consistent. First, both methods restrict initiation to stages 10B and 11, and elongation to stages 12 and 13. Between stages 10B and 11, the maximum fold amplification was detected at ACE3 by realtime PCR, ORC localized to origins, and the deconvolution showed a maximum increase in bar length. During stages 12 and 13, increases in fold amplification were detected only proximal and distal to ACE3, and ORC no longer localized to origins, whereas BrdU incorporation resolved into the double bar structure. Second, the distances of fork movement are consistent. Deconvolution measurements predicted that forks were maximally 30 Ϯ 3 kb apart in stage 10B, and this correlates with the 40-kb span of peak copy number detected by realtime PCR. In stage 11, forks were measured to have progressed across a 55 Ϯ 13-kb region by deconvolution and across a 45-kb region by realtime PCR. By stage 13, deconvolution showed that replication forks were maximally separated by 74 Ϯ 7 kb, whereas realtime PCR measured a 75-kb span.
The convergence of the three lines of data argues against two alternative explanations for the immunofluorescence results. One alternate hypothesis is that ORC remains localized after stage 11, yet is not detectable because protein levels drop below detectable limits or epitopes become inaccessible. This is unlikely, as we observed the elongation factors PCNA, MCM2-7, and even DUP/Cdt1 change from a focus to double bar structure without a change in staining intensity. In contrast, during stage 11 ORC staining intensity decreased at origins, concomitant with a rise in nuclear and cytoplasmic levels. A second alternate hypothesis is that the double bar structures do not represent fork movement but result from firings of unidentified origins to either side of the ACE3/ori␤ origin region. If this were the case, initiation events after stage 11 would occur independently of ORC, and the gradient profile from realtime PCR would be much different. As a result of these additional origins firing, the stage 12 graphs would show peaks of increased copy to either side of ACE3/ori␤, and by stage 13 the new forks would broaden the area of maximum copy number into a plateau.
The quantitative analysis of the amplification gradient provides insight into mechanisms affecting fork movement and termination and suggests that the onionskin structure (Botchan et al., 1979;Osheim et al., 1988) impedes fork movement. We calculated the maximal rate of fork movement during amplification to be 90 bp/min on average, well within the 50-100-bp/min range calculated previously (Spradling and Leys, 1988). (By quantitative realtime PCR, the furthest a replication fork could travel is 40 kb between stages 10B and 13, a period of 7.5 h.) In comparison, replication forks in the polytene larval salivary glands travel at 003ف bp/min (Steinemann, 1981), whereas rates of fork movement in both diploid Drosophila cell culture and embryo syncytial divisions are 6.2ف kb/min (Blumenthal et al., 1973). From these rates, it seems that polyteny hinders replication fork movement, an effect even more pronounced in amplification, given that the chorion cluster has a rate of fork movement three times less than polytene salivary glands. The fact that by stage 13 there is a gradient of copy number, and not a plateau further demonstrates the inefficiency of fork movement along the chorion cluster.
There do not seem to be specific termination sites to stop forks either along or at the ends of the chorion region, but fork movement may display some sequence or chromatin preference. The gradient of decreasing copy number implies that forks stop at a range of sites, as we would expect the presence of specific termination points along the region to cause steep drops in copy number. Despite this lack of specific termination sites, during stages 12 and 13 we see a greater increase in copy number to one side of ACE3 (Fig. 2, graphs, right), and often observe by immunofluorescence that one of the two bars is shorter. This suggests that the sequence or chromatin structure to the other side of ACE3 hinders fork movement, and as fewer forks move out, less BrdU incorporation occurs and a shorter bar results.
In contrast to other systems (Maiorano et al., 2000;Nishitani et al., 2000;Tada et al., 2001;Devault et al., 2002;Tanaka and Diffley, 2002), our results reveal that DUP/ Cdt1 travels with replication forks during amplification. Although it could be argued that DUP/Cdt1 simply spreads along the chromatin as amplification proceeds, this is unlikely. DUP/Cdt1 and ORC2 colocalization studies show that although ORC2 remains at origins, the DUP/Cdt1 signal decreases at origins and subsequently flanks the ORC2 signal. Furthermore, during elongation DUP/Cdt1 does not spread across the entire chorion region. Rather, there is a gap between the double bars of DUP/Cdt1 staining which increases from 300 Ϯ 30 nm in stage 10B to 740 Ϯ 70 nm in stage 13.
The presence of DUP/Cdt1 at forks during elongation strongly suggests it has a role in this phase of replication. Why might DUP/Cdt1 be required during elongation in this system? Chorion amplification is unique because replication forks chase forks, instead of converging as in normal eukaryotic replication. Given this peculiarity of amplification, and considering the steric constraints that arise and impede forks, DUP/Cdt1 may be necessary to maintain MCM2-7 at these lethargic forks. DUP/Cdt1 could function as a processivity factor for the MCM2-7 complex, holding it on the DNA, or it could continuously reload new MCM2-7 as they fall off the progressing replication forks. It is formally possible that although DUP/Cdt1 travels with the forks it does not perform a function. DUP/Cdt1 could simply not be expelled from the replication machinery upon initiation and then be dragged along during elongation. Although we do not favor this possibility, definitively proving that the DUP/Cdt1 at forks is necessary for elongation will require the use of a currently unavailable conditional allele. Such a mutation would permit inactivation of DUP/Cdt1 after initiation and allow a functional test for a role in elongation.
These studies highlight the complex regulation of chorion gene amplification. How are the number of origin firings restricted to the proper developmental time? It is known that the number of rounds of origin firing at the chorion amplicons is limited by the action of Rb, E2F1, and DP (Bosco et al., 2001). Perhaps DUP and MCM2-7 are also a part of this regulation, with origins firing only when MCM2-7 are properly loaded. It will also be interesting to decipher the regulation of DUP/Cdt1 during amplification. Recent studies have demonstrated that a Drosophila homologue of the metazoan re-replication inhibitor, Geminin, exists and interacts biochemically and genetically with DUP/Cdt1 (Quinn et al., 2001;Mihaylov et al., 2002). Female-sterile mutations in geminin result in increased BrdU incorporation during amplification (Quinn et al., 2001), raising the possibility that Geminin acts on DUP/Cdt1 at the chorion loci to limit origin firing. In addition to permitting the delineation of the regulatory circuitry controlling origin firing, the ability to distinguish initiation from elongation developmentally provides a powerful tool for the analysis of the properties of metazoan replication factors in vivo.

Fly strains
Ovary stainings were performed on the Oregon-R wild-type strain unless otherwise noted. The dup mutant allele, dup PA77 was described previously (Underwood et al., 1990;Whittaker et al., 2000).

Immunofluorescence and BrdU labeling
Double labeling of Drosophila ovaries with anti-ORC2 and BrdU was performed as described previously (Royzman et al., 1999), with the following changes: BrdU was used at 6.4 g/ml; secondary detection of ORC2 was with donkey anti-rabbit Rhodamine-RedX at 1:200; secondary detection of BrdU was with goat anti-mouse FITC at 1:200; and ovaries were mounted in Slowfade (Molecular Probes). For anti-ORC1 staining, a rat antibody obtained from Maki Asano (Duke University Medical Center, Durham, NC) was used at 1:350 and detected with donkey anti-rat Cy-3 (Jackson Immu-noResearch Laboratories) at 1:200 (Asano and Wharton, 1999).
Double labeling of Drosophila ovaries with anti-PCNA obtained from Daryl Henderson (SUNY at Stony Brook, Stony Brook, NY) (Henderson et al., 2000) and BrdU was performed as per anti-ORC2/BrdU, but incubating ovaries with anti-PCNA at 1:1,000 and mounting in Vectashield (Vector Labs).
Double labeling of Drosophila ovaries with anti-DUP and BrdU was performed as per anti-ORC2/BrdU labeling (above), but incubating ovaries with anti-DUP (Whittaker et al., 2000) at 1:1,000 for 48 h at 4ЊC. Secondary detection of DUP was performed with donkey anti-guinea pig Rhodamine-RedX at 1:200. Slides were mounted in Vectashield.
Anti-DUP, anti-ORC2 double labeling, was performed as described previously (Whittaker et al., 2000) but with the following changes: the primary antibody incubation was performed for 48 h at 4ЊC, and secondary detection was with donkey anti-guinea pig Rhodamine-RedX at 1:200 for anti-DUP and goat anti-rabbit FITC at 1:200 for anti-ORC2. Ovaries were mounted in Slowfade.
For some ovary samples, TOTO (Molecular Probes) was used to stain the DNA. These samples were treated as described above, but were treated with 1 mg/ml RNase A (Sigma-Aldrich) for 1 h at room temperature, and then were incubated with a 1:2,000 dilution of TOTO (Molecular Probes) for 10 min. All secondary antibodies were from Jackson Immu-noResearch. All confocal imaging was performed using a Ziess Axiovert 100 M with LSM510 software, using 63ϫ Plan Neofluar or 100ϫ Plan Neofluar objectives and with filters set according to the manufacturer's parameters.

Deconvolution microscopy
Fluorescence data was collected using a Zeiss Axiovert 100 M Meta confocal microscope with LSM510 software. Excitation of FITC, rhodamine, and TOTO-1 dyes used the 488, 543, and 633 nm lasers, respectively. Emission filters were tuned to minimize bleedthrough between channels. Voxels were collected at 45 nm lateral and 1,000 nm axial intervals. Deconvolution was carried out using the cMLE algorithm of Huygens2.3-professional (Scientific Volume Imaging) on an Silicon Graphics Origin 3400 server (SGI). Rendering and analysis of three-dimensional data was carried using the MeasurementPro module of Imaris3 Surpass 3.2 (Bitplane).

Fluorescent in situ hybridization
ORC2 and ACE3-FISH colabeling was performed as follows. Ovaries were stained for ORC2 as described (Royzman et al., 1999). Secondary detection of ORC2 was with donkey anti-rabbit Cy3 at 1:250. After staining for ORC2, ovaries were fixed (as per the ORC2/BrdU double labeling protocol), and were then processed for whole mount FISH as described (Calvi et al., 1998). The probe used for the third chromosome chorion locus was a 3.8-kb SalI fragment from the plasmid pT2, containing both ACE3 and ori␤. The hybridized probe was detected with goat anti-DIG FITC at 1:200. Samples were mounted in Vectashield.

Isolation of Drosophila DNA for quantitative realtime PCR
Egg chamber staging was performed based on morphological markers as described (Spradling, 1993). Pools of 004ف or 500 egg chambers of each stage 10B, 11, 12, 13, and 130 ovaries of stage 1-8 were isolated from fattened Oregon-R females. DNA was isolated from the pools of egg chambers as described (Royzman et al., 1999), with the addition of RnaseA treatment (1 mg/sample; Sigma-Aldrich) during the Proteinase K step.
Embryo genomic DNA was generated for use as standard curves in the realtime PCR reactions according to standard techniques (Ashburner, 1989).

Quantitative realtime PCR
Quantitative realtime PCR was performed using the ABI Prism 7000 Sequence Detection System with QIAGEN SYBR Green PCR mix. Thermocycling was done for 35 cycles.
Primer sets spanning 50 kb on either side of ACE3 (denoted as distance 0) at 5-kb intervals, primers to the nonamplified rosy (ry) locus, and primers to another nonamplified intergenic region on chromosome arm 3R (located approximately at cytological position 93F2, 52ف kb upstream of the pol␣ locus) were generated using Primer 3 software. Primers were designed to be 22 bp on average, with an optimum Tm of 65ЊC, and yielding products of 85 bp on average. Primers were supplied by IDT, and primer sequences are available upon request.
Each experimental reaction (per egg chamber stage, per primer set) was performed in triplicate, alongside four tenfold dilutions of standard DNA (embryo genomic DNA) and no-template control reactions (all in triplicate). The same embryo genomic DNA samples were used in all control reactions for internal consistency. Each experimental reaction contained DNA from approximately one to one half of an egg chamber, and was done in 25 l total volume (12.5 l SYBR Green 2ϫ Master Mix, 10 l dH20, 2 l DNA, 0.25 l each 25 nmolar primer). Relative fluorescence was measured per sample in comparison to standard curves and standard deviations of the triplicate reactions were calculated by the ABI Prism 7000 software. Fold amplification was calculated by dividing relative fluorescence for one of the third chromosome amplicon products by the relative fluorescence of either the ry or the 3R non-amplified control product for a given stage. Error is expressed in terms of standard deviation, where the standard deviation of the ratio A/C ϭ (FA/FC) *{[(SA/FA)^2 ϩ (SC/ FC)^2]^.5}. A, amplicon locus; C, control locus; FA, relative fluorescence from amplicon locus; FC, relative fluorescence from control locus (ry or 3R); SA, standard deviation from same amplicon locus; SC, standard deviation from same control locus.

Online supplemental material
Online supplemental materials are available at http://www.jcb.org/cgi/ content/full/jcb.200207046/DC1. Video 1 accompanies Fig. 1, A and B, and shows a three-dimensional volume rendering of ORC2 (green) in relation to BrdU (red) and DNA (blue). Video 2 accompanies Fig. 5, A and B, and shows a three-dimensional volume rendering of DUP/Cdt1 (red) in relation to ORC2 (green) and DNA (blue).