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Women with epilepsy face an uncomfortable position when they decide to have children, as either seizures or the medications to prevent them are suspected of hurting the fetus. But a new study gives some potentially reassuring news.
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A study from researchers at the Karolinska Institute in Sweden examined the records of 1,235 children born to mothers with epilepsy between 1973 and 1986. The researchers looked at the treatments the mothers received and the school records at age 16 for the children.
The researchers found the children of mothers receiving polytherapy — a combination of drugs — had 3 times greater odds of not finishing school, compared with their peers. However, the children of mothers who were only taking a single medication did not seem to be more likely not to complete school than their peers, although they were less likely to "pass with excellence."
"The real important finding is the one that women on one drug, who have mild epilepsy, are having normal kids," said Dr. Shlomo Shinnar, a member of the American Academy of Neurology and a neurologist at the Albert Einstein College of Medicine in New York. Shinnar was not involved with the study.
Childbearing is an important consideration when prescribing drugs to treat epilepsy, because of concerns about the drugs' effects.
"We have already had, for a long time, concern over treating women who are pregnant with anti-epileptic drugs," Shinnar told My Health News Daily. "Conversely, there's also concern about women who are pregnant having seizures."
Story: Excuses, excuses: 11 health cop-outs that hold you backSeizures can injure the fetus, and so need to be treated in pregnant women.
"This is one of those diseases where it's not an option to tell people that drugs are no good and not to take drugs," Shinnar said.
Certain medications are recognized as being problematic for pregnant women (they were not specifically examined by the Karolinska study), and so, Shinnar said, physicians consider other drugs when first treating a girl or young woman – whether she is planning on having children or not – so that she will not need to switch medications once she becomes pregnant.
But problems may arise if a woman's epilepsy does not respond to treatment with those drugs.
"If one doesn't control your seizures, and the other does, it's unclear, because having seizures worsens your outcome," Shinnar said.
Women who do not respond to any single drug may face a problem, as polytherapy has been associated with problems in the past, which were confirmed by this study. But the cause and effect is unclear.
"It's difficult to tease out how much of it is polytherapy and how much of it is that people with worse epilepsy are on polytherapy," said Shinnar.
A strength of the study noted by outside researchers was that it was on a population level — meaning it involved a large groups of children and looked at their average risk for having problems in school. However, that gave it some limitations.
When it came to measuring an individual child's risk, "We can't understand what the risk is, what the individual factors contributing are," said Dr. Kimbor Meador, director of the Emory Epilepsy Center in Atlanta.
Story: Therapy for women prone to miscarriage questionedWithout individual medical records, he said, it is difficult to tease out factors such as the number of seizures the mother may have had during pregnancy, or the effects of certain medications that were given, which may have caused problems on their own. The researchers looked at only two medications separately from the group of patients taking a single drug and while those drugs are still in widespread use, more have come into use since the study ended.
But researchers said the study should be reassuring to women who are worried their medication presented a risk to their baby.
"I think the research shows there's a risk, but it shouldn't be emphasized such that the women think they can't have kids that are normal," Meador said.
The study was published today (Nov. 4) in the journal Epilepsia.
Epilepsy Meds Linked to Infertility
Pregnant Moms Who Eat Peanuts Could Raise Baby's Allergy Risk
Epilepsy: Symptoms and Treatment


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ello mates'
This blog is intended to provide Kputnick with a character for his zombie survival story, I have been super excited to do this since we talked about it a couple days ago. I love this idea because....srsly? must I actually have to explain myself as to why I love this idea? nuff said.
Character name: I dont know if I use my real name or a made up name here sooo..* Insert name*
Backstory: She was born and raised in Portland, Oregon. A city known for its retro, Eco friendly and idealistic community where "weird" has never been more popular. Always sticking to herself socially she grew up surfing the inter webs and spending countless hours in front of screens playing video games. Constantly being creative and seeking knowledge she would often spend her time learning things from the web and implementing them in day to day life...
When she hit 18, she joined the military and spent the next 8 years advancing in marksmanship, weapons, stealth and hand to hand combat, so much in fact that she had been selected for various stealth and operative missions. At the peak of her career she was on a recon mission with a four man team in Nicaragua scouting enemy camps and gathering intelligence when her and the other three members were captured. Held for months, tortured for information and on the brink of death she hit an epiphany moment where she had 2 choices...."Stay and die" or "Escape to live". She did what was necessary to live, unfortunately her team had long been dead and unable to recover. After she was back in the states she was given an honorable discharge and a medal to accompany, however she still had many sleepless nights with reoccurring nightmares...
Expertise: Erica's expertise is mostly in stealth, weapons and hand to hand combat. She is able to negotiate, plan, analyze and adapt at a very fast rate as well as carry out plans of action in very efficient ways to optimize results.
Well yeah....thats it....not the best but Im so burnt out right now...ugh.

subset: commoncrawl


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US Youth Have Serious Mental Health Issues 818
Ant writes "Google News carries a Canadian Press report that 'a new study has found that five times as many high school and college students in the United States are dealing with anxiety and other mental health issues than youth of the same age who were studied in the Great Depression era. ... Pulling together the data for the study was no small task. Led by [San Diego State University psychology professor Jean Twenge], researchers at five universities analyzed the responses of 77,576 high school or college students who, from 1938 through 2007, took the Minnesota Multiphasic Personality Inventory, or MMPI. The results will be published in a future issue of the Clinical Psychology Review. Overall, an average of five times as many students in 2007 surpassed thresholds in one or more mental health categories, compared with those who did so in 1938. A few individual categories increased at an even greater rate — with six times as many scoring high in two areas: 'hypomania,' a measure of anxiety and unrealistic optimism (from 5 per cent of students in 1938 to 31 per cent in 2007), and depression (from 1 per cent to 6 per cent).'"


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Rebels finish the regular season 18-13, 9-9. We'll know SEC tournament seeding information later this evening, probably between the 6 and 8 seeds.
Nice finish for Henderson and the Rebels at home.
In one of his rare visits to the media room he'd obviously been crying. Then he loosened up. Good stuff here:
Vanderbilt's Dai-jon Parker did a good job of fighting through screens and guarding Henderson. Henderson in turn did a good job of getting to the rim.
I mentioned on Twitter middle of the second half that Ole Miss would not win unless Henderson or Summers — or both — hit a couple of big shots. Henderson ultimately did but they were at the free throw line.
The Rebels were at one point below 60 percent at the free throw line for the game, but they hit their last six — four of them by Henderson — who was 6-for-7 for the game.
He hit two with 3:36 left for a 61-60 Ole Miss lead, the Rebels' first since 27-26 at 5:21 in the first half.
Ole Miss was 24-for-39 from the line for the game.
Henderson at the free throw line was basically a matter of taking what the defense was giving.
So, Henderson finishes with 18 points on 5-for-19 floor shooting, 2-for-13 from 3. Summers was 4-for-9 from the floor, 3-for-4 from the free throw line and had 12 points. Below average for both of them, but they'll take the win over the numbers.
Here's Kennedy
In many ways it was a typical Ole Miss game. There was energy and effort from the bigs. They're just not very good. A rebound margin that I think reached minus-12 during the game finished at minus-3. Probably has to do in part to Vanderbilt's depth, but Ole Miss did rebound better down the stretch.
Anthony Perez didn't score. A great talent. Sometimes he shows up, sometimes he doesn't.
The most painful part of the status quo is the bigs' inability to make shots at the rim. You heard Kennedy describe it as "agonizing, deflating." That has to be fixed if the Rebels are going to have any sort of success next season.
The energy is there, the production is not. Henderson leads Ole Miss with seven points but is only 2-for-8 from the floor, 1-for-5 from 3.
Vanderbilt was getting really good looks in the paint early and knocking down shots. The Rebels used pressure mid-court defense to go on an 11-0 run and take the lead, but Vanderbilt has surged back ahead.
Vanderbilt is 10-for-10 at the free throw line, Ole Miss 11-for-19. Vandy is plus-8 on the glass.
Ole Miss, as has become the norm this season, has missed repeated shots at the rim.
Final regular season game, and the home finale for Marshall Henderson who was just honored in a Senior Day tribute:
Game updates on Twitter.


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The revelation of the payment to her parents Sue and Gary Woodward, confirmed yesterday by the Daily Mail's managing editor, follows a categorical denial by Woodward last week that she had received any payment for her story.
The money was paid last November during the height of the trial drama unfolding in Boston, Massachusetts. On 30 October, Woodward was found guilty by a jury of the murder of baby Matthew Eappen but four days later the Daily Mail ran an exclusive interview with her parents, prompting speculation that money had changed hands.
At this point, Woodward was (and remains) a convicted criminal in the eyes of the law and a payment would be in breach of the Press Complaints Commission's code of practice which forbids payment to convicted criminals except in cases where it may be in the public interest.
The official fund for Woodward was set up on 7 December last year as the donations from wellwishers rapidly swelled to around pounds 200,000. Before that, a more informal fund set up by a family friend, Jean Jones, had handled the money, most of which flooded in to the Rigger public house in Woodward's home village of Elton in Cheshire and which was designed to fund her defence and family visits to the United States.
It remains unclear just where the money from the Daily Mail went. None of the original fund organisers, nor those who replaced them in December, can say whether it was put into the general pot of donations handled by Mrs Jones, whether it was used to pay off some of Woodward's bills or whether her parents retained it. Woodward's mother, Sue, has consistently denied receiving any money from newspapers.
In Elton, the Rev Tim Barker, speaking on behalf of the Rev Ken Davy, chairman of the Woodward Trust Fund, said yesterday: "No money from a newspaper or any other media has been paid to the trust since it was formed on 7 December last year."
He said that donations had come from all over the world and there may well have been a proper record kept. But, he added the trustees were not able to comment on payments made before the trust was set up.
Mrs Jones, who wrote a scathing letter to the trustees when the new trust fund was set up in December, was unavailable for comment. However, earlier this month she said that money "became God" to Sue Woodward as the worldwide appeal gathered momentum.
She alleged that while initially Mrs Woodward's sole concern was for her daughter, later, her perspective changed and said her former friend had ridden "roughshod" over those who had helped her.
A week earlier, Mrs Woodward denied an allegation that she had forged an invoice from her daughter's former solicitor, Elaine Whitfield Sharp.
Sandra McCabe, a neighbour and key member of the support group, said that she did not know the facts of the latest row and did not want to discuss it. Yesterday she was reported as saying: "Sue always said she was not going to get anything in all of this."
The payment was confirmed by the Daily Mail's managing editor, Lawrence Sear, who said it was made because the family "considered it necessary to receive payment in order to help fund their legal battle".
The Woodwards have consistently denied seeking to profit or receiving payment in relation to the case. On 12 November, two days after the guilty verdict was commuted to one of manslaughter, Woodward said: "I have heard rumours that I have sold my story or rights to my story to the news media. I want to set the record straight. I have done no such thing."


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Attorney & Exactly how They Work out Injury Settlements
If you have lately been hurt in a crash via no mistake of your very own, you might discover on your own in a sea of legal representatives without recognizing which one to pick. After being harmed, several individuals count on attorneys for assistance in safeguarding their rights and helping to guarantee they are relatively made up for shed salaries, clinical bills and pain and also suffering.
Depending upon the accident, clinical attention could or may not be needed. In a car crash, for example, the individual that caused the accident will certainly be accountable for any sort of repair services to the target's auto, along with the formerly pointed out settlement. Many people employ an attorney not long after the accident occurs although settlement agreements will usually not begin until after clinical therapy has ended.
After concluding medical treatment, attorneys like Gatti, Keltner, Bienvenu & Montesi will certainly begin to negotiate with the insurance coverage firm representing the individual that triggered the mishap. Throughout this time around, lawyers will evaluate the quantity of medical costs, medical professional credit reports and medical diagnosis, shed wages, problems and also will certainly try to approximate a fair amount of pain and suffering. After gaining your approval, personal injury lawyers will certainly send a proposition to the insurance coverage adjuster for consideration. It normally takes One Month for a reaction, which is anticipated to be a counteroffer. At this time, lawyers will certainly talk about the offer with their customer and also see if a reasonable settlement could be reached. If not, the attorney will prepare another letter clarifying that the insurer's offer is not appropriate as well as will, once more, apply for that the instance be settled for a slightly lower amount compared to the original suggested amount of money. This situation is an examination of each side as the insurer wishes to resolve the case for as little as feasible while the legal representatives intend to see to it their customers are correctly compensated. After numerous letters back and forth, as well as perhaps some telephone calls, the legal representatives will certainly advise their customer when a reasonable offer has been gotten to. While they can not instruct their client on what to do, they can suggest them on the justness of the deal and also how that number might transform if the instance were to proceed to trial.
For factors of both time and expense, the majority of injury situations are cleared up from court. Once a test is given, lawyer costs as well as court boost dramatically. A lawyer obtains a pre-negotiated amount of the customer's negotiation, yet gets a bigger percent if the situation moves to test. There are a number of attorneys who handle car accident situations and it is constantly vital to choose one that is most sincere and also seems most well-informed concerning the process.
The details in this short article is to be used for informative purposes only. It ought to not be considered as, or used instead of, specialist lawful guidance. Individuals that need lawful assistance ought to seek advice from among the attorneys in their area that can assist them with their inquiries as well as issues.


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Chateau Z Vineyard
Contact Information
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Lynchburg, VA 24504 - Web
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- Mon-Sun: No hours specified
- NOT OPEN TO THE PUBLIC. Wine can ONLY be obtained at the Lynchburg Community Market.
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Wineries / Vineyards Near Chateau Z Vineyard
De Vault Family Winery - Concord, VA - 10.3 miles
Neala Estate Vineyards - Madison, VA - 12.9 miles
Lazy Days Winery - Amherst, VA - 15.6 miles
Leo Grande Vineyards & Winery - Goode, VA - 15.7 miles
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This winery information last updated: Jul 8, 2015


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"All growth depends upon activity. There is not development physically or intellectually without effort, and effort means work."
-Calvin Coolidge
Former United States President Calvin Coolidge knew a thing or two about growth. From a personal perspective, his life embodied steady growth of achievement. His first foray into politics began in the Massachusetts House of Representatives in 1907. He followed this up as Mayor of Northhampton, then became a member of the Massachusetts Senate, and after a couple of more stops became Governor of Massachusetts.
"Silent Cal", as he was called due to his quiet demeanor, was then added to the Republican ticket as Vice President in 1920 and he and his running mate, Senator Harding of Ohio, went on to win in a landslide. On August 2nd, 1923, President Harding died while on a speaking tour and Coolidge became President. Coolidge then stood for election as President in 1924 and won his first official term as President.
From an economic perspective, largely based on a series of broad tax cuts, Coolidge oversaw a very economically prosperous time in U.S. economic history known as the "roaring 20s". Interestingly, as well, as the top tax rates were cut from 58% in 1922 to 25% in 1929, the economy grew and the share of the tax burden of the wealthiest Americans, those making more than $100,000 per year, also grew from 35% to 63%.
In addition to the economic growth he oversaw, Coolidge also eventually outgrew his introverted personality. In one his most outspoken moments, he said of his eventual successor Herbert Hoover, "for six years that man has given me unsolicited advice—all of it bad." Needless to say, the United States was not as economically fortunate under the stewardship of his successor President Hoover.
Back to the global macro grind . . .
Yesterday was a classic one for the ongoing debate over whether economic growth in the U.S. is accelerating or stagnating. The Purchasing Managers Index (PMI) reading from Institute for Supply Chain Management (ISM) came in at a contraction indicating 49.0. The internals of the report were negative across the board, as well, with new orders coming in at 48.8 and production coming in at 48.6. The employment component of the index was still in expansion mode, albeit only marginally at 50.1.
The key read through from this reading is that as it relates to growth in the industrial sector in the U.S., headwinds remain. On a relative basis, the United States is still faring better than the Eurozone where a 48.3 was reported in its latest PMI report, the latest in almost two straight years of factory output contraction in Europe. The Chinese economy is also struggling on the industrial front as the HSBC PMI came in at 49.2 most recently.
Another data point that came out yesterday that supported the slowing growth case was government spending on construction. Public construction spending dropped 1.2% in April to the lowest level since 2006 and down 5.7% from October. This decline is obviously due to sequestration that is being implemented at the federal level. In the short term, this may be an economic headwind, though the offset is that the deficit is declining much faster than expected. In its most recent update the non-partisan Congressional Budget Office (CBO) projected that the deficit for fiscal year 2013 will fall to $624 billion, or about 4% of GDP, and almost $200 billion less than the CBOs estimate from three months before.
Our view of economic growth in the U.S. continues to be underscored by the consumer side of the economy. On this front, the economic data released yesterday was positive as auto sales for May came in at an annualized rate of 15.3 million. This was the fourth straight month of sales over 15.0 million and continues to show strong growth over the 14.5 million in auto sales from last year. Certainly, auto sales are being driven by compelling financing programs, but as a proxy for consumer demand, they remain a positive indicator.
A key emerging American growth industry that will be a key tailwind for strong car production numbers is the U.S. energy industry. In the Chart of the Day, we highlight this in a chart of U.S. oil production going back twenty years. As the chart shows, largely thanks to technology advances, U.S. daily oil production is hitting 20-year highs. This of course follows decades of production declines starting in the 1970s.
The International Energy Administration recently published a report that projected the North American oil supplies will grow by 3.9 million barrels by 2018. This is almost 2/3rds of the non-OPEC production growth projected over that period. If accurate, this will also reduce American imports by almost 40% over that period. If the IEA is correct and this growth in production leads to a global "supply shock", in coming years, the upside to global growth may be dramatic with declining oil prices.
Turning back to the shorter term and the U.S. stock market, another key positive we see relating to growth is expectations. Currently, consensus aggregate SP500 revenue growth for the next three quarters is 0.5%, 3.4% and 2.3% respectively. This is an expected revenue growth rate of just over 2.0% in the projected period and less than half the average reported year-over-year growth rate of north of 4% in the prior three quarters. For U.S. stock market bulls, these low expectations are very supportive. For U.S. stock market bears, these expectations may well be, as Shakespeare said, "the root of all heart ache."
Our immediate-term Risk Ranges for Gold, Oil (Brent), US Dollar, USD/YEN, UST 10yr Yield, VIX, and the SP500 are now $1, $100.27-103.29, $82.48-83.74, 99.98-103.24, 2.07-2.23%, 14.63-16.59, and 1, respectively.
Daryl G. Jones
Director of Research
SJM STILL LEADER OF CASINO MARKET SHARE Macau Business
May GGR shares: SJM: 23%, LVS: 21%, Galaxy: 19%, MPEL: 14%, WYNN: 12%, and MGM: 11%.
Galaxy says it is looking for acquisitions in Macau. Francis Lui Yiu-tung, the group's deputy chairman, said most of the HK$3.6 billion additional budget (HK$ 19.6 billion in total) for the second-phase expansion of Galaxy Macau was used for upgrading decor and facilities to "better cater to market demand".
Lui said the company was adding more slot machines for the mass market. For Grand Waldo, which the group recently acquired for HK$3.25 billion, Lui said it would be positioned differently from Galaxy. He added that the company had not yet decided whether Grand Waldo would focus on the mass market while Galaxy Macau catered to high-end gamblers. The acquisition is set to be completed in the next quarter.
Galaxy group is also considering other purchases. "Land is the most difficult thing to come by. If we find any good investment opportunity around Galaxy facilities in Cotai, we will expand," said Lui.
Tokyo Governor Naoki Inose said he intends to set up a casino in the capital's waterfront district, even though operating such gambling establishments is currently illegal in Japan. "I expect the Diet to revise the law as soon as possible," he said at the metropolitan assembly session.
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This note was originally published at 8am on May 21, 2013 for Hedgeye subscribers.
"A system can become locally ordered at the expense of a global increase in entropy."
-Eric Chaisson
First, my family's thoughts and prayers go out to the those personally affected by the natural disaster in Oklahoma.
Last week I wrote a note titled Sovereign Yield Risk that generated a lot of feedback. Since we put the Hedgeye platform at the heart of a wide open global network, feedback has become our greatest asset. Our research team has its own internal pipes of communication, but they don't work unless we connect them to our client pipes and the new highway of dynamic information flow: #Twitter.
Both information and asset allocation flows matter to us, big time. Alongside price and volatility, they are critical factors that help us risk weight the probability of new bursts of entropy into the Global Macro matrix. Japanese Government Bond Yields breaking out above our long-term TAIL risk line would qualify as a new burst; so would a move toward 2.4% in 10yr US Treasury Yields.
Back to the Global Macro Grind…
The recent 1-month move in both JGBs (we're short them) and US Treasury Yields are 2 of the 3 most important things in my notebook this morning. The 3rd is gold. And all 3 of these major macro factors are interconnected to a causal factor with a catalyst.
Let's review what I am looking at this morning:
- Japanese Government Bond Yields (10yr JGBs) = up another +5 bps to 0.89% this morning; +31bps in the last month
- US Treasury Yields (10yr) = up +1 basis point this morning to 1.96%; +25bps in the last month
- Gold continues to crash from its 2011 #BernankeBubble top, backing off -0.5% this morning after a 1-day dead cat bounce
As always, contextualizing these moves across our multi-duration model matters too:
- JGB long-term TAIL risk line = 0.81% (so we're breaking out above that)
- UST 10yr long-term TAIL risk line = 1.82%
- Gold snapped its long-term TAIL risk line of $1681 in January (not new)
You can ignore the entropy associated with 1 or 2 of these TAIL lines snapping (I hope you didn't ignore our Gold signal 6 months ago), but it's really hard to ignore all 3 of them; especially when the mother of all bursts of entropy (#StrongDollar) is in motion.
What matters most in macro is what happens on the margin. That's why Ben Bernanke acknowledging what we have been signaling on employment, housing, and consumption #GrowthAccelerating will matter in his testimony to Congress tomorrow. That's your catalyst.
To be fair to the #EOW (end of the world) guys, their thesis remains what ours was during Bernanke's 2010-2012 QE marketing campaign. Consensus doesn't think we will ever have real (inflation adjusted) growth in the USA again primarily because QE didn't deliver it.
Ironically, but not surprisingly, the end of QE is the economic catalyst we've all been waiting for. #StrongDollar, Strong America.
To review the flow show:
1. Expectations for incremental QE fade
2. #StrongDollar manifests; Gold crashes
3. Bond Yields rise
Like the thermodynamics of water flowing toward (and over) a damn, the flow show is happening in a locally ordered pattern – and the global burst of entropy (the waterfall) is going to be very hard to stop.
I don't think mother Merrill explains flows this way, but that's cool – I just want them to keep telling their clients to sell Gold, Treasuries, and Japanese Government Bonds so that they don't get run-over by the only centrally planned bubbles that are left.
Mr. Macro Market gets this – look at the most recent burst of immediate-term entropy (3 week correlations):
- US Dollar vs SP500 = +0.91
- US Dollar vs 10yr UST Yield = +0.92
- US Dollar vs Gold = -0.87
And since 3 weeks don't matter to "long-term" investors, what if you contextualize 3 weeks within a 6 month TREND?
- US Dollar vs SP500 correlation (on a 6 month duration) = +0.79
- US Dollar vs 10yr UST Yield correlation (6 months) = +0.11
- US Dollar vs Gold correlation (6 months) = -0.78
In other words, one of these 3 things (UST Treasury Yields) does not look like the others (SP500 and Gold) on a 6-month duration, yet. But that's precisely the risk management point – the probability of US Treasuries and JGBs correlating with #StrongDollar at an accelerating rate is now going up, not down. That's new.
Can Bernanke not acknowledge both the economic growth stabilization of the last 6 months and the recent acceleration in US employment, housing, and consumption growth? Sure. I can have some IRS dude tell me the sun doesn't rise in the East too – but that doesn't mean I (or the market) has to believe them.
Our immediate-term Risk Ranges for Gold, Oil (Brent), US Dollar, USD/YEN, UST 10yr Yield, VIX, Russell2000, and the SP500 are now $1339-1421, $101.27-105.31, $83.49-84.75, 101.42-104.48, 1.90-2.02%, 12.22-13.79, 980-1004, and 1647-1678, respectively.
Best of luck out there today,
Keith R. McCullough
Chief Executive Officer
Takeaway: 30Y Mortgage rates backed up 35 bps in the last week and 70bps in the last month. Refi activity and affordability have taken a hit.
This note was originally published June 03, 2013 at 13:56 in Macro
Last week saw a significant back up in 30Y Residential Mortgage Rates as the national average rose 35 basis points W/W to 4.10% from 3.75% the week prior. From the near historical low of 3.40% reached back on May 1st, we've seen an expedited 70 bps backup in rates over the last month.
The move in rates holds a few notable impacts for housing. First, the increase in mortgage rates should have a (unsurprisingly) significant, negative impact on refinancing activity – something that has already manifest in the MBA mortgage application data with refi activity down 12.3% in the last week and 23% over the last month. This contrasts with Purchase Activity, which was actually up 2.6% in the latest week and down just 2.3% over the last month.
While we believe the positive Giffen cycle in housing (see Here for fuller discussion) should continue to predominate with demand chasing higher prices in a reflexive fashion, higher interest rates have a direct, negative impact on housing affordability.
Previously, we have shown (Here, slide 49) that under standard median income and DTI assumptions for a 30Y Freddie Mac Mortgage loan, a 10bps change in rates equates to an approximate 1.0% change in affordability. A continued rise in interest rates would serve as a headwind to a further acceleration in home values, particularly as HPI growth comparison's get steeper.
While we would view the breakout in treasury yields alongside the material sector level performance divergences (XLF +6.1%, XLU -9.0% in May) as pro-growth signals, with the housing recovery a key tenet underpinning our domestic growth outlook, we'll certainly be monitoring rate impacts on affordability closely here.
Christian B. Drake
Senior Analyst
TODAY'S S&P 500 SET-UP – June 4, 2013
As we look at today's setup for the S&P 500, the range is 21 points or 0.64% downside to 1630 and 0.64% upside to 1651.
- YIELD CURVE: 1.83 from 1.83
- VIX closed at 16.28 1 day percent change of -0.12%
MACRO DATA POINTS (Bloomberg Estimates):
- 7:45am/8:55am: ICSC/Redbook weekly retail sales
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- National Transportation Safety Board considers safety study on single-unit truck accidents, 9:30am
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- Royalty Pharma takeover of Elan temporarily blocked by judge
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- Ackman reaping 3-fold gain will shrink Canadian Pacific stake
- Australia's central bank said still has room to cut benchmark interest rate
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- U.K. construction unexpectedly resumes expansion last month
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Michael Winner is an English film director and producer, active in both Europe and the United States, also known as the food critic for the Sunday Times.
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(9 quotes found)
"A team effort is a lot of people doing what I say."
Michael Winner
"Calm down dear, its a commercial."
"The truth of the matter is that muggers are very interesting people."
"The truth of the matter is that muggers are very interesting people"
"During the Sixties, the Americans thought I was the greatest thing in the history of cinema."
"I think it's rather an insult to dogs. Men are awful: arrogant, nasty creatures."
"What? What!!!! Where is Rupert, I must call him."
"He was a very good actor but he wasn't used as an actor as much as he should have been because he became famous as Peter Ustinov,"
"took me to Hollywood"


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FedEx® International Priority Freight
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Senior Research Associate
School of Computer Sc, Engineering & Mathematics
Aaron has a strong interest in the application of electronics in biomedical devices and instrumentation. He has previously been involved in teaching courses in the field of electronics and computer architecture at the University of South Australia, where his involvement spanned 6 years and included teaching/supervising at undergraduate and postgraduate levels.
His interests cover electronic circuits, intelligent robotic control via microcontrollers and the application of microelectronic circuitry in biomedical devices.
PhD, University of South Australia 2009. Thesis title: A Remote Laboratory for Testing Microelectronic Circuits on a Silicon Wafer.
BEng (Electronics and Microengineering)(Hons), University of South Australia 2006.
Aaron currently works as a research associate at MDPP (Medical Device Partnering Program). His role involves research and development of biomedical devices and instrumentation at various stages in their product life cycle.
Project areas that he has worked in include:
Team TREV (Two-seater Renewable Energy Vehicle)
Volunteer: Motor/driver instrumentation/lighting control and CAN communication (2009 - current)
South Australian Multicultural Youth Ambassadors (SAMYA)
Chairman/Public Officer/Founding member (2009 - 2011)
South Australian Multicultural and Ethnic Affairs Commission (SAMEAC), Youth Advisory Committee
Committee member (2007 - 2011)
Students in Free Enterprise (SIFE)
Presenter/Faculty advisor (2005 - 2008)
Robotics Peer Mentoring (RPM)
Mentor/Mentor trainer (2003 - 2007)
UniSA IEEE Student Branch
Chairman (2005)
Mawson Lakes Rostrum Club
Secretary/President (2003 - 2005)
Phone: | +61 8 82017763 |
Email: | |
Location: | Tonsley (5.07) |
Postal address: | GPO Box 2100, Adelaide 5001, South Australia |


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I am from Batavia, OH and I've been a Club Horse member for 4 years, 1 month and 21 days
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Vertex China SAU-55-WB Sausilito Saucer For Sau-54/Sau-54C - 3 doz
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Conceived and designed the experiments: SP CRH. Performed the experiments: SP LP KK JDT NNJ CRH. Analyzed the data: SP LP KK JDT NNJ CRH. Wrote the paper: CRH.
CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake.
In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria.
Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.
Several human-restricted pathogens target surface receptors of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family to contact their host [1]. In particular, CEACAM1, CEACAM3, CEA (the product of the CEACAM5 gene), and CEACAM6 can serve as microbial receptors. As CEACAM-recognition has evolved independently in multiple Gram-negative bacteria including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae and N. meningitidis, as well as uropathogenic Escherichia coli, this trait seems to confer a selective advantage to these microbes in vivo [2]. All CEACAM-binding bacteria colonize the mucosal surface of the human nasopharynx, intestine or urogenital tract, where CEACAM family members are expressed on the apical membrane of polarized epithelial cells [3]. In vivo experiments employing a humanized mouse model have demonstrated that CEACAM recognition can strongly promote the initial establishment of microorganisms on the mucosa [4]. In this context, bacterial stimulation of CEACAMs enhances the adhesiveness of the infected host cells, thereby suppressing the exfoliation of the superficial epithelial layer [4], [5].
On the other hand, CEACAM family members such as CEACAM1, CEACAM3, or CEACAM6 are found on various hematopoietic cells. A common splice variant of CEACAM1, which is present on mature B-cells and CD4-positive T-cells, contains two immunoreceptor tyrosine-based inhibition motifs (ITIMs) and has been reported to interfere with antibody production by B-cells [6], [7] or CD4 T-cell proliferation, respectively, upon stimulation by CEACAM-binding bacteria [8]. Though this report has been challenged by recent observations that both CEACAM-binding and non-binding N. gonorrhoeae stimulate T-cell proliferation and cytokine secretion to the same extent [9], engagement of CEACAM1 on immune cells by pathogenic microbes might interfere with some effector functions of hematopoietic cells and thereby provide a selective advantage [10]. Interestingly, human granulocytes express a peculiar member of the CEACAM family, CEACAM3, which shares a high degree of homology in its extracellular, bacteria-binding part with CEACAM1, CEA, and CEACAM6 [11]. In contrast to CEACAM1, the CEACAM3 cytoplasmic domain encompasses a tyrosine-based sequence that is reminiscent of an immunoreceptor tyrosine-based activation motif (ITAM). Canonical ITAM sequences are found in the cytoplasmic parts of T-cell and B-cell receptor as well as Fc gamma receptor (FcγR) subunits [12]. ITAMs are critical for transducing stimulatory signals and, in the case of phagocytes, promote bactericidal activities of these effector cells [13]. Interestingly, engagement of CEACAM3 by bacteria has been shown to result in rapid phagocytosis and elimination of the bacteria in an acid intracellular compartment [14], [15]. Therefore, CEACAM3-mediated recognition and opsonin-independent phagocytosis might be a specific adaptation of the human innate immune system to balance the colonization of mucosal surfaces by CEACAM-binding microbes. In line with the idea that CEACAM3-mediated engulfment by granulocytes is to the disadvantage of the recognized microbes, several studies have demonstrated that this uptake process is mechanistically distinct from CEACAM1, CEACAM6 or CEA-mediated internalisation [16]–[19]. For example, CEACAM3-mediated internalisation strictly depends on actin cytoskeleton dynamics and does not involve cholesterol- and sphingolipid-rich membrane microdomains [18], [19]. Moreover, whereas CEA and CEACAM6 are glycosylphosphatidyl-inositol-anchored proteins and CEACAM1 lacking the cytoplasmic domain is competent for bacterial uptake, cytoskeletal rearrangements and efficient phagocytosis via CEACAM3 depend on the integrity of the cytoplasmic ITAM-like sequence [15], [16], [19], [20]. Starting with receptor clustering by multivalent bacteria, several steps in the CEACAM3-initiated signalling pathway have been delineated in primary granulocytes and CEACAM3-transfected cell lines. Most importantly, tyrosine phosphorylation of the ITAM-like sequence by Src-family protein tyrosine kinases (PTKs) appears as the initial event, which guides the assembly of a transient signalling complex and which is blocked by pharmacological inhibitors of Src family kinases [17], [18], [21]. Upon phosphorylation of tyrosine residue Y230 within the ITAM-like sequence, this phospho-tyrosine serves as a docking site for the guanine nucleotide exchange factor (GEF) Vav [22]. Direct association of the CEACAM3 ITAM with the SH2 domain of Vav provides a short-cut between receptor engagement and GTP-loading of the small G-protein Rac, which is a critical regulator of actin polymerization. Accordingly, dominant-negative versions of Rac, but not the closely related G-protein Cdc42, severely reduce opsonin-independent phagocytosis of CEACAM-binding bacteria by primary human granulocytes [15]. In line with a central role of Rac in this process, a rapid increase in GTP-loaded Rac as well as the formation of large lamellipodial protrusions is observed in granulocytes infected with CEACAM-binding gonococci [15], [23]. However, which Rac-dependent effectors contribute to actin polymerization in the vicinity of CEACAM3-bound bacteria and how these effectors are recruited to the sites of bacterial uptake is currently unknown.
In this study, we identified the adaptor molecules Nck1 and Nck2 as novel interacting partners of CEACAM3. Biochemical analyses demonstrated that Nck1 and Nck2, but not other adapter molecules such as Grb2, CrkL, or SLP-76, bound to the ITAM-like sequence of CEACAM3 in a phosphorylation-dependent manner. In microscopic investigations, Nck1 clustered together with the receptor at sites of bacterial uptake and overexpression of the isolated SH2-domain of Nck1, siRNA-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfered with CEACAM3-mediated phagocytosis and the generation of lamellipodial protrusions. Interestingly, Nck was found in a CEACAM3-containing complex together with the Rac effector WAVE2, which promoted f-actin polymerization during uptake of CEACAM3-associated bacteria. Interference with WAVE complex function by RNAi or overexpression of dominant-negative WAVE2 reduced CEACAM3-mediated internalization. These results suggest that CEACAM3-associated Nck helps to bring together Rac effector molecules such as WAVE with receptor-bound Rac activators such as the GEF Vav to promote the local actin cytoskeleton rearrangements necessary for the opsonin-independent internalisation of CEACAM-binding bacteria.
Neisseria gonorrhoeae strain MS11 lacking pili and constitutively expressing OpaCEA (Opa52; strain N309) was kindly provided by Thomas Meyer (Max-Planck-Institut für Infektionsbiologie, Berlin, Germany). Gonococci were cultured as described previously [15]. For gentamicin protection assays, overnight grown bacteria were taken from GC-agar plates supplemented with vitamins. For microscopy or uptake assays by flow cytometry, bacteria were labelled as indicated with either 0.2 µg/ml Pacific Blue-succinimidylester, Carboxyfluorescein-succinimidylester (CFSE), Rhodamine-Red-succinimidylester, or AlexaFluor647- succinimidylester (Invitrogen, Carlsbad, CA) as described [22].
Human embryonic kidney epithelial 293T cells (293 cells; ACC-635, German collection of microorganisms and cell cultures, DSMZ, Braunschweig, Germany) were cultured in DMEM supplemented with 10% calf serum (CS). HeLa cells stably expressing CEACAM3 [22] were provided by W. Zimmermann (Tumor Immunology Laboratory, LMU München, Germany) and cultured in DMEM supplemented with 10% fetal calf serum (FCS). Nck1 and Nck2-deficient mouse fibroblasts (Nck1−/−Nck2−/−cells) and the heterozygous control cells (Nck1+/−Nck2+/−cells) were described previously [24] and were provided by T. Pawson (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada). Fibroblasts were cultured in DMEM supplemented with 10% fetal calf serum (FCS) and non-essential amino acids on gelatine-coated culture dishes. All cells were subcultured every 2–3 days. Cells were counted and assayed for viability (>90%) with a Casy Cell Counter (Innovatis, Bielefeld, Germany). 2.5×105 cells were seeded into each well of a gelatine-coated 48 well plate for gentamicin protection assays. For confocal laser scanning microscopy, 7.5×104 cells were seeded in 24 well plates on glass cover slides coated with a mixture of fibronectin (4 µg/ml) and poly-L-lysine (10 µg/ml) in PBS.
Plasmids encoding CEACAM3-HA wildtype (WT) or the CEACAM3 mutant lacking the complete cytoplasmic domain (CEACAM3 ΔCT) and CEACAM3 WT-RFP were described previously [15], [22]. The HA-tagged CEACAM3 WT or CEACAM3 ΔCT were amplified using primers CEACAM3-IF-sense 5′-GAAGTTATCAGTCGATACCATGGGGCCCCCCTCAGCC-3′ and CEACAM3-IF-antisense 5′-ATGGTCTAGAAAGCTTGCAGCGTAATCTGGAACGTCATATGG-3′ and cloned with the InFusion cloning kit into pDNR-dual (Clontech, Mountain View, CA). CEACAM3 variants with an additional carboxy-terminal green-fluorescent protein (GFP), mCerulean, or mKate tag were generated by Cre/lox recombination from pDNR-dual into pLPS3'EGFP, pLPS3'mCerulean, or pLPS3'mKate as described [21]. pLPS3'mCerulean-loxp and pLPS3'mKate-loxp were constructed by replacement of the EGFP coding sequence in pLPS3'EGFP (Clontech, Mountain View, CA). mCerulean cDNA (kindly provided by D. Piston, Department of Molecular Physiology and Biophysics, Vanderbilt University, TN) was amplified using primers 5′-ACTACCGGTCGTGGTGAGCAAGGGCGAG-3′ and 5′-ACTGCGGCCGCTTATTTGTACAGTTCGTCC-3′, whereas mKate cDNA (kindly provided by D. Chudakov, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) was amplified using primers 5′-ACTACCGGTCGTGTCTAAGGGCGAAGAG-3′ and 5′-ATCGCGGCCGCTTAATTAAGTTTGTGCCCCAG-3′. The PCR fragments were inserted into the AgeI/NotI sites of pLPS3'EGFP (Clontech, Mountain View, CA).
All SH2 domain containing constructs were cloned as PCR fragments into pDNR-Dual with the InFusion cloning-Kit and subsequently transferred to pEGFP-loxp or pGEX4T1-loxp by Cre/lox recombination. Clones encoding human cDNAs of Nck1 (hNck1, IMAGp958E061170Q2), Nck2 (hNck2, IMAGp958A12182Q), hCrkL (IMAGp998N1813292Q3), hGrb2 (IMAGp958J21133Q), hSLP-76 (IMAGp998E1110303Q3) and c-Src (IMAGp958B161238Q2) were obtained from Imagenes (Berlin, Germany). The SH2 domain of Nck1 (hNck1-SH2) was amplified using 5′-GAAGTTATCAGTCGACAAGTTTGCTGGCAATCCTTGG-3′ and 5′-ATGGTCTAGAAAGCTTCAGCAGTATCATGATAAATGCTTGAC-3′. Point mutation for hNck1R308K-SH2 was introduced by site-directed mutagenesis using primers 5′-GAAGGGGATTTCCTTATAAAAGATAGTGAATCTTCGCCAAATG-3′ and 5′-CGAAGATTCACTATCTTTTATAAGGAAATCCCCTTCATGTCC-3′. Full length hNck1 was amplified using 5′-GAAGTTATCAGTCGACATGGCAGAAGAAGTGGTGGTAG-3′ and 5′-ATGGTCTAGAAAGCTTCAGCAGTATCATGATAAATGCTTGAC-3′ and inserted into pDNR-Dual for subsequent recombination into pLP-CMV-myc (myc-Nck1) and pEGFP-loxp (GFP-Nck1). hNck2-SH2 domain was amplified using primers 5′-GAAGTTATCAGTCGACCCCTCGTCCAGCGG-3′ and 5′-ATGGTCTAGAAAGCTTACTGCAGGGCCCTGACG-3′, hGrb2-SH2 domain using primers 5′-GAAGTTATCAGTCGACCCCAAGAACTACATAGAAATG-3′ and 5′-ATGGTCTAGAAAGCTTAGTATGTCGGCTGCTGTG-3′, and hCrKL-SH2 domain using primers 5′-GAAGTTATCAGTCGACATGTCCTCCGCCAGGTTCG-3′ and 5′-ATGGTCTAGAAAGCTTGCAATCACTCCTTTTCATCTGGG-3′. Cloning of human c-Src and SLP-76 SH2 domains was described [18], [22].
Human WAVE2 cDNA was kindly provided by T. Stradal (Helmholtz Centre for Infection Research, Braunschweig, Germany). WAVE2 was amplified using primers WAVE2-IF-sense 5′- GAAGTTATCAGTCGACATGCCGTTAGTAACGAGGAACATCG-3′ and WAVE2-IF-anti 5′-ATGGTCTAGAAAGCTTTATTGGTCGGACCAGTCGTCCTC-3′. WAVE2ΔVCA was amplified using primers WAVE-IF-sense together with WAVE2ΔVCA-IF-anti 5′-ATGGTCTAGAAAGCTTTAGGCATCGCTCACGGCAGGC-3′. PCR fragments were cloned into pDNR-dual and the transferred by Cre/lox recombination into pEGFP-loxp (GFP-WAVE2-WT, GFP-WAVE2-ΔVCA) or pLP-CMV-myc (myc-WAVE2-WT, myc-WAVE2-ΔVCA), respectively.
Monoclonal antibody (mAb) against CEACAMs (clone IH4Fc) was from Immunotools (Friesoythe, Germany), mAb against GFP (clone JL-8) was from Clontech (Mountain View, CA), mAbs against v-Src (clone EC10) and against phospho-tyrosine (4G10) were from Upstate Biotechnology (Lake Placid, NY), mAb against GST (clone B-14) was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against Nck1/2 (clone 108) was from BD Transduction Laboratories (Heidelberg, Germany), rabbit monoclonal antibody against WAVE (clone D2C8) was from Cell Signalling Technology (Danvers, MA). Polyclonal rabbit antibody against GFP was produced in the local animal facility at the University of Konstanz, rabbit polyclonal antibody against Nap1 (Hem2, NckAP1) was from Abcam (Cambridge, UK). Polyclonal antibody IG511 against pathogenic Neisseria has been described previously [22]. MAbs against the HA-tag (clone 12CA5), myc-tag (clone 9E10), and tubulin (clone E-7) were purified from hybridoma cell supernatants obtained from DSHB (University of Iowa, IA). GST and GST-fused SH2-domains used were all expressed in E. coli BL-21 and purified using GSTrap FF (Amersham Biosciences, Freiburg, Germany). Protein A/G sepharose was obtained from Santa Cruz Biotechnology. Streptavidin-Alexafluor647 and Phalloidin-Alexafluor546 were obtained from Molecular Probes/Invitrogen (Carlsbad, CA). Secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA). NHS-Biotin was obtained from Pierce Biotechnology (Rockford, IL).
293 cells were transfected by calcium phosphate precipitation using 3 µg of CEACAM constructs or empty vector control. For co-transfection, 5 µg of co-transfected constructs together with 3 µg of CEACAM constructs were used and in all samples total DNA was adjusted to 8 µg using the empty control vector. Cell lysis and Western blotting were performed as described [15]. CEACAM3-expressing HeLa cells were transfected with a combination of two distinct siRNAs directed against human Nck1 at 5 nM (Mission predesigned siRNA, Sigma Aldrich, St. Louis, MO) using Turbofect reagent (Fermentas, St. Leon-Rot, Germany) according to manufacturers protocol. For plasmid transfection, HeLa cells and murine fibroblasts were transfected with Lipofectamin 2000 plus according to manufacturers recommendations (Invitrogen, Carlsbad, CA) and used in assays 48 h after transfection.
Gentamicin protection assays were conducted as described [15]. Cells were seeded in gelatine-coated 48 well dishes at 2.5×105 cells/well. A multiplicity of infection of 20 bacteria per cell was routinely used and after 30 minutes of infection, extracellular bacteria were killed by 45 min incubation in 50 µg/ml gentamicin in DMEM. Binding of bacteria to the cells was determined after 30 min of infection and three washes with PBS. Cells were lysed with 1% saponin in PBS for 15 min. The samples were diluted with PBS and the number of viable bacteria was determined by plating suitable dilutions in duplicate on vitamin supplemented GC-agar.
For GST-pull downs, 3 µg of purified GST or GST-fusion protein attached to glutathione-sepharose were added to WCLs and incubated for 4 h at 4°C. After four washes with RIPA buffer, precipitates were boiled in SDS sample buffer, before SDS-PAGE and Western blot analysis. For co-immunoprecipitations, 293 cells were transfected with the indicated combination of constructs and lysed after 48 h. For precipitation, lysates were incubated with 3 µg of polyclonal rabbit anti-GFP antibody over night followed by 1 h incubation with protein A/G sepharose, all at 4°C. After three washes with Triton buffer, precipitates were boiled in SDS sample buffer before SDS-PAGE and Western blot analysis.
Cells transfected with the indicated constructs were grown on glass coverslips in 24-well plates and infected for 30 min with PacificBlue-labelled and biotinylated (NHS-Biotin) OpaCEA-expressing N. gonorrhoeae at an MOI of 40. Samples were fixed with 4% paraformaldehyde in PBS and washed three times with PBS++ (PBS with additional Ca2+ and Mg2+), prior to incubation in blocking buffer (PBS++, 10% FCS) for five minutes. Biotinylated bacteria were stained with streptavidin-Alexafluor647 prior to permeabilization. Epitope-tagged constructs were visualized in a 2-step process including detection with specific mAbs (against HA-tag or myc-tag) and staining with Cy5-coupled secondary antibody (goat-anti-mouse-Cy5). Samples were rinsed twice with PBS++ and cells permeabilized with blocking buffer containing 0.2% saponin before incubation with antibodies. Visualisation of the actin cytoskeleton was achieved using Phalloidin-Alexafluor546. Samples were viewed with a Leica SP5 laser scanning confocal microscope (Leica, Wetzlar, Germany) using a 63×, 1.3 NA Plan Neofluar oil-immersion objective. Fluorescence signals of multicolored specimens were serially recorded with appropriate excitation and emission settings to avoid bleed-through. Images were digitally processed with ImageJ and merged to yield pseudo-coloured pictures.
Nck1+/−Nck2+/−cells or Nck1−/−Nck2−/−cells were transfected with CEACAM3-GFP encoding plasmid. Transfected cells were grown on CeLLocate coverslips (Eppendorf, Hamburg, Germany) in 24-well plates and infected for 30 min with PacificBlue-labelled OpaCEA-expressing N. gonorrhoeae at an MOI of 40. Samples were fixed overnight at 4°C in 2% glutaraldehyde, 3% formaldehyde in 0.1 M sodium-cacodylate buffer (pH 6.9) containing 0.09 M sucrose, 0.01 M CaCl2, 0.01 M MgCl2. The fixed samples were washed with 0.1 M sodium-cacodylate buffer and quickly analyzed with a Leica SP5 laser scanning confocal microscope (Leica, Wetzlar, Germany) as described above. Subsequently, the samples were dehydrated in a graded series of ethanol followed by critical point drying with CO2 and sputter-coating with 5 nm gold-palladium in a BAL-TEC SCD 030. The regions of interest were re-located according to the grid on CeLLocate coverslips and analyzed on a Zeiss Auriga CrossBeam Workstation operating at 15 kV using the secondary electron detector. Correlation of confocal and SEM images was achieved using Adobe Photoshop CS5.
Recombinant lentiviral particles were generated using the plasmids pLKO.1, pLKO.1-shScrambled (containing a non-targeting control shRNA-sequence), pMD2.G, and psPAX2 provided by Addgene (www.addgene.org) and maintained in E. coli STBL4 (Invitrogen, Carlsbad, CA). Using the algorithm AAGN18TT (available online at http://jura.wi.mit.edu/bioc/siRNAext/) we identified sequences that could silence expression of human WAVE2 or Nap1 (NCKAP1, HEM2), respectively. According to this prediction, complementary primers were synthesized: hWAVE2-shRNA-sense 5′- CCGGAAGCAACCAAAGATATATCCACTCGAGTGGATATATCTTTGGTTGCTTTTTTTG-3′ and hWAVE2-shRNA-anti 5′- AATTCAAAAAAAGCAACCAAAGATATATCCACTCGAGTGGATATATCTTTGGTTGCTT-3′; Nap1: hNap1-shRNA-sense 5′-CCGGAAGAGCAGCAGCATATGTTGTCTCGAGACAACATATGCTGCTGCTCTTTTTTTG-3′ and hNap1-shRNA-anti 5′-AATTCAAAAAAAGAGCAGCAGCATATGTTGTCTCGAGACAACATATGCTGCTGCTCTT-3′. The oligos were annealed and cloned into the AgeI and EcoRI site of pLKO.1 generating pLKO.1-shWAVE2 and pLKO.1-shNap1, respectively. The correct insertion of the shRNA cassette was verified by sequencing.
For lentiviral production, 2×106 293 cells were transiently transfected with 14 µg of the respective pLKO.1 vector together with 10 µg of packaging plasmid psPAX2 and 7 µg of envelope-coding plasmid pMD2.G. 48 h later, the virus-containing cell culture supernatant was collected, centrifuged at 2000 rpm at 4°C for 7 min and filtered through a 0.45 µm pore-size filter. 5 ml of the cleared viral supernatant was used to transduce 293 cells in 10 cm culture dishes. After 24 h, puromycin (0.4 µg/ml) was added and the puromycin-resistant stable cell population was used in experiments after 6 days of selection.
Uptake of bacteria was quantified by flow cytometric analysis as described previously [25]. Briefly, 293 cells with stable knock down of WAVE2, HEM2 or control transduced cells (shScramble) were transfected with HA-tagged CEACAM3 and 1×106 of the transfected cells were seeded in 6 well-plates the day before infection. Cells were infected at a MOI (multiplicity of infection) of 20 with CFSE-labeled non-opaque or OpaCEA-expressing N. gonorrhoeae for 1 h. After infection, cells were suspended in PBS, 1% heat-inactivated FCS, 2 mg/ml trypan blue and analysed on a LSRII (Becton Dickinson). The percentage of fluorescein-positive cells was multiplied by the mean fluorescence of these cells to obtain an estimate of the total amount of phagocytosed bacteria (uptake index). In parallel, CEACAM expression by the transfected knock-down cells was determined by flow cytometry with a monoclonal anti-CEACAM antibody. The mean fluorescence intensity of CEACAM3 staining was used to normalize the uptake index to correct for differences in the transfection efficiency of the three knock-down cell populations.
Phosphotyrosine-based signalling complexes often involve SH2-domain containing adapter molecules. To identify adapter molecules potentially interacting with the ITAM-like sequence of CEACAM3, we examined a panel of SH2 domains derived from different adaptor proteins for their capacity to interact with phosphorylated CEACAM3. In a pull-down assay, GST-fusion proteins of SH2 domains derived from Grb2, CrkL, Nck1, or, as a positive control, the PTK c-Src were added to cell lysates containing phosphorylated full-length CEACAM3. Phosphorylation of CEACAM3 in the lysates was ensured by co-expression of vSrc, a constitutive active viral form of the cellular PTK c-Src, which phosphorylates CEACAM3 at tyrosine residues in the cytoplasmic ITAM-like sequence [18], [20] (Fig. 1A). As expected, the recombinant c-Src SH2 domain fused to GST precipitated CEACAM3 from the lysate (Fig. 1B). This is in line with previous reports that show co-localization and a direct association of Src family PTK SH2 domains with tyrosine phosphorylated CEACAM3 [18], [21]. In contrast, GST alone or the SH2 domains of the adaptor proteins CrkL and Grb2 were not able to precipitate CEACAM3. Importantly, the SH2 domain of Nck1 interacted strongly with CEACAM3 (Fig. 1B). To analyse if the specific binding of Nck1 involves the cytoplasmic domain of CEACAM3, additional pull-down assays were performed using full-length CEACAM3 (CEACAM3 WT) and a deletion mutant of the receptor that lacks the complete cytoplasmic domain (CEACAM3 ΔCT). Upon transfection of 293 cells and preparation of lysates, CEACAM3 WT or CEACAM3 ΔCT were present at similar levels (Fig. 1C). However, the Nck1 SH2 domain associated only with full-length, but not with truncated CEACAM3 (Fig. 1D). Again, the association was specific for the SH2 domain of Nck1 and was not observed for GST alone or the SH2 domain of Grb2 (Fig. 1D).
SH2-domain-mediated interactions require an intact phospho-tyrosine binding pocket. In Nck1, a crucial arginine residue is located in this binding pocket at position 308 (R308) of the full length Nck1 protein. Accordingly, we performed site-directed mutagenesis and replaced R308 for lysine. In contrast to the wildtype Nck1 SH2 domain, the resulting Nck1-R308K-SH2 domain lost its ability to associate with CEACAM3 (Fig. 1E), demonstrating that the observed interaction is mediated by a phospho-tyrosine-directed binding of the Nck1 SH2 domain.
In humans, two Nck homologues, Nck1 and Nck2, are expressed in most cell types [25]. To analyze the ability of either of these two proteins to associate with CEACAM3, we employed GST fusion proteins of the SH2 domains of either Nck1 or Nck2. Lysates were prepared from 293 cells transfected with GFP, CEACAM3 WT-GFP, or CEACAM3 ΔCT-GFP in the presence or absence of v-Src (Fig. 2A). Using these lysates in pull-down analyses, both the Nck1 as well as the Nck2 SH2 domain were able to bind to full-length CEACAM3, but not to CEACAM3 ΔCT (Fig. 2B). Again, binding of the Nck1 and Nck2 SH2 domains to CEACAM3 WT was comparable to the interaction found for the c-Src SH2 domain (Fig. 2B). In contrast to Nck, the SH2 domains of the two adapter proteins SLP76 and Grb2, or GST alone did not associate with CEACAM3 (Fig. 2B). Importantly, association of Nck1 and Nck2 with CEACAM3 depended on tyrosine phosphorylation of the cytoplasmic domain, as binding did not occur, if CEACAM3 was not co-expressed with vSrc (Fig. 2A). In all cases, GST and GST-SH2 domains were employed in comparable amounts as shown by Coomassie staining of the blotted precipitates (Fig. 2B, lower panels). Together, these results demonstrate that both Nck1 and Nck2 SH domains can specifically bind to the phosphorylated cytoplasmatic domain of CEACAM3.
Upon infection with Neisseria gonorrhoeae, CEACAM3 clustering triggers recruitment of Src-family PTKs to the bacteria-engaged receptor [21]. To localize the potential interaction of Nck with the bacteria-associated receptor, we co-transfected 293 cells with constructs encoding the red-fluorescent protein mKate fused to the carboxy-terminus of wildtype CEACAM3 (CEACAM3-mKate) together with either GFP, the GFP-tagged Nck1 SH2 domain (GFP-Nck1-SH2) or the GFP-tagged, mutated Nck1 SH2 domain (GFP-Nck1R308K-SH2). All constructs were successfully expressed at moderate levels suitable for analysis by confocal laser scanning microscopy. As expected, CEACAM3-mKate was predominantly found in cellular membranes (Fig. 3A). Following infection with CEACAM-binding gonococci, CEACAM3-mKate clustered at sites of bacterial engagement, but this did not result in re-distribution of GFP (Fig. 3A). Clearly, the GFP-fused Nck1 SH2 domain was strongly recruited to the contact sites of CEACAM3 and bacteria (Fig. 3A; arrowhead). This recruitment was evident for bacteria during the initial uptake process in the cell periphery, but not for bacteria in a perinuclear, intracellular position suggesting that Nck recruitment is transient. In contrast to the wildtype Nck1 SH2 domain, Nck1R308K-SH2, which did not interact with phosphorylated CEACAM3, did not accumulate in response to bacterial CEACAM3 engagement and remained evenly distributed throughout the cell (Fig. 3A). In line with the selective recruitment of the isolated Nck1 SH2 domain, also GFP-tagged full length Nck1 (GFP Nck1 FL) was strongly recruited to sites of bacterial attachment in 293 cells expressing a red fluorescent protein tagged CEACAM3 (Fig. 3B). The formation of a complex between Nck1 and CEACAM3 in intact cells was verified by co-immunoprecipitation of full length Nck1 together with CEACAM3 from cell lysates with co-expression of vSrc (Fig. 3C). These results provide evidence that a CEACAM3-Nck complex is formed in intact cells upon bacterial engagement of CEACAM3 and suggest that Nck could be involved in orchestrating the opsonin-independent phagocytosis of CEACAM3-bound microbes.
If the observed specific recruitment of Nck1 to clustered CEACAM3 would have a functional role for bacterial internalization, then blocking access of Nck to the cytoplasmic domain of the receptor should interfere with the uptake process. Therefore, we overexpressed the isolated Nck1 SH2 domain or the SLP-76 SH2 domain together with CEACAM3 and monitored bacterial internalization by gentamicin protection assays. In line with the idea that association of Nck1 with CEACAM3 is needed for efficient internalization of bacteria, overexpression of the Nck1, but not the SLP-76 SH2 domain reduced the uptake of CEACAM-binding gonococci significantly by about 25% (Fig. 4A). In contrast, adhesion of bacteria to CEACAM3-expressing cells was not diminished in the presence of the Nck1 SH2 domain (Fig. 4B). Though these results suggest that Nck is involved in the CEACAM3-mediated uptake of N. gonorrhoeae, the isolated SH2 domain of Nck1 could interfere with other SH2 domain-dependent processes such as recruitment of Src family PTKs to the cytoplasmic domain of the receptor. Therefore, we depleted endogenous Nck1 by siRNA in CEACAM3-expressing HeLa cells. Knock-down of Nck1 was only partial and reduced the amount of Nck1 by about 60% (Fig. 4C). However, this partial reduction led to a significant decrease in CEACAM3-mediated uptake of gonococci compared to untreated or control siRNA-treated cells (Fig. 4C). As a further control, HeLa cells, that do not express any CEACAM member endogenously, did not internalize gonococci (Fig. 4C). Together with the biochemical and microscopic analysis, these results identify a functional role for the adapter molecule Nck in regulating the CEACAM3-initiated uptake of bacteria.
In most cells, Nck1 and Nck2 have overlapping functions. Therefore, we employed fibroblasts derived from homozygote Nck1/Nck2-deficient mouse embryos (Nck1−/−Nck2−/−cells) and compared them to heterozygote control cells (derived from Nck1+/−Nck2+/−mouse embryos) [24]. The fibroblasts were transfected with a plasmid encoding GFP-tagged CEACAM3 and infected with biotinylated, Pacific blue-labelled OpaCEA-expressing N. gonorrhoeae. After 60 minutes infection, the fixed samples were stained for extracellular bacteria with streptavidin- Alexafluor647 allowing detection of cell-associated versus intracellular bacteria (Fig. 5A). Though intracellular bacteria were found in all CEACAM3-expressing fibroblasts, the amount of intracellular bacteria in Nck1−/−Nck2−/−cells was strongly diminished compared to Nck1+/−Nck2+/−cells (Fig. 5A). Quantification of intracellular bacteria revealed that Nck-deficiency reduced bacterial uptake by about 60% (Fig. 5B). Western blotting confirmed the lack of intact Nck1 and Nck2 in cell lysates of Nck1−/−Nck2−/−cells (Fig. 5C). Together, these data support the idea that the adaptor proteins Nck1 and Nck2 contribute to the efficient CEACAM3-mediated phagocytosis of bacteria.
Nck adapter proteins are often help to locally organize dynamic changes in the actin cytoskeleton upon stimulation of membrane receptors. As CEACAM3-initiated uptake of bacteria is accompanied by actin re-arrangements leading to large lamellipodial membrane protrusions [15], [16], we wondered whether Nck adapter proteins might be critical for this process. To address this question, we implemented correlative light-electron microscopy (CLEM) to visualize lamellipodial structures in response to bacterial CEACAM3 stimulation in the presence or absence of Nck adapter proteins. To this end, Nck1+/−Nck2+/−and Nck1−/−Nck2−/−cells were transiently transfected with CEACAM-GFP and infected with fluorescence labelled OpaCEA protein-expressing gonococci. Both, Nck-expressing and Nck-deficient fibroblasts bound equal numbers of gonococci and the OpaCEA protein-expressing bacteria led to clustering of CEACAM3-GFP, as visualized by fluorescence microscopy (Fig. 6). Upon further processing, the identical cells were re-analysed by scanning electron microscopy allowing high resolution observation of surface structures (Fig. 6). Whereas CEACAM3-bound bacteria on the surface of Nck1−/−Nck2−/−cells were surrounded by small filopodia, large lamellipodial protrusions were evident in a radius up to 6 µm in the vicinity of gonococci in contact with CEACAM3 on Nck-expressing fibroblasts (Fig. 6). Therefore, this CLEM investigation provides direct evidence that Nck adapter proteins are involved in CEACAM3-initiated, actin cytoskeleton-based subcellular structures.
CEACAM3-mediated uptake of bacteria is strictly dependent on actin polymerisation orchestrated by the small GTPase Rac [15]. A well characterized effector of GTP-bound Rac is the WAVE/Scar-complex that promotes f-actin nucleation by the Arp2/3 complex [26], [27]. One critical component of the WAVE complex, Nap1 (Nck-associated protein 1, also termed NCKAP1 or Hem2), is a known binding partner of the SH3 domain of Nck. Giving the role of Nck in CEACAM3-initiated lamellipodia formation, we wondered whether Nck could help to localize the WAVE complex to CEACAM3. Accordingly, 293 cells were transfected with GFP, CEACAM3 WT-GFP, or CEACAM3 ΔCT-GFP together with myc-tagged WAVE2 and then infected with CEACAM-binding gonococci. Confocal microscopy revealed that WAVE2 was distributed throughout the cytoplasm in cells expressing GFP irrespective of bacterial infection (Fig. 7A). However, WAVE2 was strongly recruited to sites of bacterial engagement of CEACAM3 in cells expressing the wildtype receptor (Fig. 7A). In contrast, WAVE2 was not redistributed in cells expressing truncated CEACAM3 (CEACAM3 ΔCT-GFP), even though CEACAM3 ΔCT-GFP bound bacteria similar to the wildtype receptor (Fig. 7A). Immunoprecipitation of WAVE2 from cells cotransfected or not with myc-tagged Nck and vSrc revealed a constitutive association of WAVE2 and Nck, which was not dependent on the presence of vSrc (Fig. 7B). This is in line with the idea that Nck binds to the WAVE complex in a phosphotyrosine-independent, SH3 domain-dependent manner.
To test the possibility that Nck connects phosphorylated CEACAM3 with the WAVE complex, we transfected 293 cells with combinations of CEACAM3-WT-HA, GFP-WAVE2, myc-Nck1, and vSrc. Expression of all the constructs at equivalent levels was verified by Western blotting (Fig. 7C). Upon immunoprecipitation of GFP-WAVE2, the resulting precipitates were probed for the presence of myc-Nck and CEACAM3-HA (Fig. 7D). Again, a constitutive association of Nck with WAVE2 was detected, which did not depend on the presence of CEACAM3 or v-Src (Fig. 7D). However, CEACAM3 WT was only present in WAVE2 precipitates, if the cells co-expressed myc-Nck and vSrc demonstrating that Nck orchestrates the complex formation between tyrosine phosphorylated CEACAM3 and the Rac effector WAVE2 (Fig. 7D).
Our finding of a Nck-mediated recruitment of the WAVE complex implicates that this activator of actin nucleation might be involved in actin cytoskeleton rearrangements during CEACAM3-mediated phagocytosis. To test the functional significance of the WAVE complex in bacterial uptake, we used a dominant negative variant of WAVE2 that lacks the carboxy-terminal VCA domain (WAVE2 ΔVCA). WAVE2 ΔVCA is not able to associate with the Arp2/3 complex and, therefore, disrupts WAVE-initiated actin nucleation [26]. Similar to wildtype WAVE2, WAVE2 ΔVCA was strongly recruited to sites of bacterial contact with CEACAM3 (Fig. 8A). This result suggests that the Arp2/3 complex-binding VCA domain of WAVE2 is not required for the Nck-mediated localization to phosphorylated CEACAM3. However, overexpression of WAVE2 ΔVCA severely impaired the CEACAM3-mediated uptake of N. gonorrhoeae without effecting bacterial binding to the cells (Fig. 8B). These results demonstrate that the Nck binding partner WAVE2 is involved in CEACAM3-mediated phagocytosis.
To further corroborate the functional role of WAVE2 and the WAVE complex during CEACAM3-mediated uptake of bacteria, we generated cell populations with shRNA-mediated knock-down of WAVE2 or Nap1, the Nck-binding component of the WAVE complex. Furthermore, control cells were transduced with a recombinant lentivirus encoding a non-targeting, control shRNA (shScramble). After puromycin selection, the successful knock-down was verified by Western Blotting, which demonstrated that WAVE2 and Nap1 expression were reduced by about 70% and 60%, respectively (Fig. 9A). Virally transduced cell populations were transfected with a HA-tagged CEACAM3 construct. Upon infection with non-opaque N. gonorrhoeae, neither CEACAM3-transfected cell line internalized significant amounts of bacteria (Fig. 9B). However, OpaCEA protein-expressing gonococci were efficiently internalized by the shScramble transduced cells. Strikingly, both the WAVE2- as well as the Nap1-knock-down cells showed a decreased uptake of OpaCEA protein-expressing gonococci (Fig. 9B). Though the shRNA-mediated knock-down was incomplete, the 50 to 60% decrease of bacterial phagocytosis by the WAVE2- and Nap1-knock-down cells compared to the control cell (shScramble) corresponds well to the extent of diminished expression of these two proteins. These results corroborate the functional role of the WAVE complex in CEACAM3-initiated phagocytosis of bacteria.
During CEACAM3-initiated phagocytosis, GTP-loaded Rac stimulates massive actin polymerization leading to lamellipodial protrusions [15] and our results suggested that CEACAM3-associated Nck was also involved in this phenotype. Therefore, we speculated that the Nck-associated WAVE complex might be the critical Rac effector involved in actin polymerization in the vicinity of CEACAM3-binding bacteria. To address this question, we co-transfected 293 cells with CEACAM3 and either GFP or GFP-tagged, dominant-negative WAVE2 ΔVCA. Whereas a strong accumulation of f-actin was observed in GFP-expressing cells at the sites of bacteria-initiated CEACAM3 clustering, accumulation of f-actin was absent in WAVE2 ΔVCA expressing cells (Fig. 9C).
Together, these results suggest that Nck has a functional role in orchestrating CEACAM3-initiated signalling to the actin cytoskeleton by localizing the actin polymerization promoting activity of the WAVE complex. Accordingly, the adapter molecule Nck helps to convert the cytoplasmic domain of CEACAM3 into a sophisticated signalling hub, where upon receptor stimulation Rac GEFs such as Vav are brought into close proximity to Rac effectors such as WAVE to promote efficient, opsonin-independent phagocytosis of CEACAM3-attached microbes.
Several human specific pathogens are able to engage CEACAM family members to colonize mucosal surfaces [2], [4]). On the other hand, human granulocytes are equipped with CEACAM3 to detect and eliminate CEACAM-binding bacteria in an opsonin-independent manner [11], [15].
Here we demonstrate that the adapter molecule Nck is a critical element in the CEACAM3-initiated signalling cascade that helps to connect bacterial recognition via CEACAM3 with rapid actin-dependent phagocytosis. Nck associates in a phosphotyrosine-dependent manner with the intracellular ITAM-like sequence of CEACAM3 and knock-down of Nck or overexpression of the isolated SH2 domain impair CEACAM3-mediated uptake of N. gonorrhoeae. Nck positions the WAVE complex to the clustered receptor promoting local actin polymerization and rapid engulfment of attached bacteria. These results suggest that the cytoplasmic domain of CEACAM3, by bringing together the Rac stimulator Vav as well as Rac effectors such as WAVE, functions as a specialized organizing center optimized to trigger efficient, opsonin-independent phagocytosis of CEACAM-binding bacteria (Fig. 10).
Nck adapter proteins have been recognized as important regulators of growth factor receptor- and integrin-stimulated signals that control the organization of the actin cytoskeleton [25]. For example, Nck adapter proteins are critical for the actin polymerization-driven dorsal ruffle formation upon growth factor stimulation, a process that resembles lamellipodia formation during phagocytosis [28]. Nck1 and Nck2 are co-expressed in most tissues and mediate redundant functions, as single knock-out of either gene does not impair the overall function of the organism, whereas deletion of both genes results in embryonic lethality [24]. In line with their overlapping function, Nck1 and Nck2 seem to interact with a similar set of proteins via either their SH2 or their SH3 domains [29]. Our studies also support the idea that Nck1 and Nck2 SH2 domains bind to the same target proteins, as both are able to precipitate tyrosine phosphorylated CEACAM3 in our assays. Crystallization of the Nck1 and Nck2 SH2 domains together with phosphorylated target peptides as well as biochemical binding studies have revealed that both adapters can accommodate a similar set of phospho-peptides [30]. The consensus motif for Nck-SH2 domain binding has revealed a preference for one or two acidic amino acids carboxy-terminal to the phospho-tyrosine in the form of pY-D/E-D/E-V [30]. Indeed, one of the two tyrosine residues within the ITAM-like sequence of CEACAM3 is followed by two negatively charged amino acids in the form of Y-E-E-L, which closely matches the characterized motif and supports the idea that the Nck SH2 domain directly associates with the cytoplasmic part of CEACAM3.
Importantly, Nck also participates in Fcγ receptor- and T-cell receptor (TCR)-initiated signaling events that involve ITAM phosphorylation [29], [31]. There, Nck associates with the activated receptors via additional, tyrosine-phosphorylated adapter molecules such as c-Cbl or SLP-76, respectively. In the context of T-cell stimulation, Nck is associated with the TCR complex via SLP-76 and recruits the Wiskott-Aldrich-Syndrome protein (WASP), which binds to the carboxy-terminal SH3 domain of Nck [32], [33]. Besides Nck, phosphorylated SLP-76 also binds the guanine nucleotide exchange factor Vav [34]. Interestingly, SLP-76 bears multiple Y-E-X-P motifs in its amino-terminal part that bind the SH2 domains of Nck and Vav, though they are clearly distinct from the optimal Nck SH2 (pY-D/E-D/E-V) or Vav SH2 (pY-M-E-P) recognition sequences as determined by phospho-peptide scanning [30], [35]. A similar situation exists for CEACAM3, where the Y-E-E-L motif surrounding Y-230 directly associates with the Vav SH2 domain [22] and might also be responsible for Nck binding.
By the help of Nck, SLP-76 brings into proximity a stimulator (Vav) as well as an effector (WASP) of GTP-bound small Rho GTPases to locally organize the actin cytoskeleton [33], [34]. It is enticing to speculate that a similar role is carried out by the cytoplasmic domain of CEACAM3 and thereby this receptor directly, without intermediary adaptor proteins, recruits the machinery for rapid actin polymerization. The assembly of such an actin regulating signalling complex by the cytplasmic portion of CEACAM3 might also explain why the ITAM-like sequence of this receptor can trigger efficient phagocytosis in the absence of Syk or Zap70 [36]. These two tyrosine kinases are essential for cytoskeletal rearrangements initiated by canonical ITAM motifs [13], [37]. For example, inhibition of Syk activity in macrophages compromises FcγR-mediated phagocytosis. In contrast, CEACAM3-mediated uptake of bacteria is not influenced by Syk inhibitors in primary granulocytes or by co-expression of Syk in CEACAM3-transfected cell lines ([36]; Pils and Hauck, unpublished observations). Together, these findings suggest that CEACAM3 short wires receptor stimulation with efficient phagocytosis and, similar to phosphorylated SLP-76, achieves rapid reorganization of the actin cytoskeleton by optimizing the local environment for Rho GTPase signaling.
Despite the conceptual similarities, there is one important difference in the SLP-76-mediated coordination of TCR-induced actin rearrangements at the immunological synapse and CEACAM3-induced actin polymerization during phagocytosis. Upon TCR stimulation, the small GTPase Cdc42 is thought to trigger WASP-mediated actin cytoskeleton rearrangements [33], [38]. In contrast, CEACAM stimulation in granulocytes is tightly linked to Rac GTP loading, and dominant-negative versions of Cdc42 do not interfere with N. gonorrhoeae internalization in CEACAM3-transfected human cell lines or primary human granulocytes [15], [23].
How the selective recruitment and stimulation of either Cdc42 or Rac is achieved is currently unknown, as both TCR- as well as CEACAM3-initiated signalling involve the guanine nucleotide exchange factor Vav [22], [39], [40]. Nevertheless, our findings that Nck associates with phosphorylated CEACAM3 and recruits the WAVE2 complex shed light on the downstream events following Rac GTP loading. WAVE2 is part of a pentameric complex that is intrinsically inactive [41], [42]. Besides WAVE2, Abi, and Brk1, the complex contains the proteins Sra and Nap1, which seem to shield the carboxy-terminal VCA domain of WAVE2, thereby preventing WAVE-initiated Arp2/3 complex stimulation and actin nucleation [27], [43]. Though the precise details are currently unknown, the actin nucleation promoting activity of the WAVE complex appears to depend on multiple inputs including association with GTP-loaded Rac, binding to phosphoinositides, and phosphorylation of WAVE [42], [44], [45] that all seem to alter the conformation of the complex (Fig. 10).
Interestingly, one integral component of the WAVE complex, Nap1 (also termed NCKAP1 or Hem2), has been initially identified as an Nck binding partner in a yeast-two-hybrid screen [46], [47]. Interaction between Nap1 and Nck is mediated by proline-rich sequences in Nap1 and Nck SH3 domains allowing a constitutive, phosphorylation-independent association as also detected in our analysis. Though direct binding of the SH3 domain of IRSp53, a BAR-domain containing protein localized at lamellipodial membrane protrusions, has been shown to contribute to activation of the WAVE complex, it is not known, if the SH3 domain-mediated association of Nck with Nap1 can result in an allosteric activation of the complex [27]. However, the Nck-Nap1 interaction could clearly contribute to the subcellular localization of the WAVE complex, thereby directing the actin nucleation machinery to clustered and tyrosine phosphorylated CEACAM3. Moreover, Nck could affect WAVE complex activation indirectly. In particular, Nck is known to bind the cytoplasmic tyrosine kinase Abl, that phosphorylates WAVE2 at tyrosine residue Y150 providing an essential post-translational modification for full activation of the WAVE complex [45], [48]. Abl directly binds to Abi, another constituent of the WAVE complex, and additional interactions between Nck SH3 domains and Abl might facilitate or stabilize such an association.
Nck also associates with the actin nucleation promoting factor N-WASP, an effector of GTP-loaded Cdc42 [25]. Nck/N-WASP driven actin polymerization is critical for pedestal formation in mammalian cells infected with enteropathogenic E. coli and for actin-based intracellular motility driven by the vaccinia virus protein A36R [49]–[51]. Based on our data, we can not rule out an involvement of WASP family members in CEACAM3-induced lamellipodia formation. However, the dependence on Rac-GTP instead of Cdc42-GTP point to the WAVE complex instead of WASP as potential downstream elements. In addition, other cellular factors, such as Spire or formins, could contribute to the proper organization of the actin cytoskeleton during the complex process of CEACAM3-mediated internalization, either in parallel or in a sequential manner. Indeed, even in the absence of a functional ITAM-like sequence or upon deletion of the complete cytoplasmic domain, an inefficient, residual uptake of bacteria via CEACAM3 has been observed [15], [16], which might also explain the remaining uptake observed in Nck1/Nck2-deficient cells. However, based on our CLEM results on the lack of lamellipodial protrusions in Nck1/2-deficient cells and based on the severe reduction of actin polymerization in the vicinity of clustered CEACAM3 in WAVE2 ΔVCA-expressing cells we suggest that the Nck-mediated local enrichment of the WAVE complex is the major actin nucleation promoting activity during CEACAM3-mediated phagocytosis of bacteria.
Our work reveals the extremely compact organization of CEACAM3-initiated signalling that connects the opsonin-independent recognition of CEACAM-binding pathogens via the extracellular immunoglobulin domain with rapid phagocytosis initiated by the cytoplasmic domain. By recruiting in a tyrosine-phosphorylation dependent manner the guanine nucleotide exchange factor Vav as well as the Nck-WAVE module, the CEACAM3 ITAM-like sequence elegantly integrates an upstream activator and a downstream effector to provide an optimized subcellular microenvironment for Rac-dependent actin rearrangements. Indeed, CEACAM3-mediated uptake by human granulocytes is exceptionally fast and results in a large number of internalized bacteria within 15 minutes after phagocyte encounter [15], [18], [22]. The involvement of few, but ubiquitously expressed signalling components not only explains why CEACAM3 can function as an efficient phagocytic receptor in different transfected cell lines, but also points to a minimal set of proteins needed for rapid and efficient engulfment of particles by human granulocytes.
We thank D. Chudakov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) for mKate cDNA, T.F. Meyer (MPI for Infection Biology, Berlin, Germany) for the Neisseria strain used in this study, T. Pawson (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada) for Nck1 and Nck2-deficient and control mouse fibroblasts, D.W. Piston (Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA) for mCerulean cDNA, T. Stradal (Helmholz Zentrum für Infektionsforschung, Braunschweig, Germany) for WAVE2 cDNA, Wolfgang Zimmermann (Tumor Immunology Laboratory, LMU München, Germany) for HeLa cells stably expressing CEACAM3, and D. Deininger, S. Feindler-Boeckh, R. Hohenberger-Bregger, and H. Schuhwerk for expert technical assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was supported by funds from the Landesstiftung Baden-Württemberg (LS-Prot-66) and by a grant from the Ministry of Science, Research and the Arts of Baden-Württemberg to C.R.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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