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Click here to listen to WIZARD'S DUEL
And one more snippet from SigerFest for now. This is one more short story Scott wrote in his senior year of high school, and published in the Young Author's Showcase. You've heard all about Scott's travails on the football field in high school, right? I promise you haven't heard much about this. This one? It has rapping. And wizards.
Again, I'd recommend SiglerFest 2012 if you can make it. It's going to be a grand ol' time fore sure. In the meantime, take a listen to what you missed out on back in May!
AUTHOR'S NOTE: Scott here, adding in a note. This might be the most-embarassing story I have ever created. I wrote this as a senior in high school, when Run DMC, Beastie Boys, NWA, Salt-n-Peppa and Kid-N-Play were ripping off faces all over the world. I'd read some of the SPELLSINGER series by Alan Dean Foster, and felt that hip-hop would be "bitchin' " (I belive that was the word I used at the time) as a poetic form of spell-casting. I'm sure I thought this was fantastic when I wrote it. Enjoy a laugh at my expense.
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4 Comments
Can't wait to listen to this again! Highly entertaining!
[flickr-photo:id=3710325452,size=m] [flickr-photo:id=4738086914,size=m]
*Proud member of GirlCo and The Gutter Sisteren. The CBBC Crack Mamma*
HahahahahahahahahahaThis is awesome! I would love to see Hunter Hunterson hunting an evil rappin' wizard.
[flickr-photo:id=4722743287,size=m] Dragon Lady of DØØM & Co-Founder of Gutter Sistren
With Hunter, all things are possible ...
Since Hunter is my fun "this is magic so we can just do whatever without form or structure" story, you never know what might show up.
I admit...I lol'ed hard!!!
That was pretty good, I like how Scott had to work up to bustying his "Sic rhymes". lol :)
Does someone go into the park and, uh... lift up the dinosaurs' skirts?
Does someone go into the park and, uh... lift up the dinosaurs' skirts?


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I would tell my friend for sure. Although what happens after that I would keep out of as it's not my child.
I do believe in age appropriate discipline, and over 5 minutes is way too long for a child that age, let alone the distress. Chances are he probably doesn't even remember what he did at that point. So sad!!

subset: commoncrawl


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Barista Prima Coffeehouse House Blend Coffee K-CupsCoffee
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"A sprawling indictment of eight U.S. administrations. A convincing, urgent argument." Kirkus Reviews
"The descriptions of Hendon's. . . personal mission provide an intriguing story--and carry the ring of truth." Publishers Weekly
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An Enormous Crime is nothing less than shocking. Based on thousands of pages of public and previously classified documents, it makes an utterly convincing case that when the American government withdrew its forces from Vietnam, it knowingly abandoned hundreds of POWs to their fate. The product of/i>/b>/b>/i>
An Enormous Crime is nothing less than shocking. Based on thousands of pages of public and previously classified documents, it makes an utterly convincing case that when the American government withdrew its forces from Vietnam, it knowingly abandoned hundreds of POWs to their fate. The product of twenty-five years of research by former Congressman Bill Hendon and attorney Elizabeth A. Stewart, this book brilliantly reveals the reasons why these American soldiers and airmen were held back by the North Vietnamese at Operation Homecoming in 1973, what these brave men have endured, and how administration after administration of their own government has turned its back on them.
This authoritative exposé is based on open-source documents and reports, and thousands of declassified intelligence reports and satellite imagery, as well as author interviews and personal experience. An Enormous Crime is a singular work, telling a story unlike any other in our history: ugly, harrowing, and true.
"A sprawling indictment of eight U.S. administrations. A convincing, urgent argument." Kirkus Reviews
"The descriptions of Hendon's. . . personal mission provide an intriguing story--and carry the ring of truth." Publishers Weekly
An Enormous Crime
After months of training at secret bases in Guatemala, twelve hundred Cuban freedom fighters departed in ships from Puerto Cabezas, Nicaragua, in mid-April 1961 bound for the Bay of Pigs on the southern coast of Cuba. Their primary mission: Land, establish a beachhead, and hold it long enough to allow a provisional government to be put ashorea government that would then receive recognition and overt aid from the United States and from anticommunist nations throughout Latin America, and whose forces, if all went as planned, would quickly drive Fidel Castro from power. The U.S. government had trained and equipped the brigade, planned, approved, and financed the invasion, and assured the freedom fighters that U.S. air strikes would destroy the handful of planes comprising Castro's air force on the ground before the invasion was launched. Secure in this assurance that the skies above the beachhead would be theirs, the men of Brigade 2506 went ashore at 2:30 A.M. on Monday, April 17, with high hopes that the overthrow of Fidel Castro was at hand.
These hopes were dashed when the promised preinvasion bombing raids were first cut back and then canceled altogether on orders from Washington. With Castro's air force still intact, the invaders were doomed. By Tuesday, the trapped rebels were pleading by radio for U.S. air cover, telling of government tanks sending merciless crossfire into their ranks and of MiGs diving again and again to strafe men armed only with rifles, machine guns, and bazookas. No assistance was dispatched, however, and the rebels were overwhelmed.
At 5:30 Wednesday afternoon, all organized resistance ceased when the final rebel position at Playa Girón fell. Within hours, the Cuban government announced that its troops had "destroyed in less than seventy-two hours the Army which was organized during many months by the imperialist government of the United States. All the mercenaries," Havana radio declared, " ... are either dead or prisoners awaiting action by revolutionary tribunals."
The surviving prisoners were taken to Havana that Friday and paraded before the nation's television cameras. Premier Castro then took to the airwaves and delivered a four-and-a-half-hour report to the nation on the invasion. Clearly elated by the rout at Bahía de Cochinos, the victorious Castro charged that President Kennedy was personally to blame for the invasion and ridiculed the American president as an "international bully" who should be likened to Adolf Hitler. Noting that the American government was already calling for clemency for theprisoners, Castro mocked the U.S. gesture, declaring, "They should have asked clemency for the children who were killed by their bombs." All prisoners captured during the invasion, the Cuban leader declared, were considered counterrevolutionaries who "must be shot."
Operation Pluto, the U.S.-backed invasion of Cuba, had ended in total disaster.
Mid-May 1961
John Kennedy knew that Brigade 2506 was no band of cutthroats. Many of the freedom fighters, in fact, were not unlike the president himselfcharismatic, brave, patriotic, from wealthy Catholic families. Among them were Cuba's finest, grandsons of the old Spanish aristocracy and scions of assorted fortunes. As one observer noted, in capturing them it was as if Castro had captured "the entire Havana Yacht Club." But now here they wereherded around on television like cattle, charged with treason and facing possible executionand, in the minds of many, all because the American president, acting as mediator between quarreling advisors rather than as a forceful commander in chief, had personally ordered the rebels' air cover withdrawn in the critical early hours of the invasion. The fact that all this weighed heavily on the young president may explain why he moved so quickly when he heard of Castro's offer.
The Cuban premier had been addressing a farmers' rally in Havana on May 17 when he told his audience that a good way to increase agricultural output might be for Cuba to trade the approximately twelve hundred prisoners captured during the invasion to the United States in return for five hundred bulldozers or farm tractors. Within hours of being advised of Castro's remarks, Kennedy was on the phone to Eleanor Roosevelt, asking the former first lady to chair a committee to raise funds to purchase five hundred tractors for Cuba if Castro would free the twelve hundred imprisoned rebels. Mrs. Roosevelt agreed to serve, as did Walter P. Reuther, president of the United Automobile Workers Union, and Dr. Milton S. Eisenhower, president of Johns Hopkins University and brother of the former president. On May 19, only two days after Castro made his offer, committee members wired the Cuban leader to advise him of the formation of the Tractors for Freedom Committee and of their intention to undertake a nationwide drive in the United States to raise the funds necessary to purchase the tractors. "We do this," they told Castro, "as proof that free men will not desert those who risked all for what they thought was right." The three signers concluded by inviting the Cuban leader to immediately send representatives to the United States to work out details for the proposed swap.
Within hours of receiving the committee's offer, Castro paroled ten of the Bay of Pigs prisoners and dispatched them to Washington with his demand: five hundred Caterpillar Tractor Company Super D-8 bulldozers, two hundred equipped with disks for plowing and three hundred with bulldozer blades. Castro estimated that the five hundred bulldozers would cost approximately $28 million. He further informed the committee that the swap was neither an exchange nor a ransom; rather, he said, the bulldozers were to be considered as "indemnification" for war damage caused during the invasion.
Although the committee would have preferred giving Castro only light farm tractors, and found the matter of "indemnity" to be a flagrant effort to direct world attention to America's losing role in the invasion, members chose not to quibble over specifications or semantics. Instead, following their session with the paroled prisoner-representatives, the committee announced it was prepared to meet Castro's demands. Walter Reuther, committee cochair, told a news conference that the committee had given a "firm commitment" to the prisoner delegation that the specified equipment would be sent to Cuba. He also said a similar pledge had been sent to Castro. The committee estimated the cost to be as high as $20 million depending on whether or not the Cuban president continued to insist on the top-of-the-line Super D-8 Caterpillars.
As the Cuban prisoner-negotiators departed for Cuba with the deal, President Kennedy issued a direct personal appeal to all Americans to contribute toward the purchase of the tractors. Calling the prisoners "our brothers," the president expressed confidence that "every American would want to help." He stated that all contributions to the Tractors for Freedom Committee would be tax deductible, and he let it be known that he, as a private citizen, would likely make a contribution to the tractor fund himself. He further declared that the State Department would move quickly to issue previously outlawed export licenses for the tractor shipments and stated emphatically that the Logan Act, a law prohibiting private individuals from negotiating with foreign governments, would not be invoked against the Tractors for Freedom Committee.
The American people responded to the president's appeal by sending thousands upon thousands of contributions to the Tractor Committee's "Post Office Box Freedom" near UAW headquarters in Detroit. Volunteers in cities all across the country moved quickly to form local subsidiary committees to raise funds for the effort. Similar committees also sprang up in a dozen countries throughout Central and South America. In Rio de Janeiro, the president of the Roman Catholic Youth Association opened a drive to buy five tractors to be sent to Cuba in exchange for the prisoners, while in nearby São Paulo, students dressed in prison garb paraded through the streets collecting funds.
The reaction was markedly different, however, on Capitol Hill, where, according to Time magazine, a "large part of hell broke loose" over the president's remarks on the tractor swap. Members of both houses of Congress, already angry over Castro's repeated insistence that the tractors be considered reparations for damages caused during the invasion, took to the floor threatening to strip the Tractor Committee of its tax-exempt status and make committee members subject to all provisions of the Logan Act. Still others demanded the State Department halt its issuance of export licenses for the tractors. The attacks were broad-based and bipartisan, with the most serious concern being voiced over the precedent the payments would set. Eloquently addressing the matter of precedent, Democratic Senator Thomas Dodd of Connecticut warned, "Our national concern for the plight of the Cubans ... should have been evidenced by effective help on the beachhead to enable their just revolution to succeed. By paying Castro's price for a thousand good men, we give him the means to strengthen his enslavement of 6,000,000 others. The American people will, for the first time to my knowledge, be making use of ransom and tribute as an instrument of policy.If we start to pay tribute now for 1,000 of the one billion communist hostages, where will it stop?"
Former Vice President Richard Nixon, speaking at the Oklahoma State Republican Convention in Oklahoma City, declared the Kennedy plan "morally wrong" and urged his former rival to withdraw his support of the transaction. Nixon declared that America had "decided 100 years ago that human lives are not something to be considered as material or to be bartered on the slave block." To continue with the trade, the former vice president said, "would encourage every tinhorn dictator around the world to try to take advantage of America."
As the political controversy mushroomed, opponents of the plan seized upon the possibility that Castro might use the bulldozers for purposes other than those envisioned by the committee. They began asking, Why such big bulldozers? Did Castro plan to build military installations and missile sites, instead of using them to help his people grow more food? Critics and editorial writers began to liken the proposal to the infamous 1944 offer by Nazi Adolf Eichmann to trade Hungarian Jews for winterized military trucks, one hundred Jews for every truck.
On June 19, unable to defuse the explosive question of possible military use of the bulldozers, the embarrassed committee withdrew its pledge to supply the five hundred Super D-8s. In a telegram to Castro, the members bluntly informed the Cuban premier, "We are prepared to ship [five hundred] agricultural tractors and no other type." The committee then gave the Cuban leader seventy-two hours to accept their offer. Castro replied that he would be willing to forgo the bulldozers and accept the smaller tractors, but because his government considered the U.S. contribution to be indemnity for war damages, the total value of the tractors must approximate the value of the bulldozers, i.e., $28 million. Committee members rejected Castro's proposal out of hand, and when the seventy-two-hour deadline passed at noon on Friday, June 23, announced that the committee was ceasing all operations and returning all contributions received.
For the second time in as many months, John Kennedy had retreated in his high-stakes dealings with Fidel Castro.
Spring 1962
Fidel Castro put the Bay of Pigs prisoners on trial for treason in the spring of 1962. The mass trial, held in early May in the courtyard of Havana's Principe Prison, lasted only four days and resulted in guilty verdicts for all 1,179 defendants. Their punishment: thirty years' imprisonment or payment of fines ranging from $25,000 to $500,000 each, the higher amount for each of the three invasion leaders. Casting an eye toward Washington, the military tribunal ruled that payment of the fine would release the individual prisoner from having to serve his thirty-year sentence. Total fines levied: $62 million.
Within hours of the reading of the verdicts in Havana, John Kennedy jumped at this unexpected second chance to free the Bay of Pigs captives. Thistime he turned to his brother Robert, the attorney general, to raise the money and get the prisoners home.
Using a Cuban refugee organization as a front, Robert Kennedy immediately dispatched the organization's "counsel," famed New York attorney James Donovan, to Havana to negotiate a deal with Castro. Following several months of talks, Donovan, who had negotiated the release of U-2 pilot Francis Gary Powers from the Soviets, struck a deal with the Cuban leader: $53 million payable in medicines, powdered milk, and baby food in return for all the prisoners.
Before the deal could be consummated, the Cuban missile crisis intervened and contact between the two sides was suspended. But when the missile crisis subsided in November, Castro sent word that he was still interested and contact was reestablished. With that, Robert Kennedy and a group of trusted advisors went to work raising the money, with the goal of getting the prisoners home by Christmas. Operating out of offices in the Justice Department, the group made hundreds of calls to executives of pharmaceutical, medical, surgical supply, and food products companies seeking donations for the prisoner deal. On dozens of occasions, Robert Kennedy met personally at the Justice Department with company executives to seek their assistance. At one such meeting, the attorney general reportedly told pharmaceutical executives "my brother made a mistake" at the Bay of Pigs and implied that the prisoner exchange would help rectify that. He followed his remarks with a strong appeal for donations of medicines. Similar appeals for assistance were made to executives of the nation's airline, railroad, and steamship companies. Sometimes the persuasion was less than gentle. The Minneapolis Tribune reported that a spokesman for one large corporation facing a government lawsuit said his company received a call from the Department of Justice directing it to supply specific items, plus a specific amount of cash. "We knew we were being blackjacked," the spokesman said, "but there was nothing we could do about it."
Though some of his tactics may have been open to question, the results of Robert Kennedy's efforts were nothing short of astounding. As described in U.S. News & World Report at the time:
The first shipment of the Castro ransom, 32,000 pounds of medicines, was flown from New York to Miami during the night of December 17 ... . After that first shipment, the floodgates were down. By air, truck, and railroad, hundreds of thousands of pounds of baby food, canned goods, medicine [and] medical supplies poured into Florida from every corner of the U.S ... . Eight domestic airlines began flying 600,000 pounds of supplies to Florida; 19 railroads soon had 80 boxcars on this special run; eight trucking firms were moving 420,000 pounds of supplies from distant points, and 15 shipping companies had put up a ship and the money to move its cargo to Havana ... . It was a logistical operation almost without parallel except in wartime.
Castro released the prisoners just in time for Christmas. The first four planeloads of freedom fighters arrived to a tumultuous welcome at Homestead Air Force Base outside Miami on the evening of December 23, and by late Christmas Eve all surviving prisoners from Brigade 2506 were back in America. Two days after Christmas, the president and Jacqueline Kennedy met privately with the leadership of the brigade at the winter White House in Palm Beach. Then, on December 28, the Kennedys joined the entire brigade and forty thousand wildly cheering Cuban exiles at a welcome home rally at Miami's Orange Bowl. Thunderous cheers rocked the stadium as first the president and then his wife praised the men for their bravery and commitment to freedom. The crowd repeatedly interrupted the president with shouts of "Viva Kennedy" and "Libertad por Cuba," especially when he pledged to see the rebels return to liberate their homeland. Mrs. Kennedy's remarks, delivered in Spanish, evoked equal enthusiasm, with the crowd shouting "Viva Jackie, viva Jackie" and "Jack-k-leen, Jack-k-leen, Jack-k-leen."
After personally greeting many of the freed prisoners who were in formation near the podium, the president and first lady got into their open-topped limousine to depart. As they rode slowly along the sidelines, standing in the back of the open car and shaking the outstretched hands of the exiles, few doubted that John Kennedy had fulfilled his responsibility to those he sent to the Bay of Pigs. The ransomed men of Brigade 2506 were finally home.
Within a week of the Orange Bowl rally, Communist leaders from throughout the world gathered in Havana for ceremonies marking the fourth anniversary of Fidel Castro's rise to power. Following a two-hour parade of military might before a reviewing stand containing some four hundred notables in the Communist world, Castro delivered the keynote address. The Cuban premier, clearly angered by Kennedy's remarks at the Orange Bowl the previous week, derided Kennedy as a "vulgar pirate chief" and openly mocked his decision to pay what Castro insisted was "indemnification" to Cuba for damages the United States caused during the Bay of Pigs invasion. "For the first time in history," Castro boasted, "imperialism has paid war indemnification. They call it ransom. We don't care what they call it. They had to agree to pay indemnification." 1
The delegation from North Vietnam listened attentively.
AN ENORMOUS CRIME. Copyright © 2007 by William M. Hendon and Elizabeth A. Stewart. All rights reserved. For information, address St. Martin's Press, 175 Fifth Avenue, New York, N.Y. 10010.
"The descriptions of Hendon's. . . personal mission provide an intriguing story—and carry the ring of truth." —Publishers Weekly
Former U.S. Rep. Bill Hendon (R-NC) served two terms on the U.S. House POW/MIA Task Force (1981–1982 and 1985–1986), as a consultant on POW/MIA affairs with an office in the Pentagon (1983), and as a full-time intelligence investigator assigned to the Senate Select Committee on POW/MIA Affairs (1991–1992). He has traveled to South and Southeast Asia thirty-three times on behalf of America's POWs and MIAs. Hendon is considered the nation's foremost authority on intelligence relating to American POWs held after Operation Homecoming and is an expert on the Vietnamese and Laotian prison systems. He lives in Washington, D.C.
Elizabeth A. Stewart's father, Col. Peter J. Stewart (USAF), is missing in action in North Vietnam. His name appears on Panel 6E, Line 12, of the Vietnam Veterans Memorial in Washington, D.C. She has spent more than two decades researching intelligence relating to American POWs and MIAs. Her efforts have taken her from Capitol Hill to Cambodia, from the South China Sea to the presidential palace in Hanoi, and to the most remote regions of northern Vietnam. An attorney, she lives in Winter Haven, Florida.
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reading this book was very disturbing to think the u.s. government could actually do this. the book was easy to read and should be read by anyone interested in the vietnam war and its so called ending.
A Long suppressed and rumored story literally continuing to stink to the ends of the firmament. Under told for obvious reasons. Ignored in all relevant quarters by tacit collusion toward burying this odious pile of human defecation for decades. Ignored by a fetid MSM that doesn't even possess a curious brain or bent or even exist for the public good. Something that should forever sully and stain the honor of American politicos of all stripes. This should be a "national read and retch," but it won't! Why? It's better to bury it and forget we could commit such a crime! God Help US. Oh I forgot! We no longer have the freedom to credibly utter such pious things!


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Hotel Scheepers, Valkenburg aan de Geul
- Free WiFi
Main amenities
- 21 smoke-free guestrooms
- Breakfast available
- Bar/lounge
- Self parking
- Airport shuttle
- Terrace
- Coffee/tea in a common area
- Air conditioning
- Daily housekeeping
- Lobby fireplace
- Front desk safe
- Snack bar/deli
For families
- Cribs/infant beds (surcharge)
- Free toiletries
- Terrace
- Daily housekeeping
- Laundry facilities
- Hair dryer
What's around
- Heart of Valkenburg aan de Geul
- Netherlands American Cemetery & Memorial (0.1 mi / 0.2 km)
- Theodoor Dorrenplein (0.1 mi / 0.2 km)
- Museum Land van Valkenburg (0.2 mi / 0.4 km)
- Valkenburg Castle (0.4 mi / 0.6 km)
- Valkenburg Cable Car (0.4 mi / 0.7 km)
Hotel Scheepers, Valkenburg aan de Geulfrom $73
- Family Room
- Double Room Single Use
- Comfort Twin Room
- Comfort Double Room, 1 Bedroom
- Comfort Double Room, Jetted Tub
- Comfort Twin Room, 1 Bedroom, Accessible, Ground Floor
- Double Room
- Triple Room
- Family Room, Jetted Tub
More great choices
At a glance
Key facts
Hotel size
- This hotel has 21 rooms
- This hotel is arranged over 2 floors
- Check-in time 2 PM-11 PM
- Check-out time is 11:30 AM
Airport shuttle service is available on request. Contact the property in advance to make arrangements.
Required at check in
- Credit card deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- Pets allowed *
- Specific rooms only *
- 1 per room (up to 10 kg)
- Free WiFi and wired Internet access in public areas
- Free WiFi in rooms
- Airport shuttle (available on request) *
- Self parking *
Other information
- Smoke-free property
- Resort fee included
Nearby Three Star Valkenburg aan de Geul hotels
In the hotel
- Breakfast (surcharge)
- Bar/lounge
- Coffee shop/café
- Snack bar/deli
- Room service (during limited hours)
- Coffee/tea in a common area
- Front desk (limited hours)
- Tours/ticket assistance
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 1
- Year Built 1835
- Elevator/lift
- ATM/banking
- Safe-deposit box at front desk
- Terrace
- Fireplace in lobby
- Television in common areas
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- Air conditioning
- Individually decorated
- Designer toiletries
- Hair dryer
- Flat-screen TV
- Digital TV channels
- Desk
- Free WiFi
- Phone
- Daily housekeeping
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Also popular with travelers from the USA
Special features
On Site
- Bowling alley on site
- Miniature golf on site
- Horse riding/rental on site
- Mountain biking on site
- Bicycle rentals nearby
- Golf lessons available nearby
- Golfing nearby
- Hiking/biking trails nearby
- Scooter/moped rentals nearby
- Segway rental/tour nearby
Awards and affiliations
- This hotel is listed in the 2014 Condé Nast Traveler Gold List of the world's best places to stay and the 2014 Travel + Leisure 500.
Hotel Scheepers, Valkenburg aan de Geul's small print
Also known as
- Hotel Scheepers Valkenburg aan de Geul
- Scheepers Valkenburg aan de Geul
Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation.
This property's policy is to refuse certain bookings for the purpose of group events or parties, including pre-wedding stag/bachelor and hen/bachelorette parties.
Only registered guests are allowed in the guestrooms.
For transfers, guests must contact the property before arrival at the number on the booking confirmation.
The name on the credit card used at check-in to pay for incidentals must be the primary name on the guestroom reservation.
Special cancellation policies or charges may apply for group reservations (more than 8 rooms for the same property / stay dates).
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tax is imposed by the city: EUR 1.76 per person, per night. This tax does not apply to children under 7 years of age.
Pet deposit: EUR 5 per night
The charges below are included in the total price displayed:
- Resort fee
Optional extras
Late check-out can be arranged for an extra charge of EUR 12.50
Self parking costs EUR 7.50 per night
Breakfast is offered for an extra charge of EUR 10.50 for adults and EUR 9.50 for children (approximately)
Cribs/Infant beds are available for EUR 12.50 per stay
Pets are allowed for an extra charge of EUR 7.50 per pet, per night
Airport shuttle service is offered for an extra charge of EUR 15.00 per vehicle (one way)
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.2 /5 from 40 reviews
Hotel is 5 minute walk to train station. 15 minute train ride to Maastricht. Close to shops and restaurants. Great rooms. Friendly staff. Highly recommend.
Lovely place with very friendly staff. Will come again!
2 nights in Valkenburg. Lots of restaurants and historic town. Great place to stay fir Andre Rieu concert. Cheap train to Maastrich takes an out 20 mins and 4 trains an hour.
Hotel and staff we great, great choice at breakfast. We were above the bar so it was quite noisy at times but all went quiet at 11.00 when bar closed. Quite central everything within walking distance.
ideal spot ,very friendly staff
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The boat arrived in time and we got a nice private trip to the airport, I should have booked the same thing for the arrival.
It was extremely easy and convenient to get us from our hotel in Lido right to the airport at 6 a.m. Also a very beautiful boat and ride! Our driver was quick and on time! Highly recommend this service.
A quick and easy way to transfer from the Airport to Venice, my teenage children and I enjoyed it.
Julie Ann G Australia
We received a message to our hotel the day before departure as stated for departure time. Our water taxi left on time.
Erkki L Finland
The only right way for come to the city...... :
A hassle free transfer - airport to hotel water-taxi. Takes the stress out of figuring out how to get from the airport to your hotel. Will definitely use them again.
We booked the transfer from our home in the U.S. and the viator rep and vendor in Venice were superb in confirming our reservation and even have a message left for us the day before departure at our hotel so that we know where to be to wait for the private water taxi to transport us to the airport!!! great service and worth the investment!!! Love the service and will book again anywhere they service
Excellent service, found the desk where we met the agent, he showed us where to collect the transfer water taxi. Very helpful. Had a fax the day before departure letting us know time and where the transfer would be. Very good service, would recommend.
it was the only way to get from the airport to the hotel, the ride was nice got to see allot of Venice as we traveled to our hotel.
Right on time, three other friendly couples enjoyed the trip with. Highly rated!!!
Excellent, pick up on time and lovely way to leave the islands in Venice.
This was a seamless way to get airport.
This was a shared ride in a water taxi from the city of Venice. We were 4 couples who were picked-up at different hotels and were taken to the Venice airport . Service was prompt and well coordinated, with the help of the hotel concierge who printed the voucher with the appointed pick-up time. The ride lasted about 1 hour. Be advised that there is perhaps 5 blocks to walk from the dock at the Marco Polo airport to reach the terminal.
Excellent Service, collected on time from our hotel and transported to airport with a nice sightseeing tour of Venice on route, good value for money and would highly recommend.
The transfer was like clockwork. A nice boat, on time, and a beautiful experience. The shared transfer is no problem only a few stops - or none - to pick up other people and then zip across the lagoon.
They were early, and helped with luggage. Easiest way to the airport.
Very good
Great service, was 10 minute early to pick up time so lucky we were early but wonderful service to book. Only downfall is long walk from water taxi stand into terminal.
Stress free way to leave Venice
The boat driver was punctual helpful and the trip was quick.
Hassle free way to get to airport.
Worth the money for a stress free end to a lovely holiday.
Would certainly use again.
I was worried about getting out to the airport in Venice. I was traveling solo, so I had to pay for two spots. They had a minimum of two to make the stop. It was still worth it.
Excellent way to get from airport to hotel. Highly recommended
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Totally Impressed with Ship: Carnival Fantasy Cruise Review by wnorman
Overall Member Rating
Totally Impressed with Ship
Destination: Bahamas
Embarkation: Charleston
I must say we have always had very friendly waiters in the dining room but I did NOT find that this time. We did anytime dining and found only the workers at the front of the restaurant were friendly. Never experienced this before. Quite disappointing that the staff were not friendly during dinner. Not that we did not get good service, because we did. Usually when we do anytime dining we find waiters who we want to continue serving us More the whole week but that was not the case this time.
Ports: was so looking forward to Grand Turk as we had never been here before. Was totally disappointed with the island - I guess I was expecting more. We got off ship and purchased hop on hop off bus for 19.00 which I thought was a great deal but really nothing to see. Margarittaville was big and busy.
Nassau: had been before but never did an island tour. Got off ship and purchased local city tour for 20 bucks each. Waited til we had 8 people and off we went. It was incredible!!!! Supposed to be 2 hours but we were gone for 3. After tour, we went to Junkanoo beach which is right by the ship. Amazing time there - got jerk chicken and beers at a stand on the beach and it was great. Much better than cabbage beach where we had been before. No hassels at all!!!!!!
Half Moon Cay - LOVED IT!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Gorgeous! Powder sand. It was my favourite. Not much to do other than enjoy the beach and water. Carnival moves the ship to the island - their drinks and barbeque lunch.
Totally enjoyed our sea days!!!!!!!!!!!!!!!!!!!! The whole week the ocean was like glass, totally calm. Have never experienced a whole week like this. Never felt ship move whole week.
Getting off ship, was good experience also. QUICK!!!! We then spent 3 days in the Historic District at the Days Inn on Meeting Street. Loved hotel location and Charleston. We could walk everywhere.
I would definitely go on Fantasy again. Less
Published 10/28/13
Cabin review:
loved location
Read Cruise Critic's Carnival Fantasy Review >>
Unimpressed on many levels aft...
An Almost Perfect Little Cruis...
Spotless big ship.
Great 5 Night Cruise on the Ca...
It was ok, not our best cruise...
Had a great time!!
Great 5 Night Cruise on the Ca...
An Almost Perfect Little Cruis...
Worst cruise EVER
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The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.
Normal inheritance of genetic traits from one cell or organismal generation to the next depends on accurate chromosome replication and segregation. Defective chromosome segregation is associated with birth defects and cancer. The centromere is a single site on the chromosome that is responsible for assembling the kinetochore, which mediates chromosome attachment to the microtubule spindle and all chromosome movements. In addition, the spindle assembly checkpoint (SAC) ensures normal inheritance by delaying entry into anaphase when chromosome–spindle attachments are defective. Previous studies suggested that SAC function required kinetochore localization of key components. This study shows that elimination of a centromere-specific histone (CID) results in an early mitotic delay. Although this delay occurs earlier than the established time of SAC function (at the metaphase–anaphase transition), it depends on the presence of an essential SAC protein (BUBR1). Furthermore, the CID-mediated early mitotic delay occurs in the absence of kinetochore formation or localization of key SAC proteins. These results suggest that the fidelity of kinetochore–microtubule attachment is also monitored early in mitosis, and in the absence of kinetochore formation and localization of SAC components.
Citation: Blower MD, Daigle T, Kaufman T, Karpen GH (2006) Drosophila CENP-A Mutations Cause a BubR1- Dependent Early Mitotic Delay without Normal Localization of Kinetochore Components. PLoS Genet 2(7): e110. doi:10.1371/journal.pgen.0020110
Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America
Received: November 9, 2005; Accepted: May 31, 2006; Published: July 14, 2006
Copyright: © 2006 Blower et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by NIH R01 GM066272 to GK.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: SAC, spindle attachment checkpoint
Proper kinetochore assembly and function is essential for the faithful transmission of chromosomes during both mitosis and meiosis. One critical function of the kinetochore is to serve as the site of the mitotic spindle attachment checkpoint (SAC), which monitors kinetochore microtubule attachment prior to anaphase onset [1,2]. It is hypothesized that the SAC monitors the attachment of kinetochore microtubules during prometaphase and metaphase, and inhibits anaphase progression until all chromosomes have achieved bipolar spindle attachment. The current model for SAC function suggests that unattached kinetochores recruit checkpoint proteins (such as components of the MAD, BUB, and ZW10/ROD protein complexes), and that these proteins are modified by unattached kinetochores to generate a diffusible signal that delays the onset of anaphase. It has recently been demonstrated that defects in SAC function result in organismal lethality and dominant haploinsufficiency defects. Haploinsufficiency for mouse MAD2, BUB3, or RAE1 results in elevated rates of chromosome missegregation, defects in SAC function, and a predisposition to cancer [3–5], demonstrating the fundamental importance of this checkpoint.
The role of kinetochore localization of SAC components in the generation of the anaphase delay [6] has come into question recently with the published disruption of several inner kinetochore proteins. Disruption of Nuf2 or Ndc80/Hec1 in human cells results in a mitotic arrest, despite the fact that several outer kinetochore components (including Mad2) are unable to sustain kinetochore localization [7,8]. Similarly, disruption of human or chicken CENP-H or CENP-I (the homolog of Schizosaccharomyces pombe Mis6) arrests cells in mitosis for hours, despite the mislocalization of a variety of outer kinetochore proteins, including some SAC components [9–11]. In contrast, disruptions of components of the SAC result in premature entry into anaphase, rather than mitotic arrest or delay. These results suggest that continuous kinetochore localization of SAC components may not be necessary to signal the cell to delay anaphase onset. However, incomplete depletion of the inner kinetochore proteins could produce partially functional kinetochores in which some outer proteins localized properly, while others were mislocalized [8,11,12]. A partially dysfunctional kinetochore could recruit sufficient amounts of SAC proteins to generate an effective checkpoint signal, consistent with a requirement for SAC component recruitment to kinetochores.
A key component of the inner kinetochore is the centromere-specific histone H3-like protein CENP-A [13]. Several recent studies have demonstrated that CENP-A proteins are present in all eukaryotes, and that these proteins are essential for both cell and organismal viability [14–20]. CENP-A proteins replace both copies of histone H3 in centromeric nucleosomes, and physically and genetically interact with all other core histones [14,21–23]. CENP-A proteins are at or near the top of the kinetochore assembly pathway, and are required for the localization of nearly all other kinetochore proteins examined to date, including all tested SAC components [13]. Therefore, cells lacking CENP-A would be expected to contain chromosomes with severely compromised kinetochores, which would be incapable of generating the SAC signal.
Here we report that mutations in the Drosophila CENP-A family member (CID, for "Centromere Identifier") [24] result in an early mitotic delay. Furthermore, cid mutants have an intact SAC response to microtubule disruption despite the absence of kinetochore localization of SAC components ROD and BUBR1. We present data that suggest that the DNA damage/repair checkpoint is not responsible for the CID-mediated early mitotic delay. In contrast, the mitotic delay of cid mutants is partially suppressed by mutation of the SAC component bubr1. We discuss models for the role of SAC proteins in monitoring aspects of kinetochore assembly early in mitosis.
CID Null Mutants Display Embryonic Lethality
In a previous study, we found that anti-CID antibody injections into syncitial embryos resulted in phenotypes expected for kinetochore disruption (failure to congress in prometaphase, metaphase arrest, and anaphase segregation defects), but also produced unusual phenotypes (interphase and prophase arrests). However, it was unclear if these phenotypes were the consequence of loss of CID function, an artifact of antibody binding to CID, or a consequence of the specialized nature of the syncitial nuclear divisions. Therefore, we examined the phenotypic consequences of cid null mutations in Drosophila embryos. The alleles examined were T11–2 (Q51 to stop), T12–1 (Q83 to stop), T21–3 (Q94 to stop), and T22–4 (Q102 to stop) (J. Cecil and T. Kaufman, unpublished observations). All of these alleles are lethal when homozygous, in trans-heterozygous combinations, and over a deficiency for the region (unpublished data); thus, the cid gene is essential for Drosophila development.
To examine the phenotypic consequences of cid disruption, crosses were made between parents heterozygous for a cid mutation and a balancer that contained an ElaV-LacZ fusion construct, which is expressed in the developing nervous system [25]. We collected embryos from these crosses and stained them for CID, LacZ, histone H3 phosphorylation at serine 10 (PH3) [26], and DNA (DAPI). cid null and heterozygous embryos were unambiguously distinguished by the absence or presence (respectively) of ElaV-LacZ expression. cid null mutant embryos died around stage 15 of embryogenesis, and displayed a phenotypic series that correlated with the temporal disappearance of maternal CID protein and the absence of newly synthesized zygotic protein. At embryonic stages 9–10, cid null embryos displayed lagging chromosomes during anaphase and unresolved chromatin bridges during telophase, which were not observed in heterozygous controls (Figure 1A and 1B). These phenotypes are the result of partial loss of CID protein; staining with anti-CID antibodies demonstrated that maternally derived CID is still present in stage 9–10 embryos, albeit at reduced levels (Figure 1A and 1B). Lagging chromosomes and chromatin bridges are entirely consistent with the phenotypes we observed after partial disruption of CID by RNAi or antibody injection [17,27].
CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different stages of development were monitored to evaluate mitotic progression and segregation defects.
(A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic defects and robust CID staining at kinetochores (inset).
(B) cid null animals (trans-heterozygous for different cid alleles, see Materials and Methods) exhibited lagging chromosomes during anaphase. Some CID staining was still visible at this stage, demonstrating that these phenotypes resulted from partial loss of CID function, due to the presence of maternal CID protein.
(C) cid/CyO stage 14–15 embryos show normal mitotic progression and normal CID staining at kinetochores (inset).
(D) cid null animals exhibited an elevated mitotic index, lower nuclear density, and little detectable CID staining in some cells at stage 14–15. The strong depletion of CID staining suggests that these phenotypes are the result of complete loss of zygotic cid function. cid null animals have a large number of presumably polyploidy cells (inset) suggesting high levels of aneuploidy due to repeated failures in cell division. Scale bar indicates 15 μm.
In later stages of development (stages 13–15), high levels of CID staining were observed in heterozygous siblings, whereas in cid homozygous mutant embryos, most cells in mitotically active tissues had no visible CID signal (Figure 1C and 1D). The PROD protein binds a satellite DNA near the Chromosomes 2 and 3 centromeres [28], and its localization is not dependent on the presence of CID [17]. Comparison of the levels of CID and PROD staining in homozygous mutant and heterozygous control embryos suggest that approximately 90%–100% of CID was depleted in stage 15 cid mutants (Figure S1, see Materials and Methods). Thus, some cells retain small amounts of maternal CID, and these alleles behave as nulls with respect to functional zygotic protein, as predicted from the early stop codons present in the mutations.
Homozygous cid null embryos displayed few defects associated with gross morphological patterning or development (unpublished data). However, defects were associated with the organization of the developing nervous tissue, consistent with the fact that few other cell types are actively dividing in later stage embryos, and that the most severe defects are also associated with the nervous tissue in other mitotic mutants that die during embryogenesis [29]. These later stage, terminal embryos displayed a high degree of disorganization of the developing nervous tissue, with obvious micronuclei, large presumably polyploid nuclei, very few true metaphase plates, and few anaphases and telophases. The overall nuclear density was much lower in the cid mutants (~1/2 of heterozygous controls), which is also consistent with the aneuploidy that results from failures in chromosome segregation and cell division. These phenotypic characteristics are very similar to a recently described mutation in Drosophila CENP-C [30], suggesting that these defects result from disruption of the inner kinetochore.
CID Disruption Results in an Early Mitotic Delay
The appearance of H3 phospho-serine 10 (PH3), destruction of the mitotic cyclins, and mitotic spindle morphology can be used to discriminate different stages of G2 and mitosis (Figure 2A). Cyclins A and B begin to accumulate in S and G2 phases [31], and PH3 begins to appear in late G2, and is used as a general marker for mitotic index [32]. Subsequently, cyclin A destruction is observed during prometaphase, cyclin B destruction occurs at the metaphase to anaphase (M:A) transition, and PH3 staining is gradually lost from chromosomes at the end of telophase.
Cell cycle progression was monitored in cid/CyO and cid/cid embryos by staining for PH3, cyclin A, cyclin B, and tubulin.
(A) Schematic diagram of the appearance and destruction of various cell cycle regulatory factors and markers. ANA, anaphase; META, metaphase; PRO, prophase; PRO-META, prometaphase; TELO, telophase.
(B) cid/cid animals displayed an elevated mitotic index, and an increased number of cells in prophase and prometaphase, compared to cid/CyO controls. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase.
(C) cid/cid animals had a 2-fold higher number of cyclin A and B positive cells than cid/cid controls. Scale bars indicate 15 μm.
To determine the effects of cid depletion on cell cycle progression, the number of cells in specific stages of mitosis was determined by staining homozygous and heterozygous mutant stage 15 embryos for tubulin, cyclin A, cyclin B, and PH3. Three observations demonstrated that cid mutants were delayed early in mitosis, predominantly in prophase/prometaphase. First, cid mutants displayed a 2.4-fold higher mitotic index (p < 0.01) and a 2-fold higher number of cells positive for cyclin A (p ≤ 0.01) and cyclin B (p ≤ 0.01), in comparison to heterozygous control siblings (Figure 2B and 2C). Second, cid mutants showed a marked increase in the number of cells in prophase and prometaphase, as judged by chromosome and spindle morphology (Figure 2B). Third, very few cells progressed to anaphase in cid mutants, suggesting that cid mutants were delayed prior to the metaphase–anaphase transition (Figure 2B). Thus, complete depletion of CID in embryos results in a mitotic delay, predominantly in prophase and prometaphase.
Inactivation of the DNA Damage Checkpoint Does Not Abrogate the cid-Mediated Mitotic Delay
The mitotic delay observed in homozygous cid mutant embryos suggested that cid depletion and failure to form a kinetochore activated a cell cycle checkpoint. A recent study in Xenopus suggested that DNA damage and repair may be involved in CENP-A assembly at centromeres [33]. Therefore, incomplete kinetochore chromatin assembly or chromosome segregation errors caused by cid mutation could result in DNA damage and activation of the DNA damage checkpoint, which would mediate the early mitotic cell cycle delay. To determine whether DNA damage phenocopies the cid null mutations, we treated cid mutant and heterozygous embryos with doxorubicin, a topoisomerase II inhibitor known to generate dsDNA breaks [34]. We found that doxorubicin treatment dramatically decreased the mitotic index of cid heterozygous embryos (Figure 3A–3C), consistent with previous studies of the effects of DNA damage on cell cycle progression [35]. Doxorubicin treatment had little effect on the mitotic index of cid homozygous mutant embryos, which likely reflects the fact that these cells were already delayed in mitosis at the time of drug addition. Importantly, the decreased mitotic index in cid heterozygotes demonstrates that a DNA damage-induced cell cycle delay results in a fundamentally different (opposite) phenotype from the increased mitotic index observed in untreated cid null embryos.
The effect of DNA damage on cell cycle progression was determined by treating stage 15 cid null and heterozygous embryos with the topoisomerase II inhibitor doxorubicin.
(A–C) cid/CyO cells dramatically decreased entry into mitosis in response to DNA damage, whereas cid/cid cells were unaffected by doxorubicin (Dox.) treatment.
(D–F) The MEI-41/ATR kinase was inhibited by treating cid homozygous and heterozygous embryos with 2 mM caffeine. Inactivation of the DNA damage checkpoint did not suppress the cid-mediated mitotic delay, as the mitotic index of cid mutants remained double that of controls, with the majority of the mitotic cells delayed in prophase or prometaphase.
A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase.
Scale bars indicate 15 μm.
We also performed the reciprocal experiment, to determine if an intact DNA damage checkpoint was necessary for the cid-mediated early mitotic delay. A central component of the DNA damage response, the MEI-41 ATR kinase [35], was inhibited by treating cid mutant and heterozygous embryos with 2 mM caffeine [34]. We found that caffeine treatment of Drosophila embryos phenocopied mei-41 and grapes maternal affect mutations, and is likely to completely inactivate the DNA damage checkpoint (unpublished data). Inactivation of MEI-41 by caffeine treatment did not suppress the cid-mediated mitotic delay. The mitotic index of cid mutants remained nearly twice that of heterozygous controls, and most of the mitotic cells were found in prophase or prometaphase, with very few cells progressing to later stages of mitosis (Figure 3D–3F). These results demonstrate that inactivation of the DNA damage checkpoint does not abrogate the cid-mediated mitotic delay, and confirms that this delay is not the result of DNA damage induced by cid mutations.
cid Mutant Cells Have an Intact SAC Response to Microtubule Disruption
The other major cell cycle checkpoint that could be responsible for the cell cycle delay observed in cid mutants is the SAC, which monitors kinetochore microtubule attachment and regulates the metaphase to anaphase transition. However, the cid-mediated mitotic delay appears temporally and phenotypically distinct from SAC-mediated cell cycle effects. When the SAC is activated (e.g., by the addition of microtubule polymerization inhibitors), cyclin A is degraded, but not cyclin B [31,36] (compare to Figure 2), and cells arrest in prometaphase/metaphase with condensed but unaligned chromosomes. Finally, the absence of normal kinetochore formation in all cases in which CENP-A proteins have been depleted or mutated [15–20,37] suggests that cid null mutant cells should not have an intact SAC, and that SAC components should not play a role in the cid-mediated mitotic delay.
To directly test for the presence of an SAC response to microtubule disruption in cid mutant animals, we treated stage 15 cid homozygous and heterozygous animals with the microtubule depolymerizing agent colcemid. We found that both homozygous and heterozygous cells were delayed in response to colcemid treatment (Figure 4), consistent with the observation that the Saccharomyces cerevisiae CENP-A homolog Cse4 is not required for SAC function [38]. First, both genotypes displayed a nearly 2-fold increase in mitotic index after 1 h of treatment (Figure 4B). Second, the increased mitotic index was accompanied by a large increase in the number of cells accumulated in prometaphase in both cid null and heterozygous animals. We conclude that cid mutant cells retain an intact SAC response to microtubule disruption; thus, SAC components could play a role in the cid-mediated early mitotic delay. In addition, the fact that some cid/cid cells accumulated in prometaphase/metaphase after colcemid treatment indicates that cells can eventually overcome the prophase delay, and that prometaphase is likely to be the terminal arrest point, similar to an SAC-mediated cell cycle arrest.
cid null and heterozygous embryos were treated with colcemid to determine if they have an intact SAC response to spindle disruption. Both cid/CyO and cid/cid cells were able to delay the cell cycle in response to spindle disruption (A), as evidenced by an approximately 2-fold increase in mitotic index and an accumulation of cells in prometaphase (B).
A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase.
Scale bars indicate 15 μm.
Mutation of the SAC Component bubr1 Partially Suppresses the cid-Mediated Mitotic Delay
To directly examine the role of the SAC in the cid-mediated mitotic delay, we determined if a mutation that inactivates the SAC can restore normal cell cycle progression. cid bubr1 double mutants were generated, and homozygous and heterozygous double mutant embryos were monitored for cell cycle progression by staining for PH3. Surprisingly, bubr1 mutations partially suppressed most of the cell cycle phenotypes associated with cid mutation (Figure 5A and 5B). The mitotic index in cid bubr1 double mutants was nearly the same as observed in heterozygous controls (1.1-fold, p = 0.6, cid bubr1/cid bubr1 compared to cid bubr1/CyO), compared to the 2.4-fold increase observed for cid/cid mutants over controls (see above). The number of cells delayed in prophase and prometaphase also decreased dramatically in cid bubr1 double mutants and was comparable to heterozygous controls, whereas the number of cells in anaphase showed a corresponding increase and was greater that controls. Note that cid bubr1 double mutants had a mitotic index nearly double that of cid single mutants, for reasons that are unclear at this time. We eliminated bias that could arise from this difference by only comparing homozygous double mutants to heterozygous double mutants (see Materials and Methods for a more detailed discussion). We conclude that inactivation of a component of the SAC relieves the cid-mediated mitotic delay, suggesting that at least one component of the SAC is involved in delaying cell cycle progression in the absence of CID.
(A) cid bubr1 double mutants were examined for mitotic progression by staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear density and number of anaphases compared to cid single mutants. Scale bar indicates 15 μm.
(B) bubr1 suppressed the high mitotic index and high number of cells delayed in prophase and prometaphase observed in cid single mutants (compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase.
CENP-C and the SAC Components BUBR1 and ROD Are Unable to Localize to Kinetochores in cid Mutants
It has been proposed that the APC (anaphase-promoting complex) inhibitory signal is generated by the rapid turnover of SAC proteins at unattached kinetochores [39–43]. We previously demonstrated that all tested outer kinetochore proteins (ROD, BUBR1, Cenp-meta, and POLO) fail to localize to kinetochores in CID-depleted tissue culture cells and CID antibody-injected embryos [17]. CENP-A disruptions in Caenorhabditis elegans, mouse, and human cells also result in failure to properly localize kinetochore components, including SAC proteins [15,17–19,37]. Disruption of kinetochore formation and SAC protein localization in cid/cid embryos was determined by staining for inner and outer kinetochore proteins. We found that the inner kinetochore protein CENP-C [30] was absent in most cid/cid cells, and occasionally was mislocalized in a diffuse pattern throughout the cell, consistent with studies in other organisms and with a severe disruption of kinetochore assembly (Figure 6). Consistent with these results, the SAC components ROD and BUBR1 were unable to localize to kinetochores in stage 15 cid null animals, whereas BUBR1 and ROD were localized to kinetochores during all stages of mitosis in heterozygous controls (Figure 6). We conclude that cid null mutations delay cells in early mitosis in the absence of sustained kinetochore localization of essential components of the SAC, despite the requirement for at least one of these SAC components (BUBR1).
Kinetochore localization of CENP-C, ROD, and BUBR1 were determined in stage 15 embryos. In cid/CyO control embryos (left), all three proteins were localized to the centromere/kinetochore during interphase (CENP-C) or the early stages of mitosis (ROD and BUBR1). All three proteins were absent from centromeres/kinetochores in cid/cid animals (right); in some cases, CENP-C was mislocalized in a diffuse pattern. Scale bars indicate 5 μm.
We have shown that null mutations in the Drosophila member of the CENP-A protein family result in embryonic lethality after depletion of maternal CID protein. CID-depleted embryonic cells display an early mitotic delay, consistent with cell cycle defects observed after CID antibody injection [17], suggesting the involvement and activation of a cell cycle checkpoint. This result is similar to a recent knockout of CENP-A in chicken DT-40 cells and a CENP-C mutation in Drosophila, both of which resulted in a mitotic delay in the absence of kinetochore assembly [20,30]. However, these studies did not determine when the delay occurred in mitosis, whether the mitotic delays involved the SAC or the DNA repair checkpoint, or whether similar responses to CENP-A depletion occurred in animals.
We addressed the possible involvement of two known cell cycle checkpoints in the CID-mediated early mitotic delay, specifically the DNA damage and SACs. A recent study suggested that DNA damage and repair may be involved in CENP-A assembly in Xenopus [33], raising the possibility that elimination of CID alters centromeric chromatin, resulting in DNA damage at the centromere. We addressed this hypothesis in two complementary ways. First, we compared the cid mutant mitotic delay phenotypes to the behavior of cells after inducing general DNA damage with doxorubicin. Induction of DNA damage resulted in a reduced mitotic index, not the increased mitotic index observed in the cid mutant embryos. Second, we disrupted the DNA damage checkpoint in cid mutant embryos using caffeine treatment, which inhibits MEI-41 (ATR), an essential component of the DNA damage response [34,35]. Caffeine treatment did not abrogate the cid-mediated mitotic delay. We conclude that DNA damage does not appear to be the signal that induces the cid-mediated early mitotic delay, and that this delay does not require an intact DNA damage checkpoint.
These results led us to address the possible involvement of the SAC in the CID-mediated early mitotic delay in animals. The SAC monitors microtubule attachments to the kinetochore; if normal bipolar attachments are not formed, activation of the SAC blocks entry into anaphase, resulting in a prometaphase/metaphase arrest [34,35]. The fact that the CID-mediated delay occurred earlier in mitosis than expected for activation of the SAC suggested that this checkpoint might not be involved. However, we observed that cid null mutant cells retained an intact SAC response to microtubule disruption by colcemid, which is similar to the response of Cse4 mutants in Sa. cerevisiae [38]. In addition, mutating an essential SAC component (BUBR1) resulted in abrogation of the CID-mediated delay. Previous studies suggested that kinetochore localization of SAC proteins (e.g. MAD2, BUBR1, ROD, and CENP-E) is absolutely required for SAC function. Nevertheless, we observed that BUBR1 and ROD, and the inner kinetochore protein CENP-C, lacked kinetochore localization in cid mutant embryos. These results suggest that the CID-mediated early mitotic delay involves the SAC, and that BUBR1 is serving a kinetochore-independent role in delaying mitotic progression, as suggested by recent studies in human and yeast cells [12,44,45].
Why Do cid Mutants Display an Early Mitotic Delay That Is BUBR1-Dependent?
Based on the previous observation of interphase/prophase arrest after CID antibody injection into embryos, we proposed that cells monitor kinetochore assembly early in mitosis, in addition to monitoring the presence of bipolar attachments later in mitosis [17]. It is also possible that cid null kinetochores may be able to recruit normal levels of SAC components at early stages of mitosis, but are unable to retain functional levels later in mitosis, as observed for disruption of human Hec1 and Nuf2 and DT-40 CENP-A [7,20].
Alternatively, mitotic arrest may occur in the absence of kinetochore localization of SAC components. Consistent with this hypothesis, the cid-mediated early mitotic delay requires at least one SAC component (BUBR1), yet occurs without sustained kinetochore localization of multiple, essential components of the SAC (reported here and in [17]). The finding that defects in kinetochore assembly lead to a BUBR1-dependent early mitotic delay is supported by several recent studies. Disruption of chicken CENP-A, CENP-H, or CENP-I, all inner kinetochore proteins, delays cells in mitosis for hours [9–11,20]. These results suggest that the SAC is able to respond to multiple types of signals and inhibit cell cycle progression.
How could SAC components contribute to cell cycle delay early in mitosis, prior to their well-established role in monitoring bipolar attachments in prometaphase/metaphase? Loss of CENP-A proteins blocks kinetochore assembly, which may generate "free" (non-kinetochore localized) SAC complexes capable of inhibiting mitotic progression (Figure 7). Since the active inhibitory complex for the SAC is present throughout the cell cycle [46], the complete absence of kinetochore assembly, or the presence of "free" SAC components, could block cells early in mitosis by chronically activating the SAC. It has recently been shown that both BUBR1 and MAD2 function in a kinetochore-independent manner to regulate the length of mitosis, in addition to monitoring kinetochore-microtubule attachments [12,45]. Furthermore, recent studies in Drosophila have revealed a role for Bub3 in G2 and early mitosis in promoting the accumulation of mitotic cyclins [47], suggesting that components can ensure normal mitotic progression by inhibiting the APC in a kinetochore-independent manner. This interpretation is also consistent with recent studies that demonstrate that SAC proteins play multiple roles in cell cycle regulation [48–50]. For example, mutations in Drosophila bubr1 have been shown to bypass the SAC, and are also able to suppress mutations that activate both the DNA damage and SAC in early embryos [48,51]. Furthermore, it has recently been demonstrated that SAC components are responsible for mediating a mitotic arrest in response to DNA damage in vertebrate cells [34], and the mitotic arrest in response to spindle malorientation in Sc. pombe [52]. These results strengthen the conclusion that the SAC can respond to more than bipolar kinetochore microtubule attachment, and suggest multiple roles for SAC components in cell cycle regulation. Therefore, the most likely explanation for the cid-mediated mitotic delay is that inhibitory SAC complexes can be formed in the absence of kinetochore localization (Figure 7).
In normal cells, CENP-A chromatin assembly is followed by the recruitment of inner and outer kinetochore proteins [17]. We propose that until kinetochore assembly is complete, free SAC components may be responsible for cell cycle inhibition (early activation of the SAC). Upon completion of kinetochore assembly, SAC components delay anaphase until all chromosomes have achieved bipolar spindle attachment. In cid null mutants, both inner and outer kinetochore proteins are free, resulting in a SAC-dependent early mitotic delay that does not depend on localization of SAC components to kinetochores.
The role of the kinetochore in cell cycle progression and the functions of SAC components are clearly more complex than previously thought. Future studies should focus on identifying the components and mechanisms responsible for the cid-mediated mitotic delay, and determining if this complex is identical to the standard SAC inhibitory complex.
Materials and Methods
cid mutant embryos were collected from interallelic crosses and stained as described using either a formaldehyde or MeOH:EGTA fixation. Trans-heterozygous combinations of the different cid alleles were generated in order to eliminate phenotypic effects of other lethal mutations present on each of the cid mutant chromosomes (unpublished data). All of the data presented were obtained for crosses between cid11–2 and cid22–4, although crosses between other alleles produced identical phenotypes. Antibodies used were cyclin A [31], cyclin B [31], LacZ (Sigma, St. Louis, Missouri, United States), tubulin (Sigma), ROD [53], BUBR1 [51], and CID [17]. For quantitation of mitotic index and cyclin abundance, all cells within the developing central nervous system were counted from at least five mutant and five control embryos. The ratios presented are the number of PH3- or cyclin-positive cells divided by total cells, in order to normalize for the lower nuclear density present in cid mutant embryos. Quantification of the stages of mitosis was performed by costaining embryos for PH3 and tubulin. The distinction between prophase and prometaphase was made as follows: Prophase was classified as chromosomes with incomplete condensation (i.e., round PH3+ nucleus), in which no individual chromosomes or chromosome arms were visible and DNA was not obviously aligning at the metaphase plate. Prophase tubulin staining showed bright centrosomal signals with little or no obvious microtubules interacting with the chromosomes. Prometaphase was classified as chromosomes with complete condensation (i.e., clearly visible individual chromosomes and chromosome arms) in which the chromosomes were clearly in the process of aligning at the metaphase plate. Tubulin staining showed a focused bipolar microtubule array that was clearly interacting with the chromosomes.
For quantification of CID levels in mitotically active cells in mutant and control embryos (Figure S1), the sum of pixel values for both CID and PROD immunofluorescence from five to seven embryos was obtained using the two-dimensional polygon finding tool in softWoRx (Applied Precision, Issaquah, Washington, United States). The pixel values were summed and presented as a ratio of CID:PROD, to provide a rough estimate of the amount of CID depletion in each embryo. Based on these ratios, 90%–100% of CID was depleted in stage 15 cid/cid mutants, relative to heterozygous controls, suggesting retention of a small amount of maternal protein in some cells. For all quantitations, standard deviations were calculated per embryo, and data were compared using the Student t test. Note that the amount of CID depletion in cid homozygotes is likely to be an underestimate (up to 2-fold) with respect to wild-type embryos, since cid mutant heterozygotes were used as the quantitation controls.
All images were acquired using a DeltaVision workstation (Applied Precision) and analyzed using softWoRx software, as described previously [17].
Drug treatments.
cid mutant and heterozygous embryos were bleach dechorionated and incubated in a 1:1 mixture of Schneider's medium (+10% heat-inactivated FBS) and octane as described in [54]. Colcemid was used at a concentration of 3 μg/ml for 1 h, caffeine was used at a concentration of 2 mM for 2 h, and doxorubicin was used at a concentration of 2 μM for 2 h. After drug treatment, embryos were fixed using formaldehyde and processed for immunofluorescence as described above.
The bubr1 allele used was k03113, and was obtained from the Bloomington Stock Center (Bloomington, Indiana, United States). cid bubr1 double mutants were generated by recombination using standard methods.
Mutations in cid (centromere identifier/CG13329) were recovered in genetic screens designed to isolate new mutant alleles of cnn (centrosomin) [55]. The cid locus is tightly linked to cnn in the 50A region of the right arm of the second chromosome in D. melanogaster. The genes in this genomic region, proximal to distal, are cnn (centrosomin/CG4832), Cbs (centrosomin's beautiful sister/CG4840), arr (arrow/CG5912), cbc (crowded by cid/CG5970), cid (centromere identifier/CG13329), bbc (b-b in a boxcar/CG6016), and drk (downstream of receptor kinase/CG6033). The initial characterization of this region included screening of cDNA libraries and expressed sequence tag (EST) collections to produce transcript profiles for each of these genes, and saturation mutagenesis screens to recover recessive lethal and sterile mutations. Breakpoint-associated mutations, principally deletions, and complementation analyses were used to localize each of the newly recovered mutations to the individual molecularly defined and computationally identified transcription units. Using primers designed from genomic and cDNA sequences the mutant alleles of each locus were sequenced and the genetic localization of the complementation groups confirmed. This screen resulted in the recovery of the four alleles of cid reported in this paper: cidt11–2 Q51 to stop, cidt12–1 Q83 to stop, cidt21–3 Q94 to stop, and cidt22–4 Q102 to stop.
Examination of cid bubr1 heterozygous mutants.
During the course of scoring the mitotic parameters of cid bubr1 double mutants, we noticed that cidbubr1 single mutants had a mitotic index nearly twice as high as cid single mutants alone, which prompted us to investigate these heterozygous mutants further for possible haploinsufficiency effects. We found no incidence of chromosome segregation defects in cid bubr1/CyO embryos despite their elevated mitotic index. We also examined mitotic tissue of cid bubr1/CyO third instar larval brains because this tissue allows a more precise karyotypic analysis and could reveal subtle defects not seen in embryonic tissue. We found that cid bubr1/CyO animals had a higher mitotic index than cid/CyO animals (1.10 [n = 315 fields] vs. 0.76 [n = 400 fields]), yet we did not find any evidence for aneuploidy or mitotic defects in any of the mitotic figures examined. We then determined whether cid bubr1 and cid heterozygous animals had a normal response to colcemid treatment by incubating brains with colcemid for 1 h. We found that cid bubr1 and cid heterozygous mutants had a normal response to colcemid treatment (cid/CyO mitotic index increased from 0.86 to 2.17 [n = 551 fields], and cid bubr1/CyO mitotic index increased from 1.10 to 2.37 [n = 264 fields]). From this data we conclude that cid and cid bubr1 mutants do not have a haploinsufficient effect on mitosis, and that there are likely to be other factors in the genetic background that lead to the increased mitotic index of cid bubr1 double mutants. To avoid interpretation artifacts that might be caused by this difference, in all cases we only compared data from cid bubr1 homozygotes to cid bubr1 heterozygotes, and cid homozygotes to cid heterozygotes.
Supporting Information
Figure S1. Quantification of the Amount of CID Depletion in cid Mutant Embryos
Stage 15 cid/cid and cid/CyO embryos were stained for CID and PROD. PROD was present in a punctate pattern in both genotypes. Estimation of the amount of CID depletion was preformed by comparing the ratio of total CID staining to PROD staining in five different mutant and heterozygous embryos. From this analysis we estimate that 90%–100% of CID protein is depleted in mutant embryos.
We thank the following for providing antibodies: Barbara Mellone (CENP-C), David Glover (cyclins A and B), Tibor Torok (PROD), Claudio Sunkel (BUBR1), and Roger Karess (ROD).
Author Contributions
MDB and GHK conceived and designed the experiments. MDB and TD performed the experiments. MDB and GHK analyzed the data. MDB and TK contributed reagents/materials/analysis tools. MDB and GHK wrote the paper.
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