Blackhawks deal Versteeg to Maple Leafs

The Toronto Maple Leafs have acquired forward Kris Versteeg from the Stanley Cup champion Chicago Blackhawks in a five-player deal.

Versteeg had 44 points in 79 regular-season games last season and …

Volunteer firefighters released from hospital

Two volunteer firefighters who were injured this week whilefighting a blaze near St. Albans were released from the hospital.

Jermaine Watson and Jeremy Buskirk of the West Side Volunteer FireDepartment were treated and released from CAMC …

Venus Has Frequent Bursts of Lightning

Nearby Venus is looking a bit more Earth-like with frequent bursts of lightning confirmed by a new European space probe.

For nearly three decades, astronomers have said Venus probably had lightning _ ever since a 1978 NASA probe showed signs of electrical activity in its atmosphere. But experts weren't sure because of signal interference.

Now a magnetic antenna on the European Space Agency's Venus Express probe proved that the lightning was real.

"We consider this to be the first definitive evidence of abundant lighting on Venus," David Grinspoon of the Denver Museum of Nature and Science told reporters Wednesday at a briefing in Paris.

…

Foreclosure possible? Help nears

Homeowners at risk of foreclosure can get help at a foreclosure prevention event Saturday presented by Neighborhood Housing Services of Chicago, the City of Chicago and Attorneys Title Guaranty Fund Inc.

The event takes place from 9:30 …

Seeking the priesthood of all believers

Intense commitment and honesty are 'God-filled times' for some but not all

The Kitchener-Waterloo House Churches were founded in 1969 by John Miller and Walter Klaassen-both professors at the time at die then-named Conrad Grebel College-along with others. Their impulse, like tiiat of other house churches founded around that time, was to return to an early church format where "the priesthood of all believers" mentioned in I Peter 2:5,9 was to be practised. This was a fundamental part of Mennonite Church Concern Movement beliefs among scholars and church leaders in the 1960s and 70s.

The KW House Churches are intense, intimate and intentional communities of worship, mutual …

Torre Says He'll 'Just Leave Memories'

RYE BROOK, N.Y. - Joe Torre heard enough. He felt insulted. He felt unappreciated. He won't even set foot in Yankee Stadium anytime soon, not even to clean out his office. "I walked out of there, I'm not going back," he said. "I just leave the memories."

A day after he turned down a one-year contract, convinced the team no longer was committed to him after 12 seasons and four World Series titles, he went out his way - grateful, yet defiant; respectful but hurt.

He didn't say goodbye in Yankee Stadium. Instead, he spoke for 67 minutes - one minute for each year of his life - in a hotel ballroom near his home in suburban New York, close to the Connecticut border.

…

Australian warships visit NY to mark centennial

Two Australian warships have arrived in New York harbor to commemorate the nearly century-old military relationship between Australia and the United States.

The HMAS Sydney and HMAS Ballarat sailed into New York Harbor on Sunday morning and are docked at a pier on Manhattan's West Side.

In 1908, the U.S. Navy's "Great White Fleet" was invited to stop at Australian ports during its round-the-world cruise _ and was greeted by nearly 250,000 people in Sydney. The Australian government …

Roger Ebert's Top 10 M ovies of 1992

It was an anno mirabilia, a year of marvels. For no particularreason, 1993 was the best year since the 1970s for new motionpictures. One after another they arrived, especially in the autumn,great and good films of all sorts, from the epic to the intimate,from thrillers to biopics, all with that special grace that leavesyou thankful for the movies.

I found myself granting "four stars" almost with abandon - morethan twice as many as in 1992. Readers wrote in asking if I hadn'tgone soft. Look at the movies, I advised them. I'm only reportingthe news.

At the head of the list were new films by the most populardirector in the world, Steven Spielberg, and the best …

UAB holds off Southeast Missouri State 78-56

BIRMINGHAM, Ala. (AP) — Cameron Moore scored 20 points and three others scored in double-figures as Alabama-Birmingham defeated Southeast Missouri State 78-56 on Saturday night in the season opener for both teams.

Moore enjoyed a hot-shooting night, making 9 of 13 field-goal attempts.

Ovie Soko scored 16 points, Aaron Johnson had 11 and Dexter Fields 10 for UAB, which won its 32nd straight nonconference game at …

Smoke forces JetBlue flight back to Puerto Rico

Puerto Rican officials say a JetBlue flight bound for Florida with 39 passengers on board was forced to return to San Juan after the pilot reported smoke in the cockpit.

Ports Authority director Alvaro Pilar said Flight 1762 was en route to Fort Lauderdale when it returned to Puerto Rico's international …

Bears poised to pull trigger on deal

The Bears are weighing trade offers from the Green Bay Packers andMiami Dolphins for quarterback Cade McNown and could complete a dealas early as today, a league source said Thursday.

Bears general manager Jerry Angelo is reluctant to trade McNown tothe Packers because they are a division …

Police: NYC subway derails, no injuries

NEW YORK (AP) — Authorities say two cars of a subway train derailed in lower Manhattan but no one was injured.

The Metropolitan Transportation Authority says the derailment occurred at 3:52 a. m. EDT Monday on the northbound No. 6 train between Brooklyn Bridge and Canal …

Bulls select Rose with No. 1 pick in NBA draft

Derrick Rose is going home, and a whole bunch of freshmen are following him to the NBA. The Chicago Bulls selected Rose, who grew up on the city's South Side, with the No. 1 pick in the NBA draft Thursday night, choosing the Memphis guard over Kansas State forward Michael Beasley.

With Beasley going second to Miami and Minnesota picking O.J. Mayo at No. 3, college freshmen made up the first three picks for the first time in draft history.

"We actually talked about this earlier," Beasley said. "We all grew up together and we all grew up playing against each other and we all made a pact together that we would all be here. Just to see it all fall into place and see it all happen is kind of crazy."

Rose led the Tigers to the national championship game in his lone college season. The Bulls opted for the point guard's playmaking ability over the scoring and rebounding of Beasley, who ranked in the top three in the nation in both categories.

Rose is the Bulls' first No. 1 overall selection since they grabbed Elton Brand in 1999. He's the second straight freshman taken with the top pick, following Portland's Greg Oden last year.

The 6-foot-3 guard put on a red Bulls cap, hugged some supporters, including Memphis coach John Calipari, and shook hands with Beasley, seated at a nearby table, before walking onto the stage to meet NBA commissioner David Stern.

Rose should be an upgrade over Kirk Hinrich, who now could be traded, and gives the Bulls another option if they don't re-sign guard Ben Gordon.

Expected to contend for a division title, the Bulls instead stumbled to a 33-49 record and eventually replaced two coaches. But with just a 1.7 percent chance, they won last month's draft lottery, giving them a chance to quickly return to the playoffs.

"It feels great to go in and compete," Rose said. "I'm just blessed to be in that position right now, because a lot of people aren't. And just knowing that we are a few pieces away from really contending as a team, it just makes me happy."

Miami settled for Beasley at No. 2, even though he wasn't sure if the Heat would go for Mayo instead. Beasley averaged 26.2 points, third in the nation, and topped Division I with 12.4 rebounds per game. But with questions about his size _ he may be 2 inches shorter than the 6-foot-10 he's listed at _ the Bulls may not have believed he could play the 4 spot in the NBA.

After Mayo's selection, UCLA guard Russell Westbrook was the first non-freshmen taken, going fourth to the Seattle SuperSonics _ with new teammate and reigning Rookie of the Year Kevin Durant standing and applauding the pick from the back.

Kevin Love gave the Bruins' consecutive picks, going to Memphis at No. 5. The New York Knicks followed with Italian forward Danilo Gallinari, whose father played with new coach Mike D'Antoni overseas. Fans in Madison Square Garden weren't impressed, booing loudly.

"It's part of the game, all the players have got to hear this," Gallinari said. "Not every time can you hear good things. It's normal."

Indiana guard Eric Gordon became the fifth freshman taken, going to the Los Angeles Clippers at No. 7. West Virginia's Joe Alexander, whose stock began to rise after a strong run at Madison Square Garden in the Big East tournament, went to Milwaukee with the next pick.

Charlotte gave new coach Larry Brown a point guard, taking D.J. Augustin of Texas with the ninth pick. New Jersey took Stanford center Brook Lopez at No. 10, and Jerryd Bayless joined fellow Pac-10 guards Mayo and Westbrook by going 11th to Indiana.

Loach slams TV

Film director Ken Loach has blasted the TV industry.

The award-winning filmmaker, who lives in Bath, said the sectorwas stifling creativity through "timeserving" middle managers. Hetold the London Film Festival that he wished "good riddance" to BBCexecutives recently made redundant and that more should lose theirjobs.

The 74-year-old Kes director said: "I'm pleased to see - we allare - that people are going to lose their jobs, albeit that theyneed a Pounds 1 million handshake to get out the door. Great, goodriddance, maybe a few more will join them. But let's start cuttingfurther down."

Charles Taylor nears end of defense testimony

Former Liberian President Charles Taylor has issued a string of denials on what is expected to be his last full day of testifying in his own defense at his war crimes trial.

Taylor has been testifying for 13 weeks, strenuously rejecting allegations that he commanded and controlled rebels who murdered and mutilated thousands of civilians during Sierra Leone's bloody 1991-2002 civil war.

On Monday, he denied a key prosecution allegation _ that he smuggled diamonds out of Sierra Leone in a mayonnaise jar. Prosecutors say he provided arms, ammunition and other support to Sierra Leone rebels in return for diamonds.

Taylor is expected to complete his testimony Tuesday, giving prosecutors their first chance to publicly question him.

US Democratic White House hopeful Biden compares Pakistan under Musharraf to 1970s' Iran

Democratic presidential hopeful Joe Biden is comparing the unrest in Pakistan to 1970s Iran, when Americans were held hostage.

Biden, who chairs the U.S. Senate's foreign relations committee and is knowledgeable on international relations, says what is happening in Pakistan is linked to broader U.S. foreign policy goals. He says U.S. policy should be shifted from one focused solely on President Pervez Musharraf to one based on Pakistan as a whole.

"Pakistan has strong democratic traditions and a large, moderate majority. But that moderate majority must have a voice in the system and an outlet with elections," Biden was to say Thursday during a speech at Saint Anselm College. "If not, moderates may find that they have no choice but to make common cause with extremists, just as the Shah's opponents did in Iran three decades ago."

"But unlike Iran, Pakistan already has nuclear weapons," Biden says in prepared remarks, which his campaign provided to The Associated Press.

Last weekend, Musharraf declared emergency rule, suspended the constitution and granted authorities sweeping powers to crush political dissent.

Musharraf been promising to restore democracy since he seized power in 1999 during a bloodless coup. His actions since assuming emergency powers Saturday have included ousting independent-minded judges, clamping down on the media and putting thousands of Pakistanis in jail or under house arrest.

Musharraf said his decision to suspend the constitution and oust the country's top judge were necessary to prevent a takeover by Islamic extremists. Biden disagreed, saying in the speech that such moves will only invite extremists, similar to those in Iran nearly three decades ago.

In 1979, hundreds of militant Iranian university students seized the U.S. Embassy in Tehran because they objected to U.S. support for their leader, Mohammed Reza Pahlavi, who was viewed by many Iranians as a U.S. lackey and came to power after a CIA-backed coup that toppled Iran's democratically elected prime minister in 1953.

About 70 Americans were held hostage for 444 days. They were released in January 1981 when Ronald Reagan was sworn in as president.

Biden said this scenario could be repeated, only this time with nuclear implications.

"It is hard to imagine a greater nightmare for America than the world's second-largest Muslim nation becoming a failed state in fundamentalist hands, with an arsenal of nuclear weapons and a population larger than those of Iran, Iraq, Afghanistan and North Korea combined," said Biden, who trails in the race for the Democratic presidential nomination.

New Mexico Gov. Bill Richardson, another Democratic candidate in next year's presidential election, also was scheduled to address the same forum on Thursday.

Tuesday's Sports Scoreboard

All Times Eastern
National Football League
No games today.
National Basketball Association
Charlotte 113, Chicago 108 F
Indiana 97, Orlando 90 F
Washington 104, Philadelphia 97 F
Milwaukee 98, New Jersey 76 F
Detroit 61, Dallas 60 -3
Denver 65, Golden State 62 -2
Memphis 0, Portland 0 -1
Phoenix 3, Sacramento 0 -1
Houston vs L.A. Lakers, 10:30 p.m.
National Hockey League
New Jersey 4, Dallas 0 F
Toronto 3, Florida 2 F
Washington 4, Montreal 2 F
Pittsburgh 5, Atlanta 2 F
Boston 4, Ottawa 1 F
Calgary 2, Nashville 0 -3
Chicago 4, Minnesota 1 -3
Phoenix 1, Edmonton 0 -2
Columbus 0, Vancouver 0 -1
Detroit 0, Anaheim 0 -1
Top 25 College Football Playoffs
Iowa (10) 14, Georgia Tech (9) 7 -2
Top 25 College Basketball
Texas (2) 96, Arkansas 85 F
Purdue (4) 79, Minnesota 60 F
Mississippi (14) 84, UCF 56 F
New Mexico (15) vs San Diego St., 10:30 p.m.
Georgia 73, Georgia Tech (20) 66 F
Top 25 Women's College Basketball
Texas (15) 84, Texas-Arlington 41 F
Xavier (16) 68, Missouri 60 F
West Virginia (22) 63, Pittsburgh 59 F

Coasts Brace for a Busy Hurricane Season

WASHINGTON - Hurricane forecasters expect more tropical storms than normal this season, and "it just takes one to make it a bad year," says Conrad Lautenbacher, head of the National Oceanic and Atmospheric Administration.

National Weather Service forecasters said Tuesday they expect 13 to 17 tropical storms, with seven to 10 of them becoming hurricanes and three to five of them in the strong category. NOAA is the parent agency of the weather service.

David Paulison, director of the Federal Emergency Management Agency, said what keeps him up at night is concern about individual preparedness.

"If are going to survive these storms it takes all of us to be ready," Paulison said, urging that the millions of residents in vulnerable areas prepare their homes for the storms and keep at least three days food and supplies on hand.

After the battering by storms Katrina and Rita in 2005 there were widespread fears last summer of another powerful storm striking, but the unexpected development of the El Nino climate phenomenon helped dampen conditions.

El Nino is a warming of the tropical Pacific Ocean that occurs every few years. The warm water affects wind patterns that guide weather movement and its effects can be seen worldwide. In El Nino years, there tend to be fewer summer hurricanes in the Atlantic Ocean.

But El Nino is over and conditions could develop that might even encourage more storms, said Bill Proenza, head of the National Hurricane Center.

Earlier this month Philip Klotzbach, a research associate at Colorado State University, and Joe Bastardi, the chief hurricane forecaster for AccuWeather Inc., said they anticipate a more active storm cycle this year.

And, almost as if to underscore their comments, a subtropical storm formed off the southeast coast and became Andrea, the first named storm of the year, well before the June 1 official beginning of hurricane season.

Hurricane season ends Nov. 30, but the strange season of 2005 ran over into late December, as well as using up all the planned alphabetical names, forcing storm watchers to switch to the Greek alphabet to continue naming storms.

Last year, there were just 10 tropical storms in the Atlantic and just two made landfall in the United States.

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On the Net:

National Hurricane Center: http://www.noaa.gov

Colorado State: http://typhoon.atmos.colostate.edu/

AccuWeather: http://home.accuweather.com/

Museum for bluesman B.B. King has strong 1st year

The thrill is gone at many attractions across the country as recession-mired tourists stay home, but in Indianola, Miss., a favorite son is packing 'em in at the B.B. King Museum.

A year after its opening, the B.B. King Museum and Delta Interpretive Center has drawn about 30,000 visitors to the Mississippi Delta town roughly 100 miles northwest of Jackson where the Grammy-winning singer/songwriter/guitarist once made his living on a cotton plantation.

Attendance exceeded a first-year projection of 25,000, despite the museum's opening last fall amid hurricanes, high gas prices, economic woes and the end of the traditional summer travel season, said Connie Gibbons, the museum's executive director.

King, who was born in nearby Itta Bena and now lives in Las Vegas, celebrates his 84th birthday Sept. 16. He said the museum's strong launch "makes me feel real good," but he's not surprised by it.

"I have to tell you that even though it bears my name, the museum is as much about the history and culture of the Mississippi Delta as it is about me," King said in an e-mail. "People from all over the world are fascinated by this area that I've called home."

King won't be at a weekend event in Indianola to mark the museum's anniversary. He's planning to celebrate his birthday with friends and family away from the stage, said Tina France, managing director of B.B. King Road Shows LLC.

The museum was carved out of a cotton gin where King worked before he became a music icon. Half its $15 million cost came from fundraising by local business and community leaders. King attended the opening and was there again recently when an AT&T Mississippi-funded learning center opened as an extension of the museum.

"My only regret is that we didn't have anything like this when I was growing up here," King said in his e-mail.

For the $10 admission fee, visitors find artifacts spanning the arc of King's life _ including a frayed quilt from the home where he lived as a young man, a recreation of his home studio and various versions of his guitar, Lucille. King, a Grammy-winner for his anthem "The Thrill is Gone" and albums such as "One Kind Favor" and "Riding With The King," donated a number of his personal items, including his draft card.

Ron Franklin, a Texas lawyer in Jackson on business, took time Thursday to wander the exhibits. Franklin, from Houston, said he'd been to museums worldwide.

He said visiting the blues museum in Indianola and feeling the Mississippi Delta helped him better grasp the phrase, "the birthplace of the blues."

The museum also houses memorabilia of other artists, including handwritten lyrics by rocker Janis Joplin and art objects she made from yarn, on loan from the Rock-n-Roll Hall of Fame in Cleveland, said Gibbons.

Admissions, memberships and donations helped the museum meet its budget of $820,000 for the first year; it beat its projection of 25,000 visitors by about 20 percent.

They're strong indicators of solid organization, said Dewey Blanton, spokesman for the American Association of Museums in Washington, D.C., which provides museum accreditation and support services.

"Sometimes niche museums like this have a difficult time, particularly on the launch because of the appeal of the exhibits," Blanton said. "It speaks to the power of the museum's theme _ B.B. King has been performing for 50 years."

The slumping economy might have presented an additional challenge, he said.

However, although tourism venues such as Las Vegas have taken a body blow, museum attendance often rises in troubled times, Blanton said. He cited a spike after the 2001 terrorist attacks.

For Indianola and Sunflower County, where nearly 40 percent of the population lives in poverty, success is measured in tax collections.

Indianola's tax on hotel occupancy and meals for July was up 17 percent from a year ago, according to the state Tax Commission. Mayor Arthur Marble said sales tax collections are up 12 percent over the past year as tourists scoop up souvenirs including T-shirts and mugs licensed with King's image.

Marble said the increased tourist traffic has spurred work on community projects, including the town's park.

The King museum's first-year visitors included more than 2,000 Europeans, testament to the global appeal of the blues.

Marble said he's in discussions with Lise Wiik, mayor of Notodden, Norway, about forming a sister-city partnership. Notodden holds an annual blues festival, and a Norwegian delegation traveled to Indianola in June for King's annual homecoming concert.

"She brought some economic development individuals from over there, along with a guy who built a throne for the King of the Blues," Marble said. "Europeans seem very much in love with something that we have a very hard time recognizing ourselves."

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On the Net:

B.B. King Museum and Delta Interpretive Center: http://www.bbkingmuseum.org/

Daley: `It takes a village to teach a child'

It takes not only a village to raise a child but also to teach children, Mayor Richard M. Daley and educators said Tuesday on the first day of school.

Whether it was his appearance at newly opened Simeon Career Academy, 8147 S. Vincennes Ave., or the new and restructured Daniel Hale Williams Multiplex School, 2710 S. Dearborn, headed by "super principal" Frances Oden, Daley's message was the same--urging youth to stay in school and do their best.

On the first day of school, CPS CEO Arne Duncan said he is still tallying the attendance figures. However, both he, Daley, CPS President Michael Scott, Ald. Dorothy Tillman (3rd), Rep. Monique D. Davis (D-27th), Senator Ken Dunkin (D-5th), CHA CEO Terry Peterson, and educators, are hoping the first day attendance will exceed last year's.

The attendance figures won't be ready until today, according to Duncan. Ringing the bell to bring in the 2003-2004 school year, Daley appeared at the $40 million Simeon school where he told the students: "We hope to have a great year in school and continue the progress we have made by staying focused on our core initiatives such as reading, back-to-basics curriculum, preschool and after-school programs.

"We'll continue working hard day after day to lift every child in every school and give them the best education possible," he said.

"And, as the school year takes off, I want to stress the importance of keeping children involved in positive activities every afternoon after school through KidStart and the many school, library, park and community programs that are offered."

When appearing at the Daniel Hale Williams school, named after the Black physician who invented open heart surgery and was the founder of Provident Hospital, Daley was asked if he supported charging parents criminally. He said that is something that should be looked at.

But, Scott and Duncan said charging parents criminally for their truant children would be a "last resort."

"Do we want to prosecute? Absolutely not. That's not the goal," Duncan said. "But, if at the end of the day parents aren't taking responsibility for their children going to school, we need to do something about that."

To reduce truancy, Duncan said he is matching students on an individual basis with churches and not-for-profit groups "that can support them."

"Our attendance is at an all-time high this past year, but, we want to continue to get better, and we want to hold parents accountable as well."

Oden, who is over the four principals that are running the Daniel Hale Williams School, was principal of Beethoven for nearly 10 years.

Oden, who agreed it takes a village to teach a child, plans to bring the same academic excellence she did at her former school to the Dearborn facility.

Article copyright Sengstacke Enterprises, Inc.

Roadblock: Executive Management; Philip Morris International has yet to choose a system for labeling and tracking its cigarette packages all the way to the retail sale.

The Obstacle

Philip Morris International has yet to choose a system for labeling and tracking its cigarette packages all the way to the retail sale. Yet, the global arm of the world's largest tobacco manufacturer is now faced with $1.25 billion in penalties from the European Union, and the possibility of more if it doesn't overhaul the way it monitors its supply chain.

Getting top management behind spending tens or hundreds of millions of dollars on a technology initiative is no easy task. Here's how technology project leaders can help business executives make the right decision.

The Response:

Talk directly to your audience. Executives, from the finance chief to the head of production, have different priorities. Highlight the points that matter most to each member of the management team.

For example, in a Radio Frequency Identification project, the chief financial officer might be most interested in the savings that result from better product tracking and tracing.

Sales and marketing managers might be more interested to learn that RFID can be used to collect valuable demographic and time-to-delivery information that can be used to generate revenue, by improved use of marketing and sales dollars.

Honestly show how a technology will change day-to-day operations. Management may appear slow-moving when it's simply trying to determine what impact a new technology will have on a business day-to-day.

It's incumbent on the project leader to explain the ramifications of implementing a new information system. For instance, if the application of microcircuit tags during the packing process will slow down a production line, show how much, and what steps can be taken to limit the impact.

Be up front about the short-term pain but also ready to prove to management that, despite the obstacles, deploying a given technology will benefit the organization over the long term.

Think three steps ahead. There's no magic bullet that's going to, in this case, eliminate contraband and counterfeit cigarettes. Even if Philip Morris and other manufacturers put microcircuit tags on every pack, counterfeiters may eventually begin manufacturing their own tags, says Tim Scannell, an RFID analyst at Shoreline Research in Quincy, Mass.

Think three steps ahead about what new problems will emerge and how you will address them. How will you recognize counterfeit tags, for instance?

Show how other companies address problems. Study Wal-Mart's tagging project, as well as those of Target, Albertson's and the Department of Defense. Check out European efforts (Tesco, etc.). Find out what Gillette first did, why it pulled back, when it will once again try to tag every razor it sells, and what the bugaboos were.

Demonstrate that you're learning from others' mistakes - and applying the lessons.

Marquez, Blanco lead Mexico against New Zealand

World Cup veterans Rafael Marquez and Cuauhtemoc Blanco will lead Mexico against New Zealand in a friendly Wednesday at the Rose Bowl.

The game is the second in a series of six friendlies in the United States for Mexico, which will open the World Cup against host South Africa on June 11. France and Uruguay also will compete in Group A with Mexico and South Africa.

"I think this is the moment for us to do something big, something historic," said Marquez, who added that the 2010 World Cup will be his last. "We have players with lots of experience and young people who have the hunger to win."

Marquez, a 31-year-old defender, played in the 2002 and 2006 World Cups and helped Barcelona become the first Spanish team to win the Champions League, Spanish first division and Copa del Rey titles in one season.

"I think we have to play up to our standards and with the mental approach that we need and want," Marquez said. "We think about attacking as much as possible."

Blanco, who spent three seasons with Major League Soccer's Chicago Fire, is second in career goals for Mexico's national team with 37. The 37-year-old forward represented Mexico in the 1998 and 2002 World Cups. He now plays for Veracruz in Mexico's second division.

Joining Blanco and Marquez are Arsenal forward Carlos Vela, midfielders Andres Guardado from Deportivo La Coruna in Spain and Giovani Dos Santos from Turkey's Galatasaray, and CD Guadalajara forward Javier Hernandez, who leads Mexico's first division with eight goals in seven games.

"This is the only game on a FIFA date when we'd all be available," said Guardado, who suffered a leg injury last month while playing in Spain. "I'm still not 100 percent yet but for me, it's important to be here."

Marquez added that Javier Aguirre, who replaced Sven-Goran Eriksson as Mexico's coach in April, is using the friendlies to evaluate players and that playing in Europe holds no advantage.

"Javier wants to make it very clear that you have to work just like every other player, whether you play in Europe or Mexico," Marquez said. "I found that to be the case in each game, in each camp. Our confidence has grown, especially since Javier took over."

New Zealand, set to play in its first World Cup this year, will face Mexico without veteran defenders Ryan Nelsen and Ivan Vicelich.

Nelsen, the captain and a member of Blackburn Rovers in England's Premier League, has a hyperextended right knee. Vicelich, who leads New Zealand in all-time appearances with 65, is suspended.

Leading New Zealand is forward Rory Fallon, a regular starter with Plymouth Argyle in England's second division. Three members of New Zealand's team have American connections: defenders Andrew Boyens (New York Red Bulls) and Tony Lochhead (UC Santa Barbara) and midfielder Simon Elliott (San Jose Earthquakes).

At the World Cup, New Zealand will compete in Group F with Italy, Paraguay and Slovakia.

Elections May Shift U.S. Policy in Iraq

WASHINGTON - Democratic control of Congress, public dislike for the Iraq war and the departure of Defense Secretary Donald H. Rumsfeld could open the door for a policy shift in the conflict, but early maneuvering for the 2008 presidential election could slam the door shut.

What happens will depend largely on how the White House, leaders of both parties and the candidates to replace President Bush in two years interpret the results of this week's voting and seek political footing for the 2008 race, analysts said.

Although both parties want to salvage political and military success in Iraq for its own sake, the war's prominence as a political issue complicates bipartisan cooperation.

That may be especially true for Democrats if they conclude that anything shy of a demand for fast withdrawal of U.S. troops looks wishy-washy, or that the modest course changes possible by cooperating with Republicans would be jumping onto a sinking ship.

Cooperation is possible if both parties see it in their interest to lower the political temperature on Iraq, making it less of a rallying cry for the next campaign, said James Carafano, senior fellow at the conservative Heritage Foundation. He argued that both parties and the country would benefit.

"There is a remarkable opportunity to change the politics, for Democrats and the president to take the Iraq issue off the table as an issue to play political pingpong with," said Carafano, who studies politics and military issues.

More than half of voters said they disapproved of the war in Iraq, wanted troops to start coming home and didn't think the war has improved security in the United States, according to exit polls conducted Tuesday for The Associated Press and the television networks. Those most unhappy with the war helped put Democrats in control of Congress.

Democrats say the first step to repairing the situation in Iraq is putting substantial pressure on its government to take more responsibility. The best way to do this, they say, is by pulling out some troops right away to signal the U.S. commitment is finite.

Democrats also have called on Bush to convene an international conference on Iraq and say the military mission should begin to switch from a leading role to a supportive one.

Other proposals the administration may be asked to consider include a regional dialogue with U.S. adversaries Iran and Syria, or remaking the Iraq political federation into three largely autonomous sectarian states.

Major changes such as a wholesale withdrawal of troops are unlikely in the near term.

Bush's reversal of fortune this week means he can entertain ideas from his own generals and advisers that would have looked like an admission of failure before the voting, conservative and liberal analysts said.

The changed political circumstances also mean Bush can look statesmanlike by adopting recommendations from Democrats or from an independent bipartisan panel headed by former Secretary of State James A. Baker III, whose report is due soon.

Democrats who take control of Congress in January say they will try using their clout to force a change in Iraq policy and demand that Bush start bringing troops home.

Though Democrats are divided over exactly what to propose, they say their effort will send a loud political signal to disgruntled U.S. voters, and to Iraqis to assume more responsibility.

Rumsfeld's departure could offer a path to compromise and an opportunity for the White House to pivot from some of his hardline positions.

Bush chose a far less divisive figure, former CIA director Robert Gates, to succeed Rumsfeld. Bush came close to conceding that Rumsfeld was the roadblock to new policy that his critics claimed.

"Secretary Rumsfeld and I agreed that sometimes it's necessary to have a fresh perspective," Bush said in the surprise announcement Wednesday.

"I think history has shown that switching one person can make a difference," said Lawrence J. Korb, assistant defense secretary under President Reagan and now senior fellow at the liberal Center for American Progress.

Korb pointed to the Vietnam War, when Clark Clifford took over for hawkish Defense Secretary Robert McNamara.

"Within a month President Johnson had basically offered to begin negotiating with the North Vietnamese," Korb said.

Gates has served on the Iraq study commission led by Baker and former Democratic Rep. Lee Hamilton of Indiana, signaling that the group could offer the White House a palatable way to shift gears.

"I think there is a real possibility for a bipartisan approach to our foreign policy," said Sen. Joseph Biden, D-Del., expected to lead the Senate Foreign Relations Committee.

"The Baker-Hamilton commission obviously would be one of the most likely places to build that consensus and give the administration the necessary ... political room to be able to make a radical change. At least I hope that's what will happen," Biden said.

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Associated Press writers Will Lester, Anne Plummer Flaherty and Pauline Jelinek contributed to this report.

Land Rover, Acura aim to entice Gen X men

NEW YORK Want to get your vehicle noticed by male Gen Xers? Justput buxom video game heroine Lara Croft behind the wheel.

That's what Land Rover is doing this summer when its priceyDefender sport-ute gets a role in the much-anticipated movie versionof the video game "Tomb Raider," starring Angelina Jolie as Croft.

The "Tomb Raider" Defender, which made its U.S. debut lastThursday at the New York International Auto Show, is just one of manyattempts by automakers to attract young up-and-comers to theirbrands.

Case in point: the new 2002 Acura RSX coupe, also unveiledThursday and scheduled to go on sale in July. The RSX, which replacesthe Integra, "will attract a whole new generation of buyers" toAcura, said Dick Colliver, executive vice president of Acura.

The target buyer is 27 years old, with a college diploma and anannual income of $60,000, he said. With the RSX, priced from $20,000to $25,000, Acura now has a "stairstep" lineup, Colliver said. Youngcustomers can start out in the RSX and move up to larger, moreexpensive Acuras.

Although the Land Rover Defender's price tag is well above theRSX, the premium small sport-ute is under $30,000. Land Rover willbring it to the United States before year's end. With the Defender,Land Rover expects almost to double its annual sales in 2002.

Still, automakers are not forgetting older generations. Infinition Thursday unveiled its 2002 Infiniti I35, a car it hopes willattract the same affluent 40-somethings who buy the car it replaces,the I30. The I35 luxury sedan goes on sale in October.

At the same time, Infiniti is eyeing a younger crowd. Next spring,it will introduce a new production car based on its recent XVLconcept sedan. Infiniti is touting XVL, with its 3.0-liter V-6engine, as a performance car at an attainable price.

"I feel pretty strongly about what we've got in the pipeline,"said Mark McNabb, vice president and general manager of the Infinitidivision.

In other unveilings: Subaru pulled the wrapping off its six-cylinder Outback sedan, complementing the Outback wagon. Kia debutedits 2002 Sedona, the South Korean automaker's first foray into theminivan segment. Aston Martin, the luxury Ford Motor Co. division,showed a new flagship model, the Vanquish. The premier $228,000vehicle packs a V-12, 460-horsepower engine and no gear shifter.

Tracking single proteins within cells

ABSTRACT We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.

INTRODUCTION

As we move toward a more integrative view of cellular function, there is an increasing need to develop an accurate picture of the underlying microenvironment within the cell. It is clear that protein localization is crucial for accurate processing of cell signals, but little is known about how molecules manage to end up at the right place at the right time. Are macromolecules able to diffuse sufficiently rapidly to explore the full volume of the cell and find their destination? For large complexes, at what size must motors or other means of directed transport be employed for efficient targeting? Do mobilities vary with position in the cell and are they modulated? Questions such as these must be addressed before cellular processes can be accurately modeled. Using fluorescence recovery after photobleaching (FRAP) (Wolf, 1989) and related techniques, it has been found that macromolecules have significantly lower intracellular diffusion constants (De) compared with the corresponding diffusion constants in free (aqueous) solution (Di). This is in contrast to the case of small molecules, which appear to have comparable mobilities inside the cell and in water (Fushimi and Verkman, 1991; Luby-Phelps et al., 1993). A number of groups have found that DJD, decreases with increasing molecular weight for a variety of macromolecules and cell types (Luby-Phelps et al., 1986, 1987; Lang et al., 1986; Arrio-Dupont et al., 1996, 2000; Lukacs et al., 2000), leading to models in which the cytoskeleton and other cellular components act as sieves (reviewed in Luby-Phelps, 1994, 2000). Other experiments, however, indicate that DID, is independent of molecular weight, with D^sub c^ - D^sub w^/4, at least for measurements over short time scales (Seksek et al., 1997; Politz et al., 1998).

Most FRAP experiments have been analyzed in terms of a single diffusion constant. However, more complex diffusive behavior will not always be apparent in photobleaching data (Gordon et al., 1995; Periasamy and Verkman, 1998). An alternative technique, which is more sensitive to variations in particle dynamics, is single particle tracking. With this technique, the increase in sensitivity is gained at the expense of statistical sample size; the number of particles analyzed in tracking experiments is far smaller than the number of particles that contribute to the average quantities measured in a typical photobleaching experiment. Single particle tracking has been applied to two-dimensional diffusion in the plasma membrane and has provided evidence for a wide range of dynamics, including normal, confined, and anomalous diffusion, as well as directed motion (reviewed in Saxton and Jacobson, 1997). Recently, single particle tracking of particles diffusing in three dimensions in colloidal systems has been used to probe dynamics near the glass transition (Kegel and van Blaaderen, 2000; Weeks et al., 2000).

In this paper we describe the tracking of single proteins in the cytoplasm and nucleoplasm of mammalian cells. We use R-phycoerythrin (RPE), which is a 240-kDa autofluorescent protein consisting of seven subunits and -30 chromophores (reviewed in Glazer, 1982, 1988). As shown by x-ray diffraction and electron microscopy, the protein has the shape of a disk with a diameter of 11 nm and a thickness of 6 nm (Ficner et al., 1992, and references therein). Because of the high quantum yield and extinction coefficient of RPE, single proteins can be imaged with a standard epifluorescence microscope. RPE has previously been used to track the motion of labeled membrane proteins (Wilson et al., 1996) and to identify membrane-protein clustering (Cherry et al., 1998). After microinjecting RPE into cells, we were able to track the proteins in two dimensions (the projection of the three-dimensional trajectory onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus, at 14 ms/frame. We determined diffusion constants from an analysis of mean squared displacements for each trajectory. By comparing our results to the distributions obtained for diffusion of RPE in a glycerol solution and from a Monte Carlo simulation, as well as to theoretical distributions, we find that the distribution of intracellular diffusion constants is too broad to be accounted for by statistical fluctuations. We conclude that over the time scales of our measurements, the motion of RPE is not described by simple diffusion with a unique diffusion constant in either the cytoplasm or the nucleoplasm. These experiments describe the first time that trajectories of single proteins have been followed within cells and demonstrate that single molecule tracking is an effective tool for exploring intracellular dynamics.

MATERIALS AND METHODS

Cell culture

TC7 cells, an African green monkey kidney epithelial cell line, were a gift from D. Dean (University of South Alabama). Cells were cultured on homemade coverslip chambers in Dulbecco's Modified Eagle's medium at 37C with 5% CO2. The coverslip chambers consisted of 60-mm polystyrene Petri dishes with - 1.5-cm holes drilled in the bottom and 22 mm x 22 mm no. 1.5 coverslips (Fisher Scientific, Pittsburgh, PA) glued over the holes with 734 RTV sealant (Dow Coming, Midland, MI). Grids were scratched into the coverslips to aid in locating injected cells. Before we viewed cells on the microscope, the medium, which was quite autofluorescent, was replaced with Hanks' balanced salt solution containing 20 mM HEPES (pH 7.4) (Freshney, 1994).

Phycoerythrin

R-Phycoerythrin (RPE) (Molecular Probes, Eugene, OR), which was obtained as an ammonium sulfate precipitate, was dialyzed against 100 mM sodium phosphate, 150 mM sodium chloride (pH 7.2) at 4C with multiple changes of the dialysis solution. The RPE solution was then spun through a 0.2-jm sterile filter and stored at 4C. The diffusion constant and monodispersity of RPE were determined with a DynaPro-801 dynamic light scattering instrument (Protein Solutions, Charlottesville, VA). The peptide CYTPPKKKRKVED, which contains the nuclear localization sequence (NLS) from the SV-40 large-T antigen, was synthesized at the Rockefeller University peptide synthesis facility. NLS was conjugated to RPE with the cross-linker Sulfo-SMCC (Pierce, Rockford, IL), following the manufacturer's protocols. The RPE-NLS product was dialyzed intol00 mM sodium phosphate, 150 mM sodium chloride (pH 7.2). From the shift of RPE-NLS relative to RPE on sodium dodecyl sulfate polyacrylamide gels, we estimate that each protein contained 10-20 NLS peptides (data not shown).

Microinjection

Micropipettes were pulled from borosilicate glass (I turn outer diameter, 0.78 mm inner diameter) (Sutter, Novato, CA) on a P-87 puller (Sutter). Pulled pipettes were back-filled with a few microliters of the injection solution and then topped off with light mineral oil (Sigma, St. Louis, MO) to prevent capillary suction of buffer into the pipette. The protein concentration in the injection solution was -30 Ng/ml. Injection pressure was controlled by a Picospritzer II (General Valve, Fairfield, NJ) and was in the range of 10-25 kPa. RPE was injected into the cytoplasm or nucleus as indicated. RPE-NLS was injected into the cytoplasm, and the cells were incubated for 1 h at 37C with 5% COZ to allow for efficient nuclear import.

RPE in glycerol solution

RPE was diluted to -0.3 gg/ml in a solution consisting of 1 mg/ml bovine serum albumin (Sigma), 100 mM sodium phosphate, 150 mM sodium chloride (pH 7.2). Glyercol was then added to a concentration of 50% (by weight), and a small amount of 0.2 jim red fluorescent microspheres (Molecular Probes) was added to aid in focusing. Three microliters of this solution was deposited on a microscope slide, and a 18 mm x 18 mm no. 1.5 coverslip (Fisher Scientific) was placed on top. The edges of the coverslip were sealed with paraffin.

Microscopy

Microscopy was carried out at room temperature (-23C) on an Olympus IX-70 inverted microscope (Olympus, Melville, NY) with a 100X UplanApo lens (NA 1.35) (Olympus), equipped with a HQ545/30 excitation, Q565LP dichroic, HQ610/75 emission filter cube (Omega, Brattleboro VT). Illumination for fluorescence was provided by a 150-W xenon lamp (Optiquip, Highland Mills, NY). Images were acquired with an I-Pentamax-512EFT intensified charged-coupled device camera (512 X 512, 15(mu)m X 15 Am pixels) (Princeton Instruments, Trenton, NJ) cooled to -25C, with the intensifier gain at 50%. A region of interest of size -73 pixels X 100 pixels was defined, and sequences consisting of 250 consecutive At = 14.09 ms exposures (71 frames/s) were acquired into memory of a PC, using the image acquisition program WinView 32 (Princeton Instruments).

Image analysis

RESULTS

Imaging single proteins of RIDE

To verify that we were detecting single proteins, we first imaged RPE adsorbed on a coverslip (Fig. 1). When images were acquired at 71 frames/s, we observed fluctuations or blinking in the fluorescence emission from single spots (Fig. 2). Similar blinking has been seen in single molecule imaging of GFP (Pierce et al., 1997; Dickson et al., 1997) and other fluorescent molecules (Trautman and Macklin, 1996; Lu and Xie, 1997) and is probably due to fluctuations in the local environment around the fluorophores. We have three lines of evidence supporting the claim that the spots in Fig. 1 are images of single proteins: 1) Histograms of peak intensities from sequences like those in Fig. 2 show a single peak (Fig. 3). This suggests that the particles have a welldefined stoichiometry. 2) The intensity from individual spots frequently jumps back and forth from maximum intensity to baseline (Fig. 2). Such transitions would be relatively rare if the spots corresponded to random protein aggregates. (Note that the fluorophores within RPE are coupled through resonance energy transfer (Glazer, 1989). It is possible that this cooperativity is partially responsible for the observed fluctuations between the maximum and zero emission.) 3) From dynamic light scattering measurements (data not shown) we found that RPE in solution consisted of particles of a single size with a Stokes radius of 5.6 nm (corresponding to a diffusion constant of 40 pa.m2/s), which is comparable to the size of RPE (Ficner et al., 1992). Furthermore, on the microscope we occasionally obtained sequences of particles moving onto the coverslip from free solution. This indicates that the particles on the coverslip were of the same form as the particles in solution. We therefore conclude that the spots in Fig. 1 are single proteins of RPE.

Tracking RPE in the cytoplasm

When RPE was injected into the cytoplasm of TC7 cells and images were acquired at 70 frames/s (14 ms/frame), sequences were obtained showing the motion of distinct proteins. Using our own image analysis software, particle positions were determined from intensity maxima, and trajectories were identified (see Materials and Methods). The analysis of these trajectories was complicated by the fact that we only measured the lateral (x-y) displacement of the proteins, corresponding to the projection of the protein trajectories onto the plane of focus. There were three factors that potentially limited the amount of time that RPE could be tracked with this approach: photobleaching, fluorescence fluctuations, and motion in the z (vertical) direction. From sequences of images of cells containing a large number of RPE proteins, we measured the total fluorescence intensity as a function of time under continuous illumination. The resulting curves are well described by an exponential decay, with a time constant of 3 s (data not shown). Thus proteins under continuous illumination from the excitation source had mean lifetimes of -3 s before they were photobleached. Another factor that could limit trajectory length was the fluctuations in RPE fluorescence emission described above. When the fluorescence from a single RPE protein dropped to a value comparable to the background fluorescence as a result of these fluctuations, the protein could no longer be detected. To get a bound on the typical time for this to happen, we used our particle-tracking program to identify (static) trajectories for RPE imaged on coverslips. The mean number of consecutive frames for which a single RPE could be detected above background was 40 (n = 87), which corresponds to 560 ms. The corresponding value for RPE in cells would be lower, however, because of the higher background fluorescence. The final factor that could limit trajectory length was the disappearance of proteins that moved too far out of the plane of focus. We were able to detect particles within a depth d above and below the plane of focus. This distance depends on the depth of field of the objective lens, which determines how rapidly the peak intensity in the image drops as the protein moves away from the plane of focus, and on the background fluorescence, which sets the minimum intensity for which we are able to detect proteins. If the proteins were characterized by a diffusion constant D, then the typical time for which they could be tracked before they moved too far from the plane of focus was -d2/D. As a rough estimate, if we take d - 1 lim and D - 3 jim2/s, we get a time of -0.3 s or -20 frames. Note that for systems consisting of particles with different diffusion constants, there will be a bias toward detecting longer trajectories for the slower particles. This is a result of the tendency for the faster particles to move away from the plane of focus more rapidly, as well as the fact that the image of the faster particles will have a lower intensity per pixel because the particles transit more pixels in the course of the 14-ms exposure for each frame.

At least for the fraction of RPE proteins that were moving sufficiently slowly for their trajectories to be detectable, we were able to obtain trajectories consisting of 12-50 frames (14 ms/frame), with an average of 17 frames. (The lower bound of 12 was chosen to ensure that each trajectory had enough points to determine the mean squared displacement for a reasonable number of time points.) This range suggests that both blinking and motion in z were responsible for limiting the number of frames for which proteins could be tracked and that photobleaching was not a significant limitation.

An analysis of the distributions of the mean velocities and of the velocity-velocity autocorrelation functions for the acquired trajectories did not show any evidence of directed or correlated motion (data not shown), which suggests that the displacement in each frame was not correlated with the displacement in the previous frame for each particle. To extract information about the mobilities of the proteins, we computed the mean squared displacement MSD(t) for each trajectory (see Materials and Methods). For long trajectories of particles undergoing simple diffusion with diffusion constant D, MSD(t) = 4Dt. (The factor of 4 applies for twodimensional trajectories, including projections of three-dimensional simple diffusion onto a plane.) For short trajectories, however, MSD(t) has relatively large statistical fluctuations. In this case, linear fits to MSD(t) will give a distribution of diffusion constants (Qian et al., 1991; Saxton, 1997). We determined the distribution of D from the slopes of linear fits to MSD(t) for 978 trajectories of RPE proteins moving in the cytoplasm (Fig. 4 a). The distribution is quite broad, with a mean value of 2.7 /imz/s. As has been emphasized by Qian et al. (1991) and Saxton (1997), to draw conclusions about particle motion from single particle tracking data, it is important to compare the results with distributions from other experimental systems or from theoretical models. We therefore also tracked single proteins of RPE in free solution and carried out Monte Carlo simulations of simple diffusion.

The Monte Carlo simulation was used to generate sequences of images of particles diffusing in a three-dimensional volume (see Materials and Methods). The intensity of each particle was taken to be maximal at z = 0 (corresponding to the plane of focus) and fell off with deviations from z = 0 with a Gaussian profile. As for the case of the experimental data, particles sufficiently far from the plane of focus had intensities below the threshold of detection. The sequences of images were analyzed with the same particle tracking software that was used for the experimentally acquired sequences. The distribution of lengths of the resulting trajectories was comparable to the distribution obtained for RPE in the cytoplasm (data not shown). However, the resulting distribution of diffusion constants (with a mean value of D = 2.7(mu)m2/s) was clearly narrower than the distribution for intracellular RPE (Fig. 4, b and cd).

To track RPE in free solution, we decreased the diffusion constant by adding glycerol to a dilute solution of the protein. We then acquired and analyzed image sequences with the same procedure that was used for intracellular RPE. In a solution of 50% glycerol the average diffusion constant for RPE (from a linear fit to MSD(t)) was 2.7 lim 2/s, which is the value we obtained in the cytoplasm. However, as was seen with the simulation, the corresponding distribution of D was narrower than the distribution for RPE in the cytoplasm (Fig. 4, c and cd). By applying the KolmogorovSmirnov test (Press et al., 1993), we find the probability that the data for RPE in the cytoplasm (Fig. 4 a) and in glycerol (Fig. 4 c) are described by different distributions is >99.9%.

The particle trajectories may be thought of as two-dimensional random walks in which each particle takes a step corresponding to a displacement Ar in each frame. For each particle, the square of the displacements averaged over a particle's trajectory is related to the diffusion constant by (Ar2 )tj = 4DAt, where At is the time between steps. Again this relation strictly holds only in the limit of infinite trajectories; for finite trajectories there will be statistical fluctuations. Although the above relation holds if the particle positions are determined from instantaneous snapshots, there is a correction for the case described here. This is because each image that we acquired had a finite (14-ms) exposure time and thus contained information about particle positions over the entire At = 14 ms and not about the positions at a single instant. If we were to use the mean position of a particle in each frame, then the correct formula would be (Ar 2),aj = 4(2D/3)At (see the Appendix). However, we determined each particle's position by using the mean of the intensity maxima in each frame. (The background fluorescence and intensifier noise were too high to determine the positions from the mean of all pixels occupied by a protein during the 14-ms exposure.) As a result, the relation will take the form (Deltar 2)^sub traj^ = 4D'Deltat, with D' between 2D/3 and D. The quantity (Ar 2)taj (or, equivalently, D') is useful because we can compute its theoretical distribution for the case of simple diffusion (see Materials and Methods). For the simulation described above, the distribution of D' is fit well by the theoretical distribution (Fig. 5 b). Note that the mean value of D' = 2.3 tkm2/s is lower than D = 2.7 /im2/s, as expected. For cytoplasmic RPE, on the other hand, the distribution of D' is much broader than the theoretical distribution (Fig. 5 a). Note in addition that the mean value of D' = 3.5 gm2/s is larger than the mean D = 2.7 Am2/s obtained from linear fits to MSD(t). For RPE in glycerol, the distribution of D' is reasonably well fit by the theoretical distribution (Fig. 5 c), at least relative to the case of cytoplasmic RPE. Again, the mean value of D' = 4.1 /.mz/s is larger than the mean D = 2.7 gm2/s obtained from linear fits to MSD(t); we will return to this point below in the Discussion.

Tracking RPE in the nucleoplasm

We were also able to track single proteins after microinjecting RPE into the nucleus. Injection into such a small volume may create severe disruptions of the nuclear environment, which could significantly affect the mobility of RPE. We therefore also took a second approach in which we chemically conjugated a nuclear localization sequence (NLS) to RPE to load the nucleus in a less disruptive manner. After microinjection into the cytoplasm, RPE-NLS accumulated in the nucleus (Fig. 6). Sequences were acquired and analyzed in the same manner as for cytoplasmic RPE. The average trajectory length was 16 frames for both nuclear RPE and RPE-NLS. We again found broad distributions for diffusion constants obtained from linear fits to MSD(t) for both RPE (Fig. 7 a) and RPE-NLS (Fig. 7 b), with mean values of D = 1.1 gm2/s and D = 1.7 /m2/s, respectively. The distributions of D' (obtained from (Ar2),raj) for nuclear RPE (Fig. 8 a) and RPE-NLS (Fig. 8 b) were also much broader than the predicted distributions.

DISCUSSION

By taking advantage of the bright fluorescence of RPE, we have been able to track single proteins moving in the cytoplasm and nucleoplasm of TC7 cells for an average of 17 (cytoplasm) or 16 (nucleoplasm) frames at 14 ms/frame. The lengths of the acquired trajectories were limited by fluctuations in the fluorescence emission from single proteins of RPE and from the motion of the proteins out of the plane of focus. For short trajectories, averaged quantities have large statistical fluctuations. It is therefore important to present distributions of diffusion constants rather than simply mean values (Saxton, 1997; Qian et al., 1991). We have used two quantities to characterize protein trajectories: the average step squared (Ar2 )tj and the slope of a linear fit to the mean squared displacement MSD(t). The former quantity is equal to MSD(At), where At is the inverse frame rate (14 ms). We use the term "simple diffusion" to describe the Brownian motion of particles with a constant mobility in the absence of any potentials or constraints. For simple diffusion in three dimensions, the projection of particle trajectories onto a plane (the plane of focus, in our case) reduces the system to simple diffusion in two dimensions. The resulting two-dimensional trajectories will satisfy MSD(t) = 4Dt (and Ar2)taj = MSD(At) = 4DAt). There are a number of ways in which systems can deviate from simple diffusion. These include systems with directed motion, confined motion, and diffusion with space- or time-dependent mobilities. In all of these cases, MSD(t) will not be linear in t, at least over some time scales (see Saxton and Jacobson, 1997, and references therein).

For RPE in the cytoplasm, we obtained distributions for diffusion constants (derived from (Ar2 )lai and slopes of MSD(t)) that were broader than the distributions obtained for RPE in a glycerol solution with the same mean diffusion constant and from simulated and theoretical distributions for simple diffusion. The distributions of diffusion constants for RPE and RPE-NLS in the nucleoplasm were similarly broader than those expected for simple diffusion. The broad distributions indicate that over the time scales in our experiments (-10-100 ms), the motion of RPE does not correspond to simple diffusion with a unique diffusion constant in either the cytoplasm or the nucleoplasm. With the present data, however, we cannot determine if the system is best characterized by a few diffusion constants, a continuous distribution, or more complex behavior, such as confined or anomalous diffusion (see, e.g., Saxton and Jacobson, 1997).

The resolution of particle position in our measurements was limited by noise from the image intensifier and fluctuations in background fluorescence. In particular, when we acquired trajectories of static RPE (on coverslips), the trajectories show fluctuations in the range of one to two pixels around fixed points (data not shown). (One pixel corresponds to a distance of 0.15 Am in the plane of focus.) A stationary subpopulation of RPE within cells would appear as a peak near zero in the histogram for D. Thus, the large number of trajectories with D near zero for cytoplasmic RPE relative to simulation and RPE in glycerol (Fig. 4 d) could in part reflect the presence of a stationary subpopulation. However, stationary RPE cannot account for the entire difference between distributions. For RPE immobilized on coverslips, the distribution is peaked at values much closer to the origin than what is observed for cytoplasmic RPE. As a result, if immobile RPE within cells played a significant role in skewing the distribution of D to low values, then there would be a second peak in the lowest bin in Fig. 4 d.

The diffusion constant of RPE has been measured by FRAP to be 4.6 1.1 p,mZ/s and 4.4 1 1.3 /im2/s in the cytoplasm and nucleoplasm, respectively (Schulz and Peters, 1987). These values are higher than the values we obtained from taking means of the single-particle data (D = 2.7 (mu)m ^sup 2^/s for cytoplasmic RPE, 1.1(mu)m^sup 2^/s for nucleoplasmic RPE, 1.7 Am2/s for nucleoplasmic RPE-NLS). The experiments of Schulz and Peters (1987) were performed on HTC cells instead of on TC7 cells, which could account for part of the discrepancy. However, the most likely explanation is that our means are lower because of the bias toward slow trajectories in our analysis. It is difficult to identify trajectories for proteins moving too quickly. Even when the protein remains within the field of view, if it has moved too far in two successive frames, then it is no longer clear whether it is the same protein. Note that this bias toward slow trajectories indicates that the actual distributions are even broader than those measured here.

As discussed in the Appendix, for trajectories acquired from images with a finite exposure time, the average value of D' = (Ar2 )taj/(4At) is lower than the average value of the diffusion constant D obtained from linear fits to MSD(t). This is evident for the case of our simulation. For both RPE in glycerol and intracellular RPE, however, D' is greater than D. This is most likely due to the fluctuations in background fluorescence and intensifier noise, which will result in fluctuations about the correct particle positions. These fluctuations average out over long time scales. Hence, a linear fit to MSD(t) will be less sensitive to these fluctuations than will (Ar2 )raj, which measures deviations on the shortest time scale At.

For diffusion in the cytoplasm, the most likely explanation for the variation in protein mobility is that it stems from interactions of RPE with intracellular constituents such as the cytoskeleton, membranes, or high concentrations of other macromolecules, mobile or immobile, within the cell (see, e.g., Luby-Phelps et al., 1986; Zimmerman and Minton, 1993; Luby-Phelps, 2000). These assemblies could act as obstacles that impede the motion of soluble proteins or, alternatively, provide a dense collection of sites to which RPE transiently binds. This would account for the fact that intracellular diffusion constants are lower than the corresponding free solution values (Jacobson and Wojcieszyn, 1984; Luby-Phelps et al., 1986, 1987; Lang et al., 1986; Arno-Dupont et al., 1996, 2000; Seksek et al., 1997; Lukacs et al., 2000).

The fact that we observe what appears to be a range of mobilities for RPE suggests that different RPE proteins experience different average environments over the length and time scales probed by our experiments. This could be due to a heterogeneous distribution of the material responsible for slowing RPE. Alternatively, the probability of RPE interacting strongly with intracellular components could be sufficiently low that the fluctuations in mobility due to these interactions are large. The situation is similar to the case of two-dimensional diffusion in the plasma membrane, where single particle tracking has also revealed a broad distribution of diffusion coefficients (Kusumi et al., 1993; Lee et al., 1993; Sako and Kusumi, 1994; Saxton and Jacobson, 1997). In this case it has been suggested that the variation in mobilities is due to interactions with the membrane skeleton, the extracellular matrix, or membrane microdomains (reviewed in Saxton and Jacobson, 1997). Models of twodimensional diffusion in the presence of obstacles and binding sites and the resulting distributions of diffusion coefficients have been explored by Saxton (1997). Models of anomalous diffusion in obstructed geometries in three dimensions have been applied to diffusion in organelles by Olveczky and Verkman (1998).

Much less is known about the structure of the nucleoplasm than about the structure of the cytoplasm (Pederson, 1998). Again, FRAP experiments demonstrate that diffusion is slower in the nucleus than in free solution (Lang et al., 1986; Schulz and Peters, 1987; Seksek et al., 1997; Politz et al., 1998). Our single particle tracking data indicate that nuclear RPE and RPE-NLS are not characterized by simple diffusion, based on a comparison with the predicted distribution for (Ar2 ).aj. Interestingly, we find that the distributions of diffusion constants for RPE and RPE-NLS are comparable. This suggests that interactions between the positively charged NLS with DNA, transport factors, or other nuclear proteins do not significantly affect the mobility within the nucleus. The mechanism for accumulation of NLS bearing proteins in the nucleus is not understood. One class of models is based on the idea of nuclear retention (Paine, 1993, and references therein), in which the NLS binds to constituents of the nucleus in such a way that diffusion of protein-NLS out of the nucleus is prevented. Although our results do not rule out these models, they indicate that any potential interactions leading to nuclear retention do not restrict the mobility of the protein-NLS.

The experiments described here provide the first example of intracellular single protein tracking. With improvements in the experimental system, this technique could provide a wealth of information on protein mobility within the cell. Increased sensitivity and frame acquisition rate, as well as particle tracking in three dimensions, will result in better statistical sampling and allow comparisons with specific models of diffusive transport. In addition, by microinjecting RPE that has been conjugated to other proteins, or possibly conjugating RPE to a protein of interest directly within the cell (Farinas and Verkman, 1999), it may be possible to track endogenous proteins. The single particle tracking experiments described here are a first step toward probing intracellular processes at the single protein level.

We thank S. Burley for the use of his light-scattering equipment and D. Dean for providing us with TO cells. We also thank G. Blobel, D. Dean, A. Libchaber, E. Siggia, Y. Chen, N. De Souza, D. Marciano, D. Peters, and J. Schmoranzer for many helpful discussions.

This work was supported by the Molecular Biophotonics Laboratory (Hamamatsu City, Japan). During part of this work, MG was a W. M. Keck Fellow.

[Reference]

REFERENCES

[Reference]

Arno-Dupont, M., S. Cribier, G. Foucault, P. F. Devaux, and A. d'Albis. 1996. Diffusion of fluorescently labeled macromolecules in cultured muscle cells. Biophys. J. 70:2327-2332.

Arno-Dupont, M., G. Foucault, M. Vacher, P. F. Devaux, and S. Cribier. 2000. Translational diffusion of globular proteins in the cytoplasm of cultured muscle cells. Biophys. J. 78:901-907.

Cherry, R. J., K. M. Wilson, K. Triantafilou, P. O'Toole, I. E. Morrison, P. R. Smith, and N. Fernandez. 1998. Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging. J. Cell Biol. 140:71-79.

Dickson, R. M., A. B. Cubitt, R. Y. Tsien, and W. E. Moerner. 1997. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature. 388:355-358.

Farinas, J., and A. S. Verkman. 1999. Receptor-mediated targeting of fluorescent probes in living cells. J. Biol. Chem. 274:7603-7606.

Ficner, R., K. Lobeck, G. Schmidt, and R. Huber. 1992. Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution. J. Mol. Biol. 228:935-950.

Freshney, R. 1. 1994. Culture of Animal Cells: A Manual of Basic Technique. Wiley-Liss, New York.

Fushimi, K., and A. S. Verkman. 1991. Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. J. Cell BioL 112:719-725.

Glazer, A. N. 1982. Phycobilisomes: structure and dynamics. Annu. Rev. Microbiol. 36:173-198.

Glazer, A. N. 1988. Phycobiliproteins. Methods Enzymol. 167:291-303.

Glazer, A. N. 1989. Light guides. Directional energy transfer in a photosynthetic antenna. J. Biol. Chem. 264:1-4.

Gordon, G. W., B. Chazotte, X. F. Wang, and B. Herman. 1995. Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components. Biophys. J. 68:766-778.

[Reference]

Hoel, P. G. 1962. Introduction to Mathematical Statistics. John Wiley and Sons, New York.

Jacobson, K., and J. Wojcieszyn. 1984:The translational mobility of substances within the cytoplasmic matrix. Proc. Natl. Acad. Sci. USA. 81:6747-6751.

Kegel, W. K. and A. van Blaaderen. 2000. Direct observation of dynamical heterogeneities in colloidal hard- sphere suspensions. Science. 287: 290-293.

Kusumi, A., Y. Sako, and M. Yamamoto. 1993. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65:2021-2040.

Lang, I., M. Scholz, and R. Peters. 1986. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J. Cell Biol. 102:1183-1190.

Lee, G. M., F. Zhang, A. Ishihara, C. L. McNeil, and K. A. Jacobson. 1993. Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids. J. Cell Biol. 120:25-35.

Lu, H. P. and X. S. Xie. 1997. Single-molecule spectral fluctuations at room temperature. Nature. 385:143-146.

Luby-Phelps, K. 1994. Physical properties of cytoplasm. Curr. Opin. Cell Biol. 6:3-9.

Luby-Phelps, K. 2000. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. 192:189-221.

Luby-Phelps, K., P. E. Castle, D. L. Taylor, and F. Lanni. 1987. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc. Natl. Acad. Sci. USA. 84:4910-4913.

Luby-Phelps, K., S. Mujumdar, R. B. Mujumdar, L. A. Ernst, W. Galbraith, and A. S. Waggoner. 1993. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65: 236-242.

Luby-Phelps, K., D. L. Taylor, and F. Lanni. 1986. Probing the structure of cytoplasm. J. Cell Biol. 102:2015-2022.

Lukacs, G. L., P. Haggie, 0. Seksek, D. Lechardeur, N. Freedman, and A. S. Verkman. 2000. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275:1625-1629.

Olveczky, B. P. and A. S. Verkman. 1998. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles. Biophys. J. 74:2722-2730.

Paine, P. 1993. Nuclear protein accumulation by facilitated transport and intranuclear binding. Trends Cell Biol. 3:325-329.

Pederson, T. 1998. Thinking about a nuclear matrix. J. Mol. Biol. 277: 147-159.

Periasamy, N., and A. S. Verkman. 1998. Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys. J. 75:557-567.

Pierce, D. W., N. Hom-Booher, and R. D. Vale. 1997. Imaging individual green fluorescent proteins. Nature. 388:338.

Politz, J. C., E. S. Browne, D. E. Wolf, and T. Pederson. 1998. Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. Proc. Natl. Acad. Sci. USA. 95:6043-6048.

Press, W. H., S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery. 1993. Numerical Recipes in C: The Art of Scientific Computing. Cambridge University Press, New York.

Qian, H., M. P. Sheetz, and E. L. Elson. 1991. Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys. J. 60:910-921.

Sako, Y., and A. Kusumi. 1994. Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometerlevel motion analysis. J. Cell Biol. 125:1251-1264.

Saxton, M. J. 1997. Single-particle tracking: the distribution of diffusion coefficients. Biophys. J. 72:1744-1753.

Schulz, B., and R. Peters. 1987. Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis. Biochim. Biophys. Acta. 930:419-431.

[Reference]

Seksek, O., J. Biwersi, and A. S. Verkman. 1997. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell Biol. 138:131-142.

Trautman, J. K., and J. J. Macklin. 1996. Time-resolved spectroscopy of single molecules using near-field and far-field optics. J. Chem. Phys. 205:221-229.

Weeks, E. R., J. C. Crocker, A. C. Levitt, A. Schofield, and D. A. Weitz. 2000. Three-dimensional direct imaging of structural relaxation near the colloidal glass transition. Science. 287:627-631.

[Reference]

Wilson, K. M., I. E. Morrison, P. R. Smith, N. Fernandez, and R. J. Cherry. 1996. Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging. J. Cell Sci. 109:2101-2109.

Wolf, D. E. 1989. Designing, building, and using a fluorescence recovery after photobleaching instrument. Methods Cell Biol. 30:271-306.

Zimmerman, S. B. and A. P. Minton. 1993. Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu. Rev. Biophys. Biomol. Struct. 22:27-65.

[Author Affiliation]

Mark Goulian and Sanford M. Simon

Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York 10021 USA

[Author Affiliation]

Received for publication 10 March 2000 and in final form 16 June 2000.

Address reprint requests to Dr. Mark Goulian, Department of Physics, University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-573-6991; Fax: 215-898-2010; E-mail: goulian@physics.up.edu.

Legal group: Gays shouldn't answer Pentagon survey

Concern about unintentional outings, discriminatory language expressed

WASHINGTON (AP) -Defense Secretary Robert Gates is urging gays in the military to answer a Pentagon survey on the policy that bans them from serving openly, but advocacy groups worry the poll may be biased against gays or that those who participate could be exposed and expelled.

"I strongly encourage gays and lesbians who are in the military to fill out these forms," Gates said of the poll e-mailed to some 400,000 soldiers, sailors, airmen and Marines. "We organized this in a way to protect their privacy and the confidentiality of their responses ... and it's important that we hear from them as well as everybody else."

Gates told a Pentagon press conference that the survey - distributed July 7 to 200,000 active duty military and 200,000 national guard and reservists - is crucial to an effort to broadly measure the views of men and women in uniform on the policy.

The Servicemembers Legal Defense Network said July 8 that the Defense Department has not agreed to grant immunity to anyone inadvertently outed during the survey, intended to help a special working group decide how repeal of the policy might be implemented and how that could affect the military.

"At this time SLDN cannot recommend that lesbian, gay, or bisexual service members participate in any survey being administered by the Department of Defense, the Pentagon Working Group or any third-party contractors," Aubrey Sarvis, the defense group's director, said in a statement.

Another group, Servicemembers United, said it was concerned about "unintentional bias" in the wording of survey questions but was satisfied it would not violate the confidentiality of participating gays. The Pentagon is not publicly releasing the survey, but some draft questions the group had learned about were homophobic, director Alexander Nicholson said in an interview. For instance, one asked troops if they would be comfortable sharing bathrooms or housing with gays and lesbians.

"Servicemembers United encourages all gay and lesbian active duty troops who received the survey to take this important opportunity to provide their views," Nicholson said in a statement.

A third group, the Palm Center, did not object to the survey, saying it was part of an agreed-to process of dismantling the "Don't Ask, Don't Tell" policy under which gays can serve as long as they don't reveal their sexual orientation.

Aaron Belkin, director of the center, said his group did not expect the survey to support repeal. "That said, we welcome the results and value the feedback of all the troops," Belkin said. "We will pay close attention to this process."

The Pentagon says the survey is being conducted by an outside contractor who will strip out all identifying data. "They cannot be outed," said Cynthia Smith, a Pentagon spokeswoman.

Smith noted that the survey doesn't ask whether a respondent is gay. It asks, among other things, about service members' experience in serving with people they believed were gay and their attitudes about how repealing the law might affect recruiting, privacy and unit cohesion.

Officials stressed that the survey is not a referendum.

Servicemembers United has released what it refers to as the complete DADT survey. It can be downloaded at http://servicemembersunited.org/survey.

[Sidebar]

Another group, Servicemembers United, said it was concerned about "unintentional bias" in the wording of survey questions but was satisfied it would not violate the confidentiality of participating gays.