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Rachie & Liz | Photo by Nir Landau | http://www.nirlandau.com/
Ashley and Kelsie made my wedding day perfect! I could not have had a more perfect day and I owe it to them. They took care of every last detail, resulting in a smooth event. My guests are still talking about what a great wedding it was. I encourage anyone thinking of getting married to hire them.
Ashley and Kelsie truly made our day seamless. I went the DIY route for our decorations and planned the day with the vendors. Ashley and Kelsie met with me in person and via videochat through the 9 month process. They asked questions and genuinely listened before providing their very sound advice. They were ultra-professional leading up to the big day and particularly on the big day. A couple issues came up on the day-of in my own planning, and Ashley and Kelsie made sure I didn't find out about them until afterwards. They took care of us just as much as they took care of the on-going flow of the day as it unfolded. Their friendliness, attention to detail, and experience made our day one we could really just enjoy and remember warmly.
Thank you so much for helping make our wedding so amazingly wonderful! You guys were saviors the day and night of the wedding! We loved all the small details you added, especially the lemonade table which everyone thought was extremely cute. We were shocked to see how clean and organized it was when we woke up the next morning. Seriously you guys were the best! We really couldn't have asked for a better wedding!
We are so grateful to Ashley and Kelsie for their coordination of our wedding day! Everything went off without a hitch, or at least not any that we could see. After making sure the plans were taken care of and helping us troubleshoot all the minutia, they were there from the early morning until things were cleaned up to handle every known and unknown detail. We were able to fully experience each part, knowing that setting up every extension chord, getting each course of food out and so many other things were taken care of. We now get to look back on the whole experience with such vivid memory - thank you!
Thank goodness I hired Blue Jar Events for my day-of coordinating! After months of planning DIY decorations and details for my wedding day, I realized I needed help bringing my plans to fruition. Ashley and Kelsie listened closely, studied my Pinterest page, and encouraged me in all of the DIY projects that I had my heart set on. I was simply thrilled with how beautiful they made my wedding ceremony and reception venue! They exceeded my expectations in every respect. Not only do they have impeccable taste, but they helped me get organized and stay calm leading up to the event. I was able to relax and enjoy a beautiful wedding day in large part because of their work.
Smooth as silk! That is how I would describe the job Blue Jar did for us. Thank you! Every detail was covered. The decorations and flowers were beautiful, and the caterer was excellent, both in food and service. Blue Jar dealt with each aspect of the party calmly. I would ask about something and they would say, 'Done!' I've never thrown a party where I didn't have to do a thing! Ashley and Kelsie, you are awesome.
The involvement of Blue Jar Events on our wedding day was a tremendous gift. Ashley and Kelsie handled the nitty-gritty day-of logistics that no one wants to be bothered with on their wedding day! They were on top of challenges that we didn't even know about until long after the event. We were able to stay in the moment and didn't worry about a thing, knowing that this talented duo were thinking on their feet with creativity and grace. Prior to the day, we had talked over many details, but others, like their arrangements of the gifts area and place cards, were lovely surprises that greeted us as we entered the reception space. It was a special day made even more unique, gorgeous and calm by Blue Jar Events.
With outside-the-box creativity, problem solving and professionalism, Blue Jar Events gave us the support and creative energy that turned our ideal wedding into a reality. Kelsie and Ashley adapted to our style and our unique situation, being a French-American couple living overseas, and integrated our personalities and story into a cleverly themed and joyous reunion of families and friends. Throughout the planning process they kept us organized with a scheduled checklist, updated vendor contacts, and time-saving solutions while maintaining the sentimentality of the event beyond all the logistics. With Blue Jar, wedding planning changed from a burden into a fun and creative process. We couldn't have done it without them!
Blue Jar Events gave us a wonderful, wonderful wedding that was so much more than we ever imagined it could be. We would never have been able to do it without their energy, creativity, thoughtfulness and effort. The flowers, decorations, and food (we had a food truck that BJE helped out with!) were all amazing, but the part the made it so wonderful was that BJE took care of everything on the day of the event - my wife and I were able to enjoy our wedding to its fullest without having to make any decisions or oversee anything. We will always remember it as one of the best events of our lives and will be forever thankful for all that BJE did to make it that way.
Leah & Dan | Photo by Kelly Benvenuto Photography | www.kellybenvenuto.com
Wedding and event planning in Boston, New England, and beyond.
Blue Jar Events | , Boston, MA


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Francis Sandal in White
Stay cool in cute style with the casual Francis sandals. Featuring a mix of tropical fruit and floral prints on upper, these easy low-heeled sandals have you covered for any casual outings. An EVA footbed for comfort also fulfills all you need for walking.
- 1" Heel height
- 0.5" Platform height
- Man made upper
- EVA footbed
- Pineapple and floral print
- Imported
Item no. 41819
Heel Height Low

subset: commoncrawl


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The one-pager is FREE and it's free to start the total PageZen package. So if you want to see how easy it can be to build a website for your business or your hobby or your cat, then please take our 14-day trial run.
You'll note in your account that your domains come with a free trial run of PageZen and SEO Tutor. Give 'em a whirl and let us know what you think. We even have some even easier Tips and Tricks to help out. Oh, and nice work Otto, Quin, Name.com staff and the baristas at Ink! We couldn't roll out top-notch production without you.


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The protection of front line services is of paramount importance within the Mid and East Antrim Borough Council, the new mayor has said.
Councillor Billy Ashe was elected as first citizen of the newly-operational authority, which officially came into being this week after almost a year in 'shadow' form.
Cllr Ashe will oversee the former boroughs of Carrickfergus, Larne and Ballymena, with Ballymena-based councillor Timothy Gaston serving as deputy.
"The old councils had been operating in much the same way for the past 45 years; the big challenge now is to bring the three separate councils together as one but to make sure that front line services continue," Cllr Ashe said.
Although Ballymena will be the "administrative" centre of the new supercouncil, residents in Carrickfergus and Larne should still be able to avail of services such as dog licensing locally. "This may mean council staff moving around," the mayor added.
The new authority will also boast extended powers which were unavailable to its smaller-scale predecessors. "There will be new planning powers, community planning and powers of wellbeing, which will cover areas such as health and the local economy," Cllr Ashe said.


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Tuesday, February 15, 2011
Chicken Fingers
14 comments:
Those chicken fingers look so crisp and fingerlicking!ReplyDelete
Chicken fingers are the best! I use the same recipe as yours but never thought of sprinkling garlic salt. That would be a tasty addition!ReplyDelete
Can I order the chicken fingers please? I mean whoa! Those look tasty and super crunchy. Can you send me some via DHL? I love the blog and sorry I am just now coming over and checking it out.ReplyDelete
Good idea about the garlic salt! These look so yummy!ReplyDelete
You are completly right, one cannot go wrong with chicken fingers! And if it is served with a bowl of french fries, who even needs salad! Thank you for sharing :)ReplyDelete
They look absolutely delicious!!!ReplyDelete
I LOVE chicken fingers. Dunk it into some honey mustard and I'm good to go!ReplyDelete
Thank you everyone for all the sweet comments!ReplyDelete
Rojo, is that an official suggestion for my next giveaway? Lol! If I could I would totally ship some to you, they were awesome!
Yes the garlic salt just makes these more awesome than they already are, I hope you all enjoy them as much as we did!
I love chicken fingers! They never get old. Yum!ReplyDelete
- AnonymousFebruary 17, 2011 at 8:46 AM
Those look so tasty!ReplyDelete
- AnonymousFebruary 17, 2011 at 1:41 PM
I've always loved chicken fingers and I think I always will. I even order them at restaurants from time to time. It may be a staple of most children, but you can't really go wrong. Crisp on the outside, moist inside, perfect for dipping into anything!ReplyDelete
I've never tried the chicken fingers with garlic salt either, but I must try that soon:) Thanks for sharing!ReplyDelete
mmmm I love the addition of the garlic salt! I use garlic salt in almost all of my dishes ... it's my secret ingredient. ;-)ReplyDelete
Wow these look good. The hubby and I adore chicken fingers. It's just one of those foods that gives you a little dose of happy. :)ReplyDelete


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Christina Ricci welcomes baby boy with James Heerdegen
Christina Ricci is a mom! The actress welcomed her first child, a baby boy. "They are thrilled and ready for a change of pace", a source told Us Weekly of Ricci and her husband James Heerdegen.
The news about Ricci's pregnancy was confirmed back in May after the actress stepped out at LAX airport in Los Angeles with a tell-tale baby bump.
Ricci, 34, and her husband met on the ABC series Pan Am in 2011. They got married on the Upper East Side in New York City in October 2013. "I love so many things about him. He's my favorite person on Earth. I can't wait to have our own little world replete with small beings!" the actress said of her hsuband in May 2013.
Christina Ricci is best known for playing Katie in Mermaids, Wednesday Addams in The Addams Family and Kat Harvey in Casper. She was also was nominated for an Emmy Award for her role as a paramedic in the ABC drama Grey's Anatomy.


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Editors' review
Download 60 Minutes - The Murder of Chauncey Bailey (February
In February 2008, "60 Minutes" reported on the murder of Oakland, Calif., journalist Chauncey Bailey, whose death has been linked to an investigation he was conducting into a controversial local business called "Your Black Muslim Bakery."
Later this month, two men charged with Bailey's murder are scheduled to go on trial for this killing. One of the key witnesses in the case is likely to be Devaughndre Broussard, a young man who had been a janitor and handyman at the Bakery.
Immediately after the killing, Broussard confessed to the murder and then recanted that admission. Months later, in his interview with 60 Minutes correspondent Anderson Cooper, Broussard says he didn't kill Chauncey Bailey and that he'd been coerced into a false confession by the head of the bakery, Yusuf Bey IV.
That was the way things stood in 2008. A year later, Broussard changed his story again and confessed to killing Bailey on Bey's orders. Broussard pled guilty to voluntary manslaughter and is now a likely prosecution witness in the upcoming murder case against Bey.
Below is Anderson Cooper's full interview with Broussard in the Alameda County jail where Broussard was being held in 2008.
ANDERSON COOPER: How did you first get to the Bakery? How did you first come to live there?
DEVAUGHNDRE BROUSSARD: I found out about the bakery in like July '06. It was -- it was the last day before I got released from San Francisco County Jail. I ran into one of my family members. And he told me if he -- if he could give me a job would I accept it? And I said, "Yeah." And he told me about the job, told me where it was at. At first, I was -- I was reluctant, because I was thinking like, "I'm not about to go work with no Muslims." But then I realized this is a chance for me to stay out of jail, so I went to go work there formally.
COOPER: What was it like at the Bakery?
BROUSSARD: It was a good place. Because I felt like, you know, like they're really here for the -- for the black people and what not. You know, they were speaking about how -- how we gotta change as a people. They were -- they were speaking about how, how we got to come together as one, you know, speaking on -- speaking on the problems as a -- as a people and how we can solve 'em.
COOPER: What was your job at the Bakery?
BROUSSARD: I was basically cleaning. Cleaning up, sweeping, mopping, washing dishes, scrubbing pans. The dirty work, basically.
COOPER: Did you always live there? Or did you --
BROUSSARD: I didn't always live there. At first when I first went there, there was -- they had -- they had a residence for people that was -- that didn't have nowhere to live and what not. And then I had somewhere to stay, but they was like, "It would be better if everybody who worked there would stay on -- would stay on the premises."
COOPER: How many people lived there?
BROUSSARD: It varied. It varied from time to time.
COOPER: But, I mean, from a handful to a couple dozen? I've never been there, so --
BROUSSARD: Like a handful. Like right around, like ten or fifteen people.
COOPER: Did you have a roommate there?
BROUSSARD: Nah, you had your own room.
COOPER: What was your average day there like? What was your schedule?
BROUSSARD: You wake up, get ready for work. Then you go to work. You stay at work. You clean up. And then at night it'd be -- it'd be -- it'd be brotherhood meetings. That's -- that's about it.
COOPER: Were you Muslim? Or are you Muslim?
BROUSSARD: At first, when I went there I wasn't -- I wasn't -- I wasn't -- I wasn't a Muslim or I wasn't practicing Islam or what not. I was just taking it as -- as it was a job. But then I realized like my -- I gotta -- gotta open up. Then I -- I start -- I started feeling what they was talking about. Like it was -- it was good stuff, what they was talking about. And then, so I started -- I started practicing. I started practicing Islam.
COOPER: Do you consider yourself Muslim now?
BROUSSARD: Yes, I do.
COOPER: So on August 3rd you told police that -- that you killed Chauncey Bailey. You then later on, a couple days later, said that you didn't. Why did you say that you killed him?
BROUSSARD: I didn't kill Chauncey Bailey. I gave -- I gave the false answers to the -- to the Oakland Police because I was overcome by emotion.
COOPER: What do you mean?
BROUSSARD: Because of what Yusuf Bey IV was telling me. He told me that -- he was telling me how -- how I was being tested by God. He was telling me I didn't realize I was being tested by God. He was telling me that --
COOPER: He said that God was testing you?
BROUSSARD: Yes, he did.
COOPER: How?
BROUSSARD: He was saying that -- that -- that at one point people gotta prove that -- you gotta -- you gotta prove your loyalty and what not.
COOPER: Loyalty to whom or to what?
BROUSSARD: You gotta prove your loyalty to the Bakery. And you gotta prove your loyalty to God. And then, so he was telling me that if I say I admit to this and I say that I did this, that they would release everybody. He was telling me that they was holding everybody that they arrested as suspects in the murder of Mr. Chauncey Bailey.
COOPER: So he told you that there were other people being held from the Bakery in Chauncey Bailey's murder?
BROUSSARD: Yes, he did.
COOPER: And that by testifying, by saying that you killed him, everyone else would go free?
BROUSSARD: He was saying that if I say I did this, that they would release everybody. Because that's -- that's what they was holding them for. He was telling me that -- he was telling me how well he knew the police officers or what not.
COOPER: How he knew what?
BROUSSARD: How well he knew the police officers. Because he said that they was like his, I guess, his friends and what not. He was telling me that --
COOPER: He said the police were his friends?
BROUSSARD: Yeah, one of the detectives, I guess, he knew -- I guess he knew him kind of well or what not. Because they was -- you know, they was -- they was saying -- there was -- there was talking -- they was talking like -- like they knew each other. He was -- he was -- then he's telling me that if I say I did this, I would get no more than a county year.
COOPER: He said you would just get a year.
BROUSSARD: He said I would get no more than a county year. He said that -- he was saying that if I said I did this, that he would get me a lawyer when he get out. And then the lawyer would get it dropped down to manslaughter. And I'd get no more than a county year.
COOPER: What did you think of Bey IV? I mean, what was your relationship with him?
BROUSSARD: At first, like I said, when I -- when I first went there, I just thought of it as a job. And then as time -- as time went on and, and I came to accept -- I came -- I came to believe what -- what -- what the Muslims was talking about was good. And then so, you know, I was like -- I was like -- I looked up to him. Because I'm like this is -- this is the leader of the Black Muslim Bakery. And so you gotta -- you gotta follow the leader.
COOPER: So when you get arrested, how did you come to be in a room with Yusuf Bey IV?
BROUSSARD: What you mean?
COOPER: I mean you're in police custody. How did you end up in a room with him?
BROUSSARD: The police put us in a room together.
COOPER: Why?
BROUSSARD: I don't know. Ask the police that.
COOPER: Did you ask to be in a room with Yusuf Bey IV?
BROUSSARD: No, I didn't.
COOPER: Because there had been some reports saying that you had requested it.
BROUSSARD: I didn't ask to be in a room with him.
COOPER: So they just bring you in a room with Yusuf Bey IV?
BROUSSARD: They just brung me in a room with him.
COOPER: What was the first thing he said to you?
BROUSSARD: He was telling me how I gotta take this case. He was persuading me to take this case. He was telling me that -- he was telling me like, you know, "You gotta do this for us. You gotta do this for the Bakery. You gotta do this for the brotherhood." He was saying -- he was saying like, you know, "Every -- everybody -- everybody got to prove theyself." He was -- he was saying, "I'm your commanding officer. You gotta follow orders. And you know -- and you know you can't -- you know you can't deny an order from your commanding officer."
COOPER: Did you think he was your commanding officer?
BROUSSARD: It was a chain of command at the -- at the Bakery. You know, like -- you know, like -- if somebody got more rank than you, you got to follow what they say. And he was -- he was at the top of the chain of command.
COOPER: It sounds more like an army than it does a bakery.
BROUSSARD: It was like the black man army.
COOPER: It was like the black man army?
BROUSSARD: The black man's army.
COOPER: So when he tells you that, you know, you gotta do this, you gotta -- you gotta plead guilty. You gotta say you killed Chauncey Bailey, what'd you think?
BROUSSARD: I thought he was full of s. I thought he -- I thought he was trippin'. I'm like, "Why would you say -- why would you want me to say something like that?"
COOPER: But you did end up saying that.
BROUSSARD: He was telling me how -- what I gotta say. He was telling -- he was telling me how -- how I gotta make it sound good. He was saying -- he was saying like -- he was telling me that if I don't make it believable, they won't release nobody.
COOPER: How long were you sitting with him? How long did you talk to Yusuf Bey?
BROUSSARD: I didn't have a watch. So I don't know how long we was in the room together. But he was -- I mean, it was mania. It was mania.
COOPER: It was what?
BROUSSARD: It was mania.
COOPER: What does that mean?
BROUSSARD: Like a lot of stuff was happening.
COOPER: Manic, okay.
BROUSSARD: A lot of stuff was happening.
COOPER: So you leave the room. And then right away did you tell police that you killed him?
BROUSSARD: When we was in the room he was telling me -- he was telling me -- he -- he was -- he was pressing the issue. He was pressing the issue. Because he was saying like, "You gotta take this case for us. You gotta." He was telling me how I gotta take this case and why I should take the case. And then I would tell him like, "Nah, I ain't going to take the case. Because if I take this case then I -- I would never get out of jail."
He was telling me how he said--he was telling me how I say -- he like -- he's like, "You know, I got money. I got money. And so, you know, if I get out, I'm going to make sure you get a good lawyer. I'm going to pay for the lawyer. And you know that -- and you know that as a brotherhood we stay together. We got unity. So we ain't -- we just not going to let one of ours fall."
COOPER: Did you believe him?
BROUSSARD: Yeah, I did.
COOPER: You believed him?
BROUSSARD: I believed him. I mean, I -- I seen the houses he had, the cars he had. I mean -- I mean, I -- I never had nothing like that. So I mean, you know, they say money can buy whatever. So I believed him.
COOPER: Did he say anything about Chauncey Bailey when you met with him?
BROUSSARD: He didn't say nothing about Chauncey Bailey. He was just -- he was just -- he was just saying like -- like -- like, "You got to take this case for me. You got to take this case for us. You gotta take the case for the brotherhood."
COOPER: He said a lot of things to you. What was it that really convinced you, "Okay, you know what? I'm going to say I killed Chauncey Bailey."
BROUSSARD: Then he start pressing the issue. So he was -- he was saying like I was being tested by God. And then, I mean, at that time like I said, I was practicing. I started -- I started believing in Islam. And I'm like, you know, I really believed. So I'm -- I'm gonna prove myself. I wanted to prove myself.
COOPER: Prove yourself to whom?
BROUSSARD: To God.
COOPER: So by proving yourself to God, by saying you killed Chauncey Bailey, that was proving yourself to God?
BROUSSARD: He was saying, "At this point in time you can prove yourself. You proving yourself to God. You being tested by God." He was saying that, "Most times people don't realize when they being tested by God. I'm helping you out. I'm telling you that you being tested by God."
COOPER: You said to police when you confessed that you wanted to be "a real strong soldier." What did you mean by that?
BROUSSARD: Like I says, it was a black man's army. So -- and so you -- you a soldier. You start out -- you start out -- you start out as a soldier. And so when, like I said, when they -- when they put us in a room together he was -- he was -- he was pressing the issues on, you feel me, on why I gotta do this, on why I should say I did this. He was telling me -- he was telling, "Well, you got to say this. You gotta say it like this. You gotta tell 'em like how this happened."
COOPER: When you met with Yusuf Bey IV, did you realize that he had already told police that you had killed Chauncey Bailey?
BROUSSARD: I didn't kill Chauncey Bailey.
COOPER: But did you know that Yusuf had said you did?
BROUSSARD: When they had me in the interview room without him, they kept coming in the room telling me how Yusuf Bey IV said I killed him. They was saying how Yusuf Bey IV said I shot him this many times. They'll leave the room. They'll come back and they'll say how Yusuf Bey IV say I shot him this place and that place. Then I was denying it. I say, "I don't know what you're talking about." Then later on they'll come back and they'll say, "Yusuf Bey IV said you was driving his van." And then I -- I started -- I started saying, "I plead the fifth." I started saying, "I plead the fifth." I was like, "I don't even want to talk."
And then the detective, he was saying like, "No, you gonna talk." He was like -- he was like -- he's like, "You ain't getting out of this. You can't let Yusuf Bey IV take the fall for this." And I was saying like -- I'm like -- I'm like, "Where my lawyer at?" They're like, "You ain't getting no lawyer." They're like, "You ain't getting no lawyer." I was like, I -- I -- I lay my head down. I'm like, "I plead the Fifth." And then he yanked me up. He like -- he like -- he like, "So say you did it." I'm like, "I didn't do nothing."
I kept trying to lay down. I started trying to ignore 'em. They left the room. They came back. They were saying, Yusuf Bey IV -- they're like -- Yusuf -- they're like, "Yusuf Bey IV gave you up. So why don't you give him up?" I'm like, "I don't know nothing about that. So I'm not -- I'm like, I don't know nothing about that. So I'm not going to say -- I'm not going to say nothing."
COOPER: Did you know that--that Yusuf Bey IV given you up?
BROUSSARD: He didn't give me up. He just told the police that I supposedly did something. That's what he did. I didn't do nothing.
COOPER: When you met with him, when you were in the room with him --
BROUSSARD: When I was in the room with him, when they brung me in the room, I looked at him. And he was, you know, he was laid back. And then I was like, "Let me talk to him alone." Because I want -- I wanted to see why he would even say something like that. Because I told him the night before that I wouldn't agree with this. And then I asked him, I said, "Why'd you even -- why would you even say that I supposedly killed this man?" And then he was telling me -- he was telling me, like, I got to take this case. He was saying like, "You gotta do this for us."
COOPER: So did he deny that he had --
BROUSSARD: He didn't deny.
COOPER: -- ratted you out?
BROUSSARD: He didn't. He didn't deny it. He said that -- he said I gotta -- he -- he was saying, "You are gonna take this case. You are gonna take this case." He was -- he was saying like, "You gonna take this case because they -- they gonna believe me before they believe you."
COOPER: What do you think now, I mean, knowing that Yusuf Bey IV said all this stuff about you to the police saying that, you know, you killed Chauncey Bailey?
BROUSSARD: How do I feel about that?
COOPER: Yeah.
BROUSSARD: I mean, he succeeded in what he wanted to do. So I can't -- I can't -- I can't -- I can't -- I can't sit on that, because it ain't go -- it ain't go -- it ain't going to change nothing. So I just gotta -- I gotta try my hardest.
COOPER: But, I mean, this is a guy you trusted.
BROUSSARD: Like they say, the people you love is the one that's going to hurt you the fastest. So he, you feel me, stabbed me.
COOPER: He stabbed you?
BROUSSARD: He got me.
COOPER: Yusuf Bey IV got you?
BROUSSARD: Got manip. Call him a nip, he nipped. Ran a good manip game. I thought he was going--you feel me, he told me that -- he told me if I said I did this, that everything would be good. Everything going to be -- everything going to be all right.
COOPER: He nipped you? He manipulated you?
BROUSSARD: Basically. And then I believed him. I mean, at first I was thinking to myself like, if I said I did this, I know I'm not going to get out of jail. But the way -- the way that him and the -- the detective dude, they was getting along like they really knew each other. The detective, he was saying like, "We can't -- we can't -- we can't let Yusuf Bey IV go down. You know, he do -- you know, he run -- he doing good in the community. He helping out black people."
COOPER: The police said that to you?
BROUSSARD: Police officer said that to me.
COOPER: Do you know which one? Who?
BROUSSARD: The black guy.
COOPER: The black police officer there said it to you that Yusuf Bey IV was doing good in the community?
BROUSSARD: Like he would say something. And then he would say something. And I kept -- I kept -- I'm like, "Nah, nah." I'm like, "Nah." He was saying like -- he like, "Nah, you going to do this." The detective, he was like, "You going to do this. You going to say you did this." And I was like, "Nah, nah, I don't know what you talking about." And then I was like, "Let me talk to him alone." And then, so I asked him. I was like -- like I told you earlier, I asked -- I said, "Why you even say I did this? I told you I said I ain't going to even -- I told you I ain't going to even rock with this." And he was saying like, "You gotta. You gotta. You gotta. You gotta. You gotta help us out. You gotta take this fall." He was saying like, "As your commanding officer, you gotta follow my orders."
COOPER: When was the first time you ever heard about Chauncey Bailey?
BROUSSARD: I never heard about Chauncey Bailey. I never met him. I never seen him. The first time I heard about Mr. Chauncey Bailey was that night I got arrested.
COOPER: You never heard of him before?
BROUSSARD: Never heard of him before.
COOPER: He'd been on TV though. He had a TV show. Apparently he was pretty well known at the Bakery.
BROUSSARD: I never heard of him. I never met him. Like I say, I heard about the Bakery a year -- a year before that. So I never heard about the Black Muslim Bakery before I went there.
COOPER: Was anyone at the Bakery upset with Chauncey Bailey that you knew about?
BROUSSARD: Not -- I don't know.
COOPER: So where were you when Chauncey Bailey was killed?
BROUSSARD: I was at work.
COOPER: What were you doing?
BROUSSARD: Cleaning.
COOPER: Didn't you tell police that you had been asleep initially?
BROUSSARD: I was working. I told -- they -- they asked me what time did I wake up? And I told 'em. I said, "I don't remember the time I woke up." Because I was -- the apartment that we was staying in was with two other people. And we were sharing an apartment, everybody that worked together.
And the person who -- and the other person that was staying in the house with us, he was the supervisor of the Bakery. And so, like I say, once you wake up, you -- you basically go to work. And so you -- and so the Bakery opens up the doors at nine o'clock. And people be up earlier than that. And then so I basic -- I basically was sleeping in. And I went -- when I -- when I get woke up - if I don't wake up on my own, somebody wake me up, and they'll tell me to get ready for -- for work.
COOPER: So did anybody see you working at the Bakery at the time that Chauncey Bailey was killed?
BROUSSARD: The employees, the other -- the other brothers and sisters that was working there.
COOPER: They saw you?
BROUSSARD: Yeah.
COOPER: Do you know -- are any of them willing to come forward?
BROUSSARD: I don't know. I would hope they will.
COOPER: You were seen throwing a shotgun out the window during the police raid.
BROUSSARD: No police officer saw me show -- throw a shotgun out the window.
COOPER: Nobody did? A police officer has said that he did though.
BROUSSARD: Nobody saw me throw a shotgun out the window.
COOPER: Did you ever have the shotgun?
BROUSSARD: They had a shotgun.
COOPER: You said they had a shotgun or --
BROUSSARD: They had a shotgun.
COOPER: How did the shotgun come to be in your possession?
BROUSSARD: The day before like -- like -- like they came -- they came and they came. Yusuf Bey IV was like -- he was like, "Here." He was like, "Here, you got to take this gun for night security." And he gave me a bag with some stuff in it. And then I just put it in my -- just put the gun on the floor and put the bag in my closet. That was it.
COOPER: So Yusuf Bey IV gave you the shotgun?
BROUSSARD: He told me -- he was like -- he was like -- he was like, "Here, hold this."
COOPER: What did he want you to do with the gun?
BROUSSARD: I don't know. He just -- he -- he just like -- he was like, "Here hold this."
COOPER: And what was in the bag of stuff that he gave you?
BROUSSARD: I never checked it, because it was -- it was -- because the day -- that same day, that Friday -- no wasn't -- I think it was Friday, yeah. The day before -- the day -- the day before we got arrested, it was a rally. And so he came and gave me -- he came and gave me the shotgun and the bag. And then he was like -- he was like -- he was like, "Come on, we gotta get ready for this rally."
COOPER: So if you were working maintenance at the Bakery --
BROUSSARD: Why would they give me a gun?
COOPER: Yeah.
BROUSSARD: Like I said, it was a black man's army, so it was more than a job. Like, you know, like you work. And then we had brotherhood meetings. And so, you know, at the brotherhood meetings they -- they would stress the importance of, you know, you got -- you gotta defend what's yours. And so everybody had to play a part.
COOPER: So had you seen guns at the Bakery before?
BROUSSARD: I mean, that -- that was the gun I saw.
COOPER: But before that had you seen other guns?
BROUSSARD: No.
COOPER: Did you think it was strange that all of a sudden Yusuf Bey IV gives you a shotgun?
BROUSSARD: I seen guns before. So I -- it wasn't -- it wasn't strange. I'm like, "Oh, I'm going to give this back to him as soon as I can." That's what I was thinking. I'm like, I'll get it as soon as the day over, I'm going to give it right back to him. One thing led to another like I never really got -- I real -- I really never got around to doing it.
COOPER: So you're denying that you threw the shotgun out the window?
BROUSSARD: Yes.
COOPER: Police are going to testify that they saw you throwing the gun away. You're saying point blank you didn't throw the gun away?
BROUSSARD: It's a lot of falsehood and a lot -- you know, like -- a person said I supposedly killed somebody. It -- it ain't no truth in that. But people believe in that. So I mean, you never know. You never know what's up. I mean --
COOPER: As you know, that's why we're asking you the questions. So we don't want to hear from other people. We want to hear from you and let you have the opportunity to tell your side of it. So if you had possession of the -- of the shotgun and then police found the shotgun not in your possession how did it -- what happened to the shotgun?
BROUSSARD: I don't know.
COOPER: So you don't know how it got from your room to where police found it? Did anyone ever mention that Chauncey Bailey was working on stories about the Bakery?
BROUSSARD: Nobody never mentioned that he was writing a story or he was starting to write a story.
COOPER: I didn't know the Bakery. So I'm trying to figure out if, like, people talked about stuff that was happening outside or -- or how much people talked about, you know, Chauncey Bailey's writing a story, and it's a bad story or anything like that. You hadn't heard any of that?
BROUSSARD: (shakes head)
COOPER: The white van. People said you were driving the white van that day. Were you?
BROUSSARD: No, I wasn't.
COOPER: You never drove any white van?
BROUSSARD: I never drove no van. I even got my license. I never drove the van.
COOPER: So people who come forward saying they saw you driving the van, they're wrong?
BROUSSARD: They're wrong. Falsehood.
COOPER: That was another thing Yusuf Bey IV had told police, that you borrowed the van from a guy named Rico.
BROUSSARD: And in the police reports it also says Rico gave the keys to Yusuf Bey IV. And he saw him drive away in the van. So you can make that conclusion on your own.
COOPER: Police said they saw a shotgun being thrown out the window. You're saying you didn't throw that shotgun out the window?
BROUSSARD: We already went over that.
COOPER: Right. I got it wrong, though. I said the police said that they saw you.
BROUSSARD: You already went over that.
COOPER: When you confessed, police were asking you about when you fired the shotgun. It didn't seem like you really knew how to repeatedly fire a shotgun.
BROUSSARD: No. I -- I said that was the first time I seen it. The day -- the day before, when he gave me the shotgun the day before in the afternoon, what was that? August 2nd. That was the first time I seen it. Then when they -- when they asked -- when they -- when they asked me how to -- how to operate it or what not, I was thinking they didn't believe me. I was thinking I failed. I was thinking, like, I tried.
COOPER: Police say, though, that you, when you confessed, that you knew things about the shooting that only the shooter would know. You know, how many times Chauncey Bailey was shot, where he was shot.
BROUSSARD: I came upon the details of the murder of Mr. Chauncey Bailey the night before when I was working and cleaning, finishing my daily duties at the Bakery.
COOPER: How did you come upon the details of it?
BROUSSARD: While I was cleaning and finishing up, I overheard some people talking about how a newspaper man got killed that day. And, you know, at least -- you know, seemed kind of happy, you know, like--and then -- then -- then when they had me in the interview room, as I -- as I told you earlier, the police -- the police kept coming in and saying how Yusuf Bey IV said I did this and I did that.
And then when -- when they put me in the room with Yusuf Bey IV, he was telling me -- he was telling me how I gotta make it good. I gotta make it believable. And then, at first, I was reluctant. I was, like, "Nah." He was saying -- he was saying, "You gotta say this. You gotta say this. You gotta say it like this. You gotta say it like that, 'cause if they don't believe it, nobody won't get out. And you know -- and you know I can't help nobody if we're all in jail."
COOPER: So did he tell you how many times Chauncey Bailey had been shot? How did you learn how many times Chauncey Bailey had been shot?
BROUSSARD: We'lls in the room and he was saying -- he was just telling me what I gotta say, saying, "Say this. Say this. Say it like that. Say this. Say that. Say this. Say that."
COOPER: So, he was telling specifics about what to say?
BROUSSARD: Yes, he was.
COOPER: So when police say, "Well, he knew how many times Chauncey Bailey was shot. He knew where Chauncey Bailey was shot." You say you learned all that from Yusuf Bey IV?
BROUSSARD: I learned what he told me to- I -- I told the police what Yusuf Bey IV told me to tell them.
COOPER: And did Yusuf Bey IV tell you how many times Chauncey Bailey got shot?
BROUSSARD: Yes, he did.
COOPER: Did he tell you where Chauncey Bailey had been shot?
BROUSSARD: Yes, he did.
COOPER: The night before you said you heard people talking about Chauncey Bailey. How did you know who Chauncey Bailey was? Did you know who Chauncey Bailey was?
BROUSSARD: No. I heard -- I heard people describing how a newspaperman got killed that day. That's what I see.
COOPER: And what were they saying about him?
BROUSSARD: I'm gonna give -- I'm gonna give those details up when I go to trial.
COOPER: You don't wanna say now?
BROUSSARD: Wait 'til I go to trial.
COOPER: Are you concerned about your own safety now?
BROUSSARD: You know, anything could happen to anybody at any time or anywhere, so I mean, it's always good to watch your back.
COOPER: You say you did not kill Chauncey Bailey?
BROUSSARD: No. I did not.
COOPER: Do you know who did?
BROUSSARD: I'm gonna give those charges -- I'm gonna give all that information up when I go to trial.
COOPER: So you're saying you do know? You do have information about who killed Chauncey Bailey?
BROUSSARD: I give the information up when I go to trial.
COOPER: Why not now? Why not before?
BROUSSARD: Because this don't decide my trial.
COOPER: But if there's somebody else out there who did kill Chauncey Bailey, wouldn't that help your case now?
BROUSSARD: Yes, it would. Yes, it would. But like I -- like I told you -- I just told you, you know, I'm gonna give -- I'm gonna give the information up when I go to trial.
COOPER: What do you think now about Yusuf Bey IV, knowing all you know?
BROUSSARD: I feel let down. I feel let down.
COOPER: Let down how?
BROUSSARD: I don't really like repeating myself. I just told you -- I told you he didn't -- you feel me? Like I -- I feel -- I feel manipulated now, you feel me? Cause I first -- I told him -- I told him I wasn't even go - told him I wasn't even gonna go help out with this. And then he went and told the police anything. And then the police -- the police, they kept -- they kept -- they kept talking -- they kept saying anything to me. And then, you feel me? I felt like, I told him "no" at first, and he still went and did it.
COOPER: Do you think he's a good Muslim?
BROUSSARD: That's not up to me -- that's not up to me to decide.
COOPER: You told police that you'd admit to killing Bailey even if you didn't, if it would help the Bakery. Is that what happened there?
BROUSSARD: Yes, it did. They asked me -- they said -- they said, "Would you -- would you -- would you lie? Would you lie for the Bakery?" I said, "Yeah." And then when they -- when they asked me that, I was thinking, like, I messed up. I didn't do a good enough job.
COOPER: You're saying that, you know, you won't talk until trial about what you know about who killed Chauncey Bailey. Are you worried if you say what you know about who killed Chauncey Bailey, that your life would be in danger?
BROUSSARD: I'm on 23-hour lockup. Anything -- if anything happened to me, you feel me, we'll know who's fault it is. So I doubt -- I doubt anything would happen. I'm in good hands, I guess.
COOPER: You're not worried about getting hurt here?
BROUSSARD: Unless they let somebody in my cell. Real.
COOPER: It's probably not gonna happen.
BROUSSARD: Not gonna happen. That's why I'm on 23-hour lockup in my cell by myself.
COOPER: I mean, you obviously have a lot of time to think. What do you think now about the Bakery? I mean, when you were there, you were saying that they're doing good things for the community.
BROUSSARD: The Bakery as I remember it was a good place to work, you know. It helped me -- it helped me become a better person. It made me mature, you know, had -- I left alone a lot of pettiness. You know, I felt like I wanted to do right, you know. Like, I want to help out my people. I mean, as far as I remember it, for me, you know, just bad stuff happened. That's it. Bad stuff happened.
COOPER: Is there anything else you want people to know? You want to tell people?
BROUSSARD: What else you want to know?
COOPER: I don't really have anything else, but I just wanted you to have the opportunity if there's something you want to say to people, or what people should know about you or should know about the case.
BROUSSARD: I don't have anything.
COOPER: What do think's gonna happen?
BROUSSARD: I don't wanna think on that.
COOPER: Try not to think about it?
BROUSSARD: I mean, I think about it a lot, you know. It's stressing me out. I never thought I would be in a position like this, you know. Never thought I'd be in a position like this. I'm only 20 years old, you feel me, looking at my life -- my life -- my life on the line right now. You know? I could say it as many times as I want. Say it as many times as I want that I didn't do this but, you feel me? That ain't gonna change nothing. That's gonna--it's gonna be up to them twelve people to decide.
COOPER: What do you think they'll decide?
BROUSSARD: I don't know what they'll decide. I don't know what they'll decide. I'll know when that day comes.
COOPER: Okay. Well, thanks for talking to us. Appreciate it.
© 2011 CBS Interactive Inc. All Rights Reserved.


text:
On their picket line outside the British Medical Association (BMA) yesterday, GMB members demanded the doctors association show respect for their own staff and increase their derisory pay offer.
The BMA has offered its staff a 1.5% basic pay rise plus a one off 0.5% Performance Related Pay that most staff will not get, says the GMB.
The GMB says this is far below offers that have been awarded in comparable organisations across the sector.
As well, the final pay offer for this year was made in the context of vastly increased workloads due to the NHS pensions issue, which the BMA is fighting on behalf of its own members.
Many additional hours and effort were put into the doctors' ballot and day of action by BMA staff.
James Edwards and Joel Turner said: 'This strike action is not just over pay, it's about getting respect for our union and at work.
'What the BMA are asking from the government for their members, is the same as what we are asking from them for ours, "meaningful negotiations" over our pay issues'.
The BMA staff are further incensed that the BMA has deducted full pay at a punitive rate for staff who took industrial action on 20 June, ignoring the many hours of unpaid additional work staff did in the weeks leading up to the doctors own industrial action, and contrary to the many deals negotiated by BMA staff which resulted in no pay deductions for doctors when they took action.
The GMB Convener said: 'The strike is going very well. Picket lines are solid across the country and we have closed several BMA offices. The main forces are in Edinburgh where the BMA are meeting.
'There has been good support from BMA members.
'We are insisting the BMA come back with a more reasonable offer than the 1.5%. Most staff will get less.'
A GMB statement said the BMA was not in a state of financial crisis and currently has its highest ever membership rate (150,000) following a determined recruitment drive by its staff.
It has over £70 million in reserves and has received 'a very substantial sum of money for the hire of meeting rooms at BMA House by an international sports brand during the Olympics, causing disruption to the working lives of BMA staff.'


text:
The Earth could be headed for a 'mini ice age' researchers have warned. A new study claims to have cracked predicting solar cycles - and says that between 2020 and 2030 solar cycles will cancel each other out. This, they say, will lead to a phenomenon known as the 'Maunder minimum' - which has previously been known as a mini ice age when it hit between 1646 and 1715, even causing London's River Thames to freeze over.
The ORIGINAL gathering place for a merry band of Three Percenters. (As denounced by Bill Clinton on CNN!)
Saturday, July 11, 2015
Better pump up that human-agency "global warming" -- "Scientists warn the sun will 'go to sleep' in 2030 and could cause temperatures to plummet."
6 comments:
Seriously... pie?
Low kinetic energy, no penetration, just noting.
- July 11, 2015 at 9:48 AM
It doesn't matter. Getting colder, or getting warmer, it will be our fault, and the government will need to control us to stop it. It's not about climate, it's about control.
- July 11, 2015 at 11:17 AM
Fuck the control freaks.
- July 11, 2015 at 3:55 PM
Personally, I hope it does get colder. A lot colder. The unforgiving winter of the north is what forced that prehistoric tropical primate to develop the intellectual and social traits that ultimately led to what we call "the 1st World."
Seems it's a lesson in need of re-learning.
- July 11, 2015 at 9:35 PM
- Sedition said...
When life gives you free ice, buy more booze.
- July 11, 2015 at 10:25 PM
Didn't Al Gore tell us in 2008 that the ice caps would all be melted by 2013? It's 2015. The Earth's ice caps are not only still there, but exactly the same as they ever were, to the limits of measurement.
In science, when a model makes predictions that are contradicted by reality, we discard the model.
Therefore what Al Gore and the "global warming" scamsters are doing isn't science, it's politics.
- July 12, 2015 at 11:08 PM


text:
The G protein‐coupled receptor (GPCR) Proteolysis Site (GPS) of cell‐adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40‐residue GPS motif represents an integral part of a much larger ∼320‐residue domain that we termed GPCR‐Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell‐adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β‐strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell‐adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine‐tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.
There is a Have you seen? (March 2012) associated with this Article.
Autoproteolysis is a widely used intramolecular post‐translational process that often converts inactive protein precursors into biologically active mature proteins. For instance, the hedgehog pathway, one of the fundamental signal transduction pathways in animal development, is activated only if the signalling domain is cleaved off from the autoproteolytic domain of hedgehog (Lee et al, 1994). Among autoproteolytic domains/motifs described in various proteins (including hedgehog, N‐terminal nucleophile (Ntn) hydrolases, nucleoporin98, protein inteins, and pyruvoyl enzymes; Perler et al, 1997), the G protein‐coupled receptor (GPCR) Proteolysis Site (GPS) is one of the few autoproteolysis‐mediating domains/motifs found in membrane proteins. The GPS motif is conserved in all 33 human cell‐adhesion GPCRs and all 5 human polycystic kidney disease (PKD) proteins (Krasnoperov et al, 1997; Hughes et al, 1999; Ichtchenko et al, 1999; Chang et al, 2003; Lin et al, 2004); representing one of the most widespread and conserved autoproteolytic domains/motifs. Recent studies indicate emerging physiological and pathological roles for these GPS‐containing membrane proteins in cancer (Kan et al, 2010), PKD (Qian et al, 2002), brain development (Piao et al, 2004), myelination of neurons (Monk et al, 2009), central nervous system angiogenesis (Kuhnert et al, 2010), attention‐deficit/hyperactivity disorder (Arcos‐Burgos et al, 2010), cell polarity in neural development (Chae et al, 1999; Usui et al, 1999; Shima et al, 2004; Langenhan et al, 2009), Usher syndrome 2 (Ebermann et al, 2009), mouse male infertility (Davies et al, 2004), and immunity (see Yona et al, 2008 for a review), but the mechanism of GPS‐mediated autoproteolysis remains unclear.
Cell‐adhesion GPCRs are characterized by long N‐terminal extracellular sequences comprising multiple domains, and constitute a poorly studied class of GPCRs that is the second largest family of GPCRs in humans (Fredriksson et al, 2003). Most cell‐adhesion GPCRs are orphan receptors with no known ligands. They are found in animals, protozoa, and alveolates, predating many other important signalling molecules. The CIRL/Latrophilins (here abbreviated as CLs, CL1–3) and brain angiogenesis inhibitors (BAIs, BAI1–3) are two distant subfamilies of cell‐adhesion GPCRs that both are primarily expressed in neurons (Krasnoperov et al, 1997; Lelianova et al, 1997; Nishimori et al, 1997; Sugita et al, 1999). CL1 is a calcium‐independent receptor for α‐latrotoxin, a black widow spider toxin that triggers massive neurotransmitter release from neurons and neuroendocrine cells (Krasnoperov et al, 1997; Lelianova et al, 1997; Sugita et al, 1998; Sudhof, 2001; Deak et al, 2009). BAIs initially received their name because an extracellular fragment of BAI1 inhibited neovascularization in experimental angiogenesis (Nishimori et al, 1997; Kaur et al, 2005). Recently, C1q‐like proteins, a family of secreted proteins, were found to bind to BAI3 and to regulate synapse formation and/or maintenance (Bolliger et al, 2011). CLs and BAIs are now known to be two of the most mutated genes in human cancers (especially in squamous lung carcinoma and lung adenocarcinoma) and may act as tumour suppressor genes (Wood et al, 2007; Jones et al, 2008; Parsons et al, 2008; Kan et al, 2010). On the other hand, PKD family proteins are not GPCRs, but they contain large extracellular sequences, multiple transmembrane regions and a GPS motif similar to cell‐adhesion GPCRs (Ponting et al, 1999). PKD1 is a 500‐kDa 11‐pass membrane protein with a large N‐terminal extracellular region that is cleaved at the GPS motif (Qian et al, 2002). It is mutated in 90% of the autosomal dominant PKD (ADPKD) patients (Rossetti et al, 2001). A mouse mutant with uncleavable PKD1 exhibits abnormal kidney development (Yu et al, 2007).
In cell‐adhesion GPCRs and PKD proteins, the GPS motif is always located at the end of their long N‐terminal extracellular regions, immediately before the first transmembrane helix of the respective protein (Krasnoperov et al, 1997; Sugita et al, 1998; Ichtchenko et al, 1999; Chang et al, 2003; Lin et al, 2004; Figure 1A). The GPS motif is a conserved sequence of ∼40 amino acids containing canonical cysteine and tryptophan residues. The GPS is referred to as a 'domain' in the Pfam database although no three‐dimensional structure is available, and it is unclear whether it folds autonomously. Most, if not all, cell‐adhesion GPCRs undergo autoproteolysis in the GPS between a conserved aliphatic residue (usually a leucine) and a threonine, serine, or cysteine residue (L↓T/S/C) (Krasnoperov et al, 1997, 2002b; Nechiporuk et al, 2001; Lin et al, 2004). Although previous studies suggested that the GPS motif on its own promotes autoproteolysis (Krasnoperov et al, 2002a), it is hard to imagine how such a short sequence could fold into a catalytically active autoproteolytic domain given the stability of peptide bonds. Interestingly, in cell‐adhesion GPCRs the GPS motif appears to be preceded by a conserved so‐called 'stalk' region, which in turn is often preceded by a Hormone Receptor (HormR) domain, but the role of these sequences remains unclear.
To shed light on the GPS‐mediated autoproteolysis of cell‐adhesion GPCRs and PKD proteins, and to explore the significance of the stalk and GPS sequences and their relation to the HormR domain, we performed structural and functional studies of fragments containing these regions. Here, we show that the GPS motif is not an autonomously folded unit but rather part of a much larger evolutionarily conserved domain that we refer to as the GPCR‐Autoproteolysis INducing (GAIN) domain. We obtained crystal structures of fragments from two distantly related cell‐adhesion GPCRs (CL1 and BAI3) that include the GAIN domain and, additionally, the HormR domain. These structures revealed a novel fold with a high degree of structural conservation of even distant GAIN domains. Functionally, the entire GAIN domain is required and sufficient for autoproteolysis. Sequence analyses showed that the GAIN domain is an ancient domain that exists in primitive ancestor organisms, and is conserved in all cell‐adhesion GPCRs and all PKD1‐related proteins. Our structures of the cleaved CL1 and uncleaved BAI3 GAIN domains, in combination with mutagenesis experiments, reveal the structural determinants for autoproteolysis and suggest a plausible mechanism of cleavage. Taken together, our data describe a newly identified, evolutionarily conserved domain that is present in 38 different human proteins and functions as an autoproteolytic fold.
Structures of CL1 and BAI3
To test the significance of the sequence arrangements of the HormR domain, the stalk region and the GPS motif, and to explore the mechanism of the GPS‐mediated autoproteolysis, we determined the structures of fragments containing these regions from two distantly related cell‐adhesion GPCRs, CL1 and BAI3 (Figure 1A). Experimental phases for the CL1 and BAI3 structures were independently obtained from sulphur and iodine single anomalous diffraction (SAD) data at 1.9 and 2.3 Å resolution, respectively (Supplementary Table S1, Materials and methods). Both structures reveal two domains (Figure 1B and C; Supplementary Figure S1A and B): at the N‐terminus is the smaller ∼70‐residue HormR domain, and at the C‐terminus is a larger ∼320‐residue domain that we refer to as the GAIN domain; both structures are similar, but with some significant differences (see below and Supplementary Figure S1C). Surprisingly, the GPS motif is an integral part of the GAIN domain (comprising the last five β‐strands). Thus, the GPS motif itself does not constitute an autonomously folded unit, but rather forms a single folded domain together with the so‐called 'stalk'.
DALI (Holm and Sander, 1993) searches (as of January 2012) indicated that the GAIN domain is a newly identified fold, and that our CL1 and BAI3 crystal structures represent the first high‐resolution structures of this domain. The N‐terminal subdomain A of the GAIN domain is composed of six α‐helices, while the C‐terminal subdomain B consists of a twisted β‐sandwich including 13 β‐strands and 2 small α‐helices. The high similarity of the GAIN domain structures (root‐mean square deviation (r.m.s.d.)=1.38 Å; Supplementary Figure S1C) from two distantly related cell‐adhesion GPCRs (24% sequence identity) suggests structural conservation of this novel domain across the entire family of cell‐adhesion GPCRs.
The GAIN domain functions as an autoproteolytic domain
To determine whether the GAIN domain is the minimally required region for autoproteolysis, we designed deletion mutants of full‐length CL1: CL1‐GPS which lacks the HormR and GAIN domains except for the GPS motif; CL1‐HormR_GPS in which all residues of the GAIN domain except for the GPS motif are deleted; CL1‐ΔSubA in which only subdomain A of the GAIN domain is deleted; and CL1‐ΔHormR in which only the HormR domain is deleted (Figure 2A). We expressed the deletion mutants in HEK293 cells, and analysed their autoproteolysis by immunoblotting of cell lysates, and their plasma membrane transport by indirect immunofluorescence of non‐permeabilized cells. Our results show that the entire GAIN domain with both subdomains A and B is required for autoproteolysis (Figure 2B and C; Supplementary Figure S2A). Consistent with the identification of the GPS motif as an integral part of the GAIN domain, the GPS motif is not functional by itself (Figure 2B and C), presumably because it is unfolded when expressed without the other GAIN domain sequences. A protein fragment composed of only the GAIN domain of CL1 underwent autoproteolysis, demonstrating that the GAIN domain is not only necessary but also sufficient for autoproteolysis in CL1 (Figures 2D and 5D; Supplementary Figure S2B). Indeed, the electron density map of the crystal structure shows that the crystallized HormR‐GAIN fragment of CL1 is cleaved at the expected position (Figure 1D; Krasnoperov et al, 1997). In addition, the GAIN domain of GPR56 (a different cell‐adhesion GPCR, mutations of which cause bilateral frontoparietal polymicrogyria (BFPP); Piao et al, 2004) is sufficient for autoproteolysis (Figure 2E and F; Supplementary Figure S2C), suggesting that the GAIN domain mediates autoproteolysis in other cell‐adhesion GPCRs as well.
The GAIN domain is an ancient conserved fold
To study the evolution of GAIN domains, we searched for the presence of GAIN domains in the genomes of primitive evolutionarily distant organisms, and identified genes encoding such domains. Strikingly, structure‐based BLAST searches revealed that the genome of Dictyostelium discoideum, an evolutionarily ancient organism that arose before animals emerged, encodes a GAIN domain (Figure 3A), although it lacks hedgehog autoproteolytic domains as well as signalling and adhesion domains found in higher eukaryotes, such as HormR, Wnt‐signalling components, cadherins, immunoglobulins, and integrins (Eichinger et al, 2005). Moreover, a unicellular ciliate, Tetrahymena thermophila, encodes more than eight GAIN domains. Thus, the GAIN domain is among the most ancient GPCR‐related extracellular domains. Computational analysis (McGuffin et al, 2000; Pei et al, 2007) of ancient GAIN domain sequences showed that their predicted secondary structure pattern is identical to that of the CL1 and BAI3 GAIN domain (Figure 3B; Supplementary Table S2). Moreover, their GPS motifs including the putative cleavage sites are well conserved (Figure 3A), whereas those of the autoproteolytic domain of nucleoporin98 (which also exists in ciliates) are not. Furthermore, sequence alignments of cell‐adhesion GPCRs and PKD proteins revealed that all cell‐adhesion GPCRs and PKD proteins are predicted to have a GAIN domain with identical predicted secondary structure patterns to that of the CL1 and BAI3 crystal structures (Figure 3B; Supplementary Table S3). Apart from the shared GAIN domain, PKD proteins are unrelated to cell‐adhesion GPCRs but also undergo autoproteolysis (Ponting et al, 1999), indicating that the GAIN domain has been utilized by at least two different membrane protein families during evolution. These results suggest that the GAIN domain evolved prior to the evolutionary emergence of animals, that it is one of the first functional autoproteolytic domains, and thus represents an ancient feature that is not directly connected to multicellularity.
Autoproteolysis does not lead to the dissociation of the cleavage products
In general, autoproteolysis domains produce two protein fragments that then dissociate (e.g., hedgehog and protein inteins). Strikingly, the cleaved β‐strand in the CL1 structure is well ordered with full occupancy and thermal factors comparable to the surrounding protein, indicating no sign of dissociation (Figure 1D). The cleaved β‐strand is tightly bound to the rest of the GAIN domain involving an extensive network of conserved interstrand hydrogen bonds and primarily hydrophobic side‐chain interactions (Figure 1F and G). Thus, we predict that dissociation of the cleaved β‐strand would be energetically unfavourable, and would conceivably lead to unfolding of the remaining part of the GAIN domain. Consistent with this structural prediction, the N‐terminal cleavage product of full‐length CL1 and CL3 in transfected HEK293 cells remained bound to the rest of the protein on the cell surface, and was not secreted into the media (Figure 1H). Thus, the GAIN domain forms a tightly associated heterodimer upon proteolysis.
Different from CL1, the electron density of the crystal structure of the BAI3 GAIN domain showed no evidence of cleavage (Figure 1E, Supplementary Figure 7B), and BAI3 expressed in transfected HEK293 cells was uncleaved (Figure 4A). To address the question whether the BAI3 GAIN domain is functional, we raised antibodies to BAI proteins and showed that endogenous BAI proteins in brain are cleaved (Figure 4C). The conditions of BAI expression (i.e., expression levels, glycosylation machinery, and reductive environment) are very different between insect cells (used for expression of the crystallized proteins) and HEK293 cells on the one hand, and neurons on the other hand. Thus, these differences in post‐translational processing and environment could be the reason why BAI3 is uncleaved when expressed in insect or HEK293 cells, but cleaved in the brain. Nevertheless, uncleaved BAI3 was efficiently transported to the plasma membrane in HEK293 cells, indicating that cleavage at the GPS is not required for surface transport of a cell‐adhesion GPCR (Figure 4B; see also below). In contrast, autoproteolysis of the autoproteolytic protein nucleoporin98 is required for its transport to the nuclear envelope (Hodel et al, 2002).
Unique structural features of the GAIN domain enable autoproteolysis
The autocatalytic scissile bond in the GPS of the GAIN domain (star, Figure 5A and B) is positioned at a sharply kinked loop between the last two β‐strands of the GAIN domain. In the structure of the uncleaved BAI3 GAIN domain, the autocatalytic scissile bond (Figure 1E) has a distorted geometry (phi=174.0, psi=−158.2 degrees). Furthermore, numerous residues contribute to the unique structural features of the GAIN domain that localize the scissile bond at the tip of the sharply kinked loop (Figure 5A and B; Supplementary Figure S3A and B). The kink is created due to three structural features of the protein: (1) Two conserved disulphide bonds (C834‐C820 and C832‐C801 in CL1) between neighbouring β‐strands (Supplementary Figure S3A) keep this β‐turn in place. (2) The cleaved β‐strand has conserved residues involved in numerous hydrophobic interactions with other conserved residues (Figure 1F). (3) The side chain of L837 in CL1 (L856 in BAI3) at the cleavage site is trapped in a conserved hydrophobic pocket (Supplementary Figure S3B). Comparison of the cleaved and uncleaved GAIN domain structures revealed a small tilt of the cleaved β‐strand upon autoproteolysis that relaxes the sharp kink (Figure 1I).
To understand the mechanism of GAIN‐domain catalysed autoproteolysis, we performed extensive mutagenesis of the CL1 GAIN domain (Figure 5C). Among others, three mutations (H836S, L837A, and T838G) impaired autoproteolysis but did not interfere with surface transport (and consequently, protein folding) (Figure 5C and D; Supplementary Figure S4), indicating that these residues are directly involved in CL1 autoproteolysis (also providing further evidence that autoproteolysis is not required for trafficking of GAIN domain‐containing proteins). Consistent with a previously proposed mechanism for GPS autoproteolysis (Lin et al, 2004), H836 may act as a general base (although the general base is likely different in BAI3 since that residue is an Arginine; Figure 5A and B). The general base then removes the hydroxyl hydrogen from the side chain of T838 (Serine in BAI3) that in turn makes a nucleophilic attack on the carbonyl carbon of L837 (conserved in both CL1 and BAI3). Note that the T838P (Krasnoperov et al, 2002b) and T838A mutants are unfolded (Figure 5D; Supplementary Figure S4), but replacing threonine with a glycine enables proper folding, presumably by decreasing rigidity at the cleavage site.
Disease‐related mutations in PKD1 and CL GAIN domains
The GAIN domain of PKD1 is a hot spot for mutations associated with ADPKD, one of the most common monogenic diseases (1 in 1000) (Qian et al, 2002; Figure 6A). In addition, CL and BAI genes are among the most frequently mutated genes in human cancers, with mutation frequencies of 3 and 12%, respectively (Kan et al, 2010). Their GAIN domains are, again, a hot spot for cancer mutations (Figure 6C; Supplementary Figure S6A). We tested the ADPKD and cancer mutations in GAIN domains for their effect on autoproteolysis and plasma membrane localization. Almost all ADPKD mutations in the GAIN domain of PKD1 abolished autoproteolysis, either by directly interfering with catalysis or by altering their membrane localization (presumably, by impairing folding) (Figure 6A and B; Supplementary Figure S5), indicating an essential role for the GAIN domain. However, cancer mutations in CL1 and CL3 did not affect autoproteolysis or membrane localization (Figure 6C and D; Supplementary Figure S6), suggesting that the mechanism by which these mutations lead to disease is different from that of the ADPKD mutations. Since most cancer mutations are located on the surface of the CL GAIN domains, they may be involved in the interaction of the GAIN domain with a ligand or with the nearby transmembrane helices. Indeed, it is the GAIN domain of CL1 that interacts with its long‐known binding partner α‐latrotoxin, a black widow spider toxin (Sudhof, 2001; Figure 7), demonstrating the ability of the CL1 GAIN domain to function as a protein‐binding domain in addition to an autoproteolytic domain.
Cell‐adhesion GPCRs exhibit structural similarity to hormone receptors
Although the HormR domain is the second most frequently observed domain in cell‐adhesion GPCRs (found in 12 of 33 human cell‐adhesion GPCRs), no hormones have yet been identified to bind cell‐adhesion GPCRs. HormR domain‐containing cell‐adhesion GPCRs are homologous to Class B GPCRs that always include a HormR domain (Lagerstrom and Schioth, 2008). Remarkably, our HormR domain crystal structures from CL1 and BAI3 revealed an unusually high structural similarity (0.7 Å r.m.s.d.) to the genuine hormone‐binding domain of the corticotrophin releasing factor receptor (CRFR), a member of the class B GPCRs, supporting the hypothesis that cell‐adhesion GPCRs may be hormone receptors (Figure 8A).
Superposition of the CL1 structure with the structure of the CRFR HormR domain bound to corticotrophin releasing factor (CRF), a 41 amino‐acid peptide hormone, reveals that a similar hormone could not bind to CL1 HormR in the conformation observed in the crystal structure since the GAIN domain is blocking the homologous hormone binding site on the HormR domain of CL1 (Figure 8B). Clearly, hormone binding would require a conformational change in CL1 to expose the putative hormone binding site. In class B GPCRs, the HormR domain precedes the transmembrane helices and is juxtaposed to the membrane. According to the two‐domain model of class B GPCR activation, the interaction of the extracellular hormone‐binding domain with the hormone promotes the interaction of the hormone with the transmembrane helices, leading to the activation of the receptor (Hoare, 2005). However, the mechanism of cell‐adhesion GPCR activation upon hormone binding may be different, because the GAIN domain lies between the HormR domain and the transmembrane helices.
In this study, we aimed to obtain a structural understanding of the autoproteolysis function of the GPS motif that is characteristically found in cell‐adhesion type GPCRs and PKD proteins. Towards this end, we here determined the crystal structures of fragments containing the GPS motif, the stalk region and the HormR domain from two distantly related cell‐adhesion GPCRs, CL1 and BAI3. Unexpectedly, we found that a single large domain termed the GAIN domain spans the GPS motif and the stalk region (Figure 1), and that the GPS motif by itself is not a functional domain, but only a small part of the much larger GAIN domain. The GAIN domain is a novel domain, and our CL1 and BAI3 crystal structures now define this domain family.
The CL1 deletion experiments showed that the GPS motif is not functional by itself (Figure 2). Instead the entire GAIN domain is both required and sufficient for autoproteolysis. Thus, the GAIN domain functions as an autoproteolytic fold. Strikingly, structure‐based database searches revealed that primitive organisms, such as Dictyostelium discoideum that arose early in evolution before animals emerged, encode GAIN domains although they lack most other autoproteolytic domains, important adhesion and signalling domains and critical signalling pathways (Figure 3; Supplementary Table S2). These ancient GAIN domains have conserved GPS motifs and are examples of one of the most ancient functional autoproteolytic domains. Computational analysis of the GAIN domain sequences showed that all cell‐adhesion GPCRs and all PKD proteins have a GAIN domain with identical predicted secondary structure patterns to that of the CL1 and BAI3 crystal structures showing that the GAIN domain is widespread and conserved in higher eukaryotes as well as in ancient organisms (Supplementary Table S3). Our cleaved and uncleaved GAIN domain structures, in combination with extensive mutagenesis of the CL1 cleavage site, reveal the unique structural features of the GAIN domain that enable self‐cleavage and shed light on the mechanism of autoproteolysis. The discovery of the GAIN domain also revealed a possible role for the GAIN domain in a variety of diseases, including cancer, ADPKD, and BFPP. We found that nearly all ADPKD mutations interfere with autoproteolysis of the GAIN domain, whereas cancer mutations have no effect on autoproteolysis or cell‐surface localization. Thus, the mechanism by which the cancer mutations lead to disease are unrelated to autoproteolysis and the GAIN domain likely has other functions (such as interacting with ligands or transmembrane helices) in addition to the autoproteolysis function.
In summary, our experiments demonstrate that the GAIN domain is an ancient autoproteolytic domain that exhibits unique properties and is present in a large number of GPCRs and PKD proteins. Although the proteolysis of membrane proteins by sheddases and regulated intramembrane proteolysis (RIP) proteases is well studied (Blobel, 2005; Wang et al, 2006) the autoproteolysis of a membrane protein is poorly understood. GAIN domain is one of the few domains to mediate self‐cleavage of a membrane protein. Two properties of the GAIN domain‐mediated autoproteolysis make it unique and intriguing: (1) All GAIN domains that we analysed (from ancient organisms to higher eukaryotes) always immediately precede the N‐terminal transmembrane helix (Figure 9). Additionally, HormR domains, when present, always precede the N‐terminus of the GAIN domain. (2) In contrast to most other autoproteolytic domains that cause the release of the cleaved mature protein, autoproteolysis of the GAIN domain does not lead to the dissociation of the cleaved extracellular sequences containing the GAIN domain from the membrane‐embedded regions of the protein. These observations naturally lead to the hypothesis that the GAIN domain is functionally and/or structurally associated with the membrane domains and that the GAIN domain‐mediated autoproteolysis has a complex mechanism of action.
The HormR/GAIN/transmembrane helix sequence arrangement is unrelated to the autoproteolysis function of the GAIN domain although it may be significant for the regulation of receptor signalling via intramolecular interactions. Indeed, deletion of the extracellular sequences of GPR56, a cell‐adhesion GPCR with an extracellular GAIN domain that is preceded only by a short non‐GAIN domain sequence (Figure 2E), causes constitutive activation of the GPCR (Paavola et al, 2011), suggesting a possible inhibitory role for the GAIN domain. In the GAIN domain, the cleaved β‐strand is separated from the first transmembrane helix by 5–6 linker residues (Figure 9), positioning the GAIN and HormR domains into close proximity to the transmembrane helices. The small conformational change that we observe upon autoproteolysis (Figure 1I) may be critical for the relative orientation of the GAIN domain with respect to the transmembrane helices. Binding of a hormone to the HormR domain or binding of an endogenous ligand to the GAIN domain may be potential regulators of signalling via the receptor. Our work here redefines the poorly studied cell‐adhesion GPCR class to share a large unique and widespread autoproteolytic domain that may be involved in downstream signalling.
Materials and methods
Protein expression and purification
The HormR and GAIN domains of rat CL1 (residues 460–849) and human BAI3 (residues 498–868) were cloned into the Xba1–Not1 and the BamH1–Not1 sites of the pAcGP67A vector, respectively (CL1=43 kDa plus 5 carbohydrates; BAI3=41.7 kDa plus 3 carbohydrates). A 8 × His tag was added at the C‐terminus for affinity purification. Spodoptera frugiperda (Sf9) cells (Invitrogen) were transfected with pAcGP67A carrying the gene and linearized AcNPV DNA (Sapphire Baculovirus DNA; Orbigen) using Cellfectin (Invitrogen). Baculovirus was amplified in Sf9 cells in 10% (v/v) fetal bovine serum containing SF900‐II medium (Gibco).
Large‐scale protein expression of CL1 and BAI3 was performed by infection of Trichoplusia ni (High Five) cells in Insect‐Xpress medium (Biowhitaker) at a cell density of 2 × 106 cells/ml with an infection course of 72 h. The media containing the secreted, glycosylated, recombinant protein was concentrated and dialysed using a tangential flow filtration system (PALL) and subsequently purified with Ni‐NTA resin (Qiagen) at 4°C. The proteins were eluted with 200 mM imidazole (pH 7.2). CL1 and BAI3 were further purified by size‐exclusion chromatography using a Superdex 200 column (Amersham Pharmacia Biotech) in buffer containing 10 mM Hepes (pH 7.2) and 150 mM NaCl. The peak corresponding to the proteins was then concentrated to 10.7 mg/ml (for CL1) and 17 mg/ml (for BAI3) in 30 kDa Centricon (Millipore) concentrators and used for crystallization trials.
α‐Latrotoxin (residues 413–1080) with N‐terminal GST and Myc tags in a pGEX‐KG vector was expressed in Origami cells. The expression and purification were described in Li et al (2005). The latrotoxin construct used for native gel experiments corresponds to the 20 ankyrin repeats of latrotoxin with a C‐terminal His tag. The protein was expressed in High Five cells and secreted protein was purified by affinity chromatography followed by size‐exclusion chromatography.
The GAIN domain of rat CL1 (residues Thr533–Ile849) was cloned into a pCMV5 vector and fused to Ig (26 kDa) at the C‐terminus and carried an HA tag at the N‐terminus. The GAIN domain of mouse GPR56 (residues Q115–E395) was cloned into the pDisplay vector with N‐terminal HA and C‐terminal myc tags and has the PDGFR single‐spanning transmembrane domain at the C‐terminus. The full‐length human PKD construct carried N‐terminal GFP and C‐terminal Flag tags. Full‐length human CL3 was synthesized by codon optimization and carried Flag and HA tags at identical positions as in CL1.
Crystallization of CL1 and BAI3
Initial screens for crystallization of CL1 were carried out using 96‐well format kits (Hampton Index, Salt and Crystal Screen HT, Hampton Research) on a Phoenix crystallization robot (Art Robbins Instruments). Automatic plate scanning by a CrystalPro 2 imaging system (TriTek Corporation) identified initial hits in the Wizard 1–2 screen (Emerald Biosystems). After optimization, crystals of CL1 were grown using hanging drop vapour diffusion at 20°C in 24‐well format. In all, 1 μl of 10.7 mg/ml protein in 10 mM HEPES pH 7.2, 150 mM NaCl was mixed with equal volume of mother liquor consisting of 1.26 M (NH4)2SO4, 0.1 M sodium acetate pH 4.5, 0.2 M NaCl, and equilibrated against 1 ml mother liquor. Crystals grew to full size within 3 days. Crystals were cryoprotected by transferring the crystals into mother liquor with 10, 20, and 28% glycerol gradually. The best crystals diffracted to dmin=1.85 Å. Crystals of CL1 formed in space group P3121 with one molecule per asymmetric unit and 67% solvent content.
Crystallization hits for BAI3 were obtained with a 96‐well format kit (Protein Complex, Nextal) at 20°C. In all, 17 mg/ml protein in 10 mM HEPES, 150 mM NaCl pH 7.2 was mixed with equal volume of mother liquor consisting of 15% PEG6000, 0.1 M sodium citrate pH 5.5. Crystals grew to full size within 2 days and were cryoprotected with 25% glycerol. Crystals diffracted to dmin=2.3 Å. Crystals of BAI3 formed in space group C2221 with two molecules per asymmetric unit and 63% solvent content.
Structure determination and refinement of CL1
For the CL1 structure, experimental phase information was obtained from sulphur SAD data. This structure was the first structure that was determined at the Stanford Synchrotron Radiation Lightsource by sulphur SAD phasing using an optimized data collection strategy for a long wavelength at 2 Å, and one of a few de‐novo structures that has been determined by sulphur SAD at this resolution. More than 40 crystals were screened to find the best diffracting large crystal. A test radiation damage experiment was performed on a different crystal to determine the effect of radiation damage and to optimize the parameters for the experiment. Three data sets, each comprising 360° and offset by 20°, were collected on a single long crystal by translating the beam along the long edge of the crystal. The crystal was placed in a helium stream operating at 65 K to minimize background scatter at long wavelength and to further limit radiation damage. Data collection was carried out in inverse beam mode with a wedge angle of 0.5° and an exposure time of 8 s. Total data collection lasted 9 h. The data were collected at a wavelength of 2 Å, which limited the resolution to 2.3 Å (diffraction was observed to 1.8 Å resolution at a wavelength of 1 Å). The resolution limit decreased from 2.3 to 2.7 Å at the end of one 360° data collection as a result of radiation damage. All data sets were indexed, integrated, scaled, and combined in HKL2000 using the scale anomalous and high absorption options (Otwinowski, 1997). The error model was not optimized during scaling. In all, 25 sulphur sites were found (17 sites within the protein and 8 in sulphate ions from the crystallization condition) by SHELXD (Schneider and Sheldrick, 2002). SHELXD used a high‐resolution cutoff of 2.7 Å, at which the I/σ was ∼50. The sulphur sites were refined using SHARP (Vonrhein et al, 2007). Density modification was performed by DM (Cowtan and Zhang, 1999). Among all space groups tested, only space group P3121 yielded easily interpretable electron density maps. The density modified electron density maps showed good connectivity of secondary structure elements and cleavage at the GPS site (Supplementary Figure S7A). The sulphur sites corresponded to the expected side chains when compared within the density modified electron density map. Automatic model building was done with Phenix Resolve (Terwilliger, 2003) for two thirds of the structure. The rest of the model building was carried out manually using Coot (Emsley and Cowtan, 2004), and refinement performed with Phenix.refine (Adams et al, 2010) using the MLHL target function with experimental phases. The resulting model was then refined against the higher resolution native data set at 1.85 Å resolution. The subsequent refinement consisted of alternating rounds of positional minimization, individual restrained thermal factor refinement, both using the MLF maximum likelihood target function, and model building. Water molecules and N‐linked carbohydrates were added. A summary of the data collection and refinement statistics is given in Supplementary Table S1 (PDB accession code 4DLQ).
Structure determination and refinement of BAI3
BAI3 crystals were soaked into three solutions containing the crystallization condition, 0.25 M sodium iodide, and 10, 20, and 30% glycerol immediately before they were frozen in liquid nitrogen. The total soak time was about 1–2 min. Only one in ten crystals survived this treatment. The presence of increased glycerol, thus viscosity, in the soak solutions prevented the crystals from cracking. Data collection was done in inverse beam mode with a wedge angle of 25° and an exposure time of 5.5 s. The derivative crystals were not isomorphous to the native crystals, so the native data set could not be used for isomorphous phasing. The diffraction data were collected at a wavelength of 1.6 Å, resulting in diffraction data to 2.7 Å resolution. Data processing was similar to that of CL1. The BAI3 structure was independently determined with experimental phases obtained by iodine‐SAD data (a density modified map of the iodine‐SAD data around the GPS site is shown in Supplementary Figure S7B, clearly indicating lack of cleavage in contrast to the CL1 structure). SHELXD used a high‐resolution cutoff of 4 Å, at which the I/σ was ∼50, and it found 6 iodine sites. There were two molecules in the asymmetric unit and non‐crystallographic symmetry (NCS) restraints were used during initial refinement cycles using Phenix.refine but were removed at later stages. A summary of the data collection and refinement statistics is given in Supplementary Table S1 (PDB accession code 4DLO).
Cleavage and plasma membrane localization experiments
Antibodies. Mouse monoclonal anti‐HA antibody was from Covance and rabbit polyclonal anti‐Flag antibody was from Sigma.
Mutagenesis and expression constructs. CL1 deletion constructs were engineered by PCR oligonucleotide amplification. pCMV‐CL1 HA Flag was generated as follows: The Flag epitope containing constructs were obtained by subcloning HindIII–XbaI PCR fragment from pCMV‐CL1 into the same site of pCMV‐NL1‐Flag. The Flag epitope was fused at Leu25 of CL1. A single HA epitope was inserted by PCR between Tyr918 and Glu919 in the second extracellular loop of CL1. Deletion constructs were generated using pCMV‐CL1 HA Flag construct as a backbone. Deletion constructs are as follow: pCMV‐CL1‐ΔHormR HA Flag was generated by deleting Phe479–Cys532. pCMV‐CL1‐GPS HA Flag was generated by deleting Phe479–Glu792. pCMV‐CL1‐GPS_HormR HA Flag was generated by deleting Pro535–Glu792. pCMV‐CL1‐ΔSubA HA Flag was generated by deleting Pro535–Val666. pCMV‐IgCL1 LEC was generated by amplifying Met1–Tyr131 PCR fragment and subcloning into EcoRI–SalI of pCMV‐Ig. pCMV‐IgCL1 GAIN was generated by subcloning the PCR‐amplified fragment of Thr533–Ile849 into SacII–SalI of pDisplay then by excising EcoRI–SalI fragment and subcloning into the same sites of pCMV‐Ig. Therefore, the resulting pCMV‐IgCL1 GAINR construct also bears an N‐terminal HA epitope. pCMV IgN1a‐1 and pCMVN1b were described elsewhere (Ushkaryov et al, 1994; Sugita et al, 2001). pCMV‐CL1 T838A and pCMV‐CL1 T838P were generated by PCR mutagenesis. PKD mutagenesis was performed by cutting out the wild‐type human PKD with BsiWI restriction enzyme, pasting the small fragment into a carrier plasmid. After site‐directed mutagenesis was performed, the mutated fragments were subcloned back into the original plasmid. CL3 and CL1 mutagenesis was performed by the Qiagen site‐directed mutagenesis kit.
Cell culture and transfection. For immunoblotting and immunocytochemistry experiments, HEK293T cells were cultured in 6‐well plates until they reached 70–80% confluency. The cells were then transfected using 4 μl of Fugene 6® (Roche Diagnostics) and 2 μg of respective CL1 DNA constructs. For immunoblotting experiments, cells were harvested 48 h post transfection. For immunocytochemistry experiments, the cells were trypsinized 24 h post transfection and splitted into 12‐well plates containing coverslips that have been previously coated with 0.5 mg/ml poly‐l‐lysine in borate buffer. The cells were grown on the coverslips for an additional 24 h before further experiments were done. For recombinant Ig‐protein expression, HEK cells were transfected using calcium phosphate with chloroquine and 20 μg of cDNA corresponding to the various Ig proteins. Media was then harvested 4 days post transfection.
Immunocytochemistry and image acquisition. Cells transfected with CL1 constructs were washed once with PBS and fixed with 4% paraformaldehyde for 10 min on ice. Cells were washed again three times with cold PBS and incubated at room temperature for 30 min in a blocking solution containing 3% BSA in PBS with or without 0.1% Triton X‐100 meaning that cells were permeabilized or not, respectively. Mouse anti‐HA antibody was then added (1:500 ratio) and the incubation was prolonged for another 2 h. Cells were washed three times with blocking solution and incubated for 1 h at room temperature with anti‐mouse Alexa‐488 fluorescent antibody to label CL1 receptors. Cells were finally washed again three times with blocking solution and once with water before mounting on slides using media containing DaPI for nuclear staining. Slides were then analysed by confocal microscopy. Images were acquired using the confocal microscope Leica TCS2. The same confocal acquisition settings were applied to all samples of the experiment. Collected z‐section images were analysed blindly using Leica confocal software.
Immunoblotting procedures. Transfected HEK cells were harvested 2 days after transfection and solubilized for 1 h at 4°C in buffer containing 20 mM Hepes‐NaOH pH 7.4, 0.1 mM EDTA, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 and 1% Triton. Insoluble material was removed by centrifugation. SDS–PAGE sample buffer was added to the supernatant and the resulting sample was loaded on 6% SDS–PAGE. Gels were then transferred onto nitrocellulose membranes and processed using standard procedures. Membranes were probed with a mouse monoclonal anti‐HA antibody or a rabbit polyclonal anti‐Flag antibody followed by Horseradish peroxidase‐coupled secondary antibody, incubated with ECL reagents and revealed on X‐ray films.
Ig‐fusion protein purification and pull‐down experiments
The soluble Neurexin and CL1 Ig‐fusion proteins were affinity purified from transfected HEK293 cell supernatant using protein‐A Sepharose 4 Fast Flow (Amersham Pharmacia Biotech AB, Sweden) to bind the human‐IgG portion and washed to remove unbound proteins. The immobilized Ig‐fusion proteins were then incubated 16 h at 4°C with the eluted 3 μg GST–myc–Latrotoxin in buffer containing 20 mM Hepes buffer, pH 7.4; 0.1 mM EDTA, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2 and 1% (v/v) Triton X‐100. The beads were then washed, incubated in boiling SDS–PAGE sample buffer and loaded on 6% SDS–PAGE. SDS–PAGE gels were either transferred onto nitrocellulose membrane and immunoblotted with anti‐myc antibody or stained using Coomassie Brilliant Blue R‐250 (Bio‐Rad).
BAI cleavage experiments
Antibodies Goat antiserum 11509 and rabbit antisera 233C and 234C were generated against a synthetic peptide (N‐CEKAGATIPLVGQDIIDLQTEV‐C; mouse BAI1). Rabbit antisera A324 and A323 were raised against recombinant GST‐fusion proteins (P1447 → V1582 of mouse BAI1 and R1151 → E1348 of human BAI3, respectively). T3743 was raised against GFP in rabbit.
Cell culture HEK293T cells were plated in 6‐well plates. At 70–80% confluency, they were transfected with 1 μg expression plasmid (FuGENE 6 from Roche), and then cultivated for 2 days. Cells were lysed in extraction buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X‐100, protease inhibitors), homogenized, and the soluble fractions were collected after centrifugation.
Immunoprecipitation Two mouse brains were homogenized in 8 ml buffer (25 mM HEPES pH 7.2, 320 mM sucrose, protease inhibitors). After centrifugation at 1500 g for 10 min, supernatant S1 was centrifuged at 150 000 g for 60 min. Pellet P2 was then homogenized in 6 ml lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1% Triton X‐100, protease inhibitors), and centrifuged again at 150 000 g for 60 min. In all, 3 ml of supernatant S3 was incubated with 18 μl antiserum 11509 for 60 min. Then, 180 μl protein G sepharose (GE Healthcare) was added, and reactions were incubated for 3 h. The beads were washed four times with lysis buffer, and the proteins were eluted in SDS buffer.
Immunoblotting Proteins were resolved on 6% acrylamide gels and transferred onto nitrocellulose membranes (Whatman). After a blocking step, membranes were incubated overnight with the primary antibody diluted in blocking solution (5% milk powder, 2.5% goat or rabbit serum, 0.1% Tween‐20, in TBS). After washing with TBS containing 0.1% Tween‐20, blots were incubated with a peroxidase‐conjugated secondary antibody (Cappel) for 45 min. After washing, immunoreactive bands were detected by ECL (GE Healthcare).
Supplementary data
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Data [emboj201226-sup-0001.pdf]
We thank E Özkan for crystallographic advice and discussion; M Etherton, X Piao, F Qian for α‐latrotoxin, GPR56, and PKD1 cDNAs. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. This study was also supported by a postdoctoral fellowship from Canadian Institutes of Health Research (CIHR, to AAB) and a grant from the NIMH (R37 MH52804‐08 to TCS). D Araç is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.
Author contributions: DA designed and performed experiments and wrote the paper. AAB, DA, MFB, JN, and MS performed experiments. TCS and ATB provided guidance and wrote the paper.
This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
- Copyright © 2012 European Molecular Biology Organization


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As it turned out, I did end up reading/listening to a Christmas book in December! And I was glad about my choice too!
Shannon Stacey has become one of my favorite authors in 2012. So when I saw this little gem for so cheap, I quickly snatched it up. It is SO worth your money, friends. This is a short story, but Shannon Stacey's characters are so well rounded that it felt like a full-length book. I loved that!
This story was completely fun, sexy, and emotional. It really had so much depth. And those smexy moments…wowzers!! The story pace was so well done it felt like a full novel to me. Also, all the characters are very well developed. I loved how well rounded this novella was.
I don't believe this audiobook is sold on its own anymore. It is now part of the Holiday Kisses anthology. In any case, MacLeod Andrews does a fantastic job reading this book. And it's a short listen, at only 2 hrs and 4 mins.
As always, thank you for always delivering such great Contemporary Romance stories Ms. Stacey. I am very much looking forward to reading more of your books.
Book Info
Mistletoe & Margaritas by Shannon Stacey
Narrator: MacLeod Andrews
Published by Carina Press
Pub Date: December 5th 2011
Length: 2 hrs, 4 mins
Format: Audiobook | Source: Purchased
Genres: Adult, Contemporary, Romance
Buy the book! | Goodreads
Heating up the Holidays
Claire Rutledge still believes in love, despite losing her husband. So, after two years, she's not surprised by the steamy dreams telling her it might be time to start moving on. But she didn't expect her friend Justin to have the starring role.
Justin McCormick has loved Claire since the moment he first saw her, but his best friend got there first. Now that Claire is ready to move on with her life, Justin is finding it harder than ever to hide his true feelings. And when they both get caught up in the holiday spirits at a party, their simmering mutual attraction boils over into a night neither of them can forget…
What Some Friends Had to Say About It…
Jac @ For Love and Books – "So if you're a romance fan? Or a Christmas fan? Or if you like Margarita's. Or if you've ever drank a Margarita? You NEED to read this!"
April @ Good Books and Good Wine – "Friends, Mistletoe & Margaritas had one of my favorite types of romance tropes — fighting the undeniable attraction."
Amy @ So Many Reads – "Mistletoe & Margaritas is a very sweet, emotional story. This is a great story to read anytime of the year, but especially at Christmastime."


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Spot Runner CEO Nick Grouf presented today at OMMA Hollywood. Spot Runner provides a simple, quick way for local businesses and national corporations with local offices to create, buy, and place highly targeted local TV advertising. If Google's M&A team is not already driving down to LA with truckloads of money to buy the company, they should. And if they don't, Microsoft or Yahoo should.
Accordingt to Nick, Spot Runner reduces the cost of creating a decent TV ad from $500,000 to $500 ($499 to be precise). It reduces the time necessary to plan and buy a TV campaign from 6 weeks-12 months to 24 hours. It allows thousands of local businesses and franchises that would never have been able to employ TV advertising in the old media world to do so–and target the campaigns by zip code.
Two readers argue (see comments) that what Spot Runner is offering is nothing new–that cheap creative has been available since the Dark Ages, that you can target by zip code, etc. The impression I got from the presentation was that Spot Runner seriously streamlined the process (and reduced costs). Any Spot Runner customers out there care to weigh in?
One reasonably happy Spot Runner customer weighs in (see comments). Any others out there?


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I interrupt this normally crafty/decorating blog to ask an important question: Who likes Thai food? It is really one of my favorites-tied with Middle Eastern-I really can't decide which one I like best but I love the blend of flavors and spices of both.
So, I haven't posted a recipe in a while-I haven't been cooking too many things with the addition of our newborn two months ago. It's been all about survival here and what's quick and easy, but as he sleeps a little more during the night, I get a little more energy to do more myself.
So one rainy day I decided it was perfect to try out my Thai cookbook with an easy recipe-chicken noodle soup with a bang. I'm not sure I'll ever go back to the old way of making it...
I promise this recipe is really simple-it was my first Thai recipe I've ever tried and now that I know what I'm doing I will try something a little more daring next time. I present to you Thai Chicken Noodle Soup:
So, I didn't actually add the kaffir lime leaves because I didn't make a special trip to an Asian food store-maybe next time? But the lemongrass and ginger paste were conveniently in my produce aisle-with a $1 off coupon next to it-making it really easy and cheap to add these ingredients.
I love my Pampered Chef cutting mats-you can just bend them and slide the food into the pan.
Here's another easy way to add fresh flavor to your foods: I found these mini cube trays of herbs at Costco by a brand called Dorot.
They are filled with tiny portions of either crushed garlic, parsley or cilantro = genius!
I had frozen fresh ground herbs before, but never had tiny trays like these 20 sections of 1 teaspoon each. I plan on refilling these trays with my own fresh herbs from my summer garden-I'm dreaming of fresh salsa and pesto...mmmm....
I discovered using chicken tenderloins speeds things up too, because they don't need to be tenderized or marinated and can be cut into cubes quickly. Where have you been all my life?
One of these days when I don't have toddlers hanging on me while I try to cook, I will make homemade broth from scratch. In the meantime, there's my favorite Better Than Bouillon quick option-no MSG, all natural goodness, easily stores in the fridge.
Yes, the weather's warming up, but it's still a great time to make soup, in my opinion. Add some ice cubes if you want. And guess what else? My whole family loved it (well, besides the newborn-but he will someday! ) It only took about 20 minutes to make. And I promise, it really wasn't a huge production=cooking success for this busy momma.
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