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"I was staring at my 1000lb safe and thinking to myself how is anyone going to move this thing. It is not only cumbersome and heavy it has to clear two flights of stairs. I instantly thought I am going to have to get a professional mover in to sort this out. I phoned 3 other companies and only one company said they would do it and it was this one. They had the right equipment to move the safe in a manner that would not cause harm to anyone or the building. They took about 3 hours but they managed to get the safe out as promised which was a huge relief. For the effort they put in to this I was beyond impressed and they deserve my full praise."
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The gold plan right here in Van Buren is one of our most popular plans. The good thing about this plan is you get our large truck and the choice of a daily flat rate moving plan. It helps because if the move is going to take a while you are on 1 set fee. You also get 3 to 4 movers to finish the job and this is best suited for 3 bedrooms and up homes.
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subset: commoncrawl


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Accept subscription and one-time payments with Stripe in your Rails apps
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Build a basic VueJS frontend application that authenticates to the server using JWT, Rails API, and LocalStorage
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Adding new cards to our Vue app, persisting them in Rails, and re-rendering our UI
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This episode we handle drag and drop of cards in their own column and also between columns in our boards
Refactoring our trello clone into sub-components and introducing a global datastore to really clean up our code
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Success is usually attained in small steps. Training sessions with your dog should last 10 to 15 minutes, two to three times per day. This is especially true for puppies because of their very short attention spans. Longer sessions can cause an adult dog to become bored. Start by teaching basic commands. Try to stick with one action per training session so your dog does not get confused.
Just as a child needs a caring parent; an athletic team needs a coach; your puppy needs a leader and a clear social hierarchy. If you do not take up the role of leader, your dog will; and you will end up with an unruly, disobedient dog. Many people try to win their new puppy's love by letting the puppy always have its way. Buckets of affection is a wonderful thing for most puppies, but it must be tempered with respect.
Seeking professional help doesn't mean ditching your DIY training program. You can find a professional dog trainer who offers private training sessions, and some trainers even offer online sessions. Many dog owners prefer to join a local dog obedience class so they will be under the supervision of a dog training instructor without the higher cost of private sessions. Plus, classes challenge your dog to learn around the distractions of other dogs.
Letting your pup outside every hour or two gets old, but it's the simplest way to prevent accidents from happening. If you've ever wondered why some people choose to get new puppies during the summer or when they're on vacation it probably has to do with potty training. If you've house training a dog before you know how much time & commitment it takes.
reed.co.uk offers a large variety of Dog training courses which you can choose from based on your learning needs and goals. The Dog training courses on offer vary in time duration and study method, with many offering tutor support. Depending on your learning outcomes, reed.co.uk also has Dog training courses which offer CPD points/hours or qualifications.
A training class is not there to train your dog. Its purpose is to teach you to train your dog so you will need to be committed to train your dog for short sessions (5 minutes) several times a day rather than just simply turn up for classes! This little bit of training everyday will be repaid with a lifetime of living with a well behaved dog. You will also learn to avoid problems before they begin as well as receive help to overcome any that you already have with your dog.
Suppose you consistently praise your puppy for their actions, while potty training. Then say your dog has an accident. Do not praise you dog immediately. Instead, take your dog outside and wait for it to go to the bathroom. When it finishes doing its business, take it inside, and keep it in a separate room while you clean up the mess. After this you should act disappointed in your dog, but only for a few minutes. Keep you and your dog motivated to potty train.
Before enrolling with a particular club contact them and ask if you can go to watch a class without your dog. This will help you decide if this is the right environment for you and your dog. Some clubs have waiting lists and you will need to book ahead, some accept people on a roll on roll off basis. Prices will vary from a joining fee and then weekly payments to a one off fee for a certain length of training.
Dogs thrive on a predictable schedule, and setting one up for your new puppy will make life easier for both of you. Feed your puppy at the same times each day to help you predict and schedule bathroom breaks. Schedule post-meal walks, as well as hourly trips outside during the initial stages of potty training. Always take your puppy out after crating, even if was only for a short time, and remember that vigorous play should always be interrupted for potty breaks.
There are several medical issues that can interfere with potty training. Dogs with a urinary tract infection (UTI) will urinate frequently in small amounts, and will not have much control. You may also notice excessive licking of their genital region. If you notice a change in the consistency of their stool, the cause could be a gastrointestinal issue. Some common causes in puppies are intestinal parasites, having eaten something not in their normal diet, or a sudden food change. If a food change is necessary, do it gradually over 5 to 7 days. If you suspect any of these issues could be a problem, you should consult with your veterinarian.[19]
Effective dog training does not require many items, but there are a few basic supplies that will help make the process more convenient and effective. Choose a dog collar or harness that is suitable and comfortable for your dog. Then decide which dog leash is best for training. A retractable leash is not appropriate for dog training. You will also need dog training treats that your dog enjoys and are easy to eat quickly so the reward is more immediate. There are plenty of great treats available at pet stores or you can also use something you make at home, like small pieces of plain cooked chicken or turkey.
It also helps to teach your puppy a "potty phrase" when you take him outside for a bathroom break. A potty phrase is a way to gently remind your puppy what he needs to do when he's outside. It's a big help during the potty training phase of puppyhood and you can continue to use for the rest of his life. Pick a phrase like "hurry up" or "go ahead" and say it softly right as your puppy eliminates. In time your dog will associate the phrase with the act of elimination, so you can say it when your puppy gets distracted and forgets what he needs to do outside.
One of the easiest ways to prevent accidents is learning to recognize when your puppy needs to go out. Most puppies will sniff the ground when they're getting ready to potty, but there are many other more signals that happen prior to sniffing. Puppies that pace, seem distracted and walk away from play are subtly signaling that they have to go out. If your puppy tries to sneak out of the room, take a potty break right away.
Letting your pup outside every hour or two gets old, but it's the simplest way to prevent accidents from happening. If you've ever wondered why some people choose to get new puppies during the summer or when they're on vacation it probably has to do with potty training. If you've house training a dog before you know how much time & commitment it takes.
Training clubs that run the Kennel Club Good Citizen Dog Scheme - the largest dog training programme in the UK are a sensible place to begin. Here you will learn about every aspect of dog ownership from the Puppy Foundation Courses through to Bronze, Silver and Gold award levels. Go to GCDS Training Clubs in your County to find one near to you or email the GCDS Team ([email protected]) or call 0207 518 1011.


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Great start to my Friday.
Brilliant effort.
Type: Posts; User: Mt Smart
Great start to my Friday.
Brilliant effort.
On the edge of my seat here . . . . .
How goods this been. Come on please.
Ash a likely Lower leg injury according to fox.
Sounds like they are taking a guess.
Great strangling of the panthers to open them up, some lovely in goal kicks
Come on Lads !
Good try.
- - - Updated - - -
Bunker cant steal that off the Don
No worries Titanic, always enjoy reading the threads here when our lots are about to face off.
It's been a long time since the Warriors and Titans played each other in an official 'top eight...
Just got home from the ground. I thought the Titans were very unlucky and in some instances robbed with the obstruction rulings. Brave effort by your men without your best. There has been a lot made...
No problem. The Agent (Inventus sports) taking the unusual step of going online this morning to rebut the rumors just shows how sensitive things are between that company and the Warriors. Several...
Konrads Agent is denying the rumor, Warriors Ceo Jim Doyle has said the Warriors are not in negotiations with the Titans over Konrad.
Doyle doesn't tell porkies.
So unless it is already a done...
Hurrells agent has been the larger part of the problem, the guys a clown and manages a number of the Warrior U20's, Hurrell is the biggest name in his stable.
Hurrells agent has tried to demand...
Shame Agnatius's identical twin Rodney isn't into footy, top bloke in his own right though.
Glad to see him realising his potential with the Titans, team looks like it is finding it's feet in the...
Really looking forward to the game.
Pleased we are playing the Titans for our ANZAC fixture.
The Games we played against Melbourne saw some epic contests, certainly the Warriors fire for the...
Bang on about Rohan. He poached him for a reason....seriously Agnatius was a stand out for him...won some NSW cup games for him with massive performances.
All he needs is some patience shown by an...
Hi guys I'm not sure where to post this but I see you have signed Agnatius Paasi.
I know his family. I've not met him but I know his siblings and have followed his league path with interest, he is...
Missed !!!!
Can't take much more of this come on !!!
How goods this ?
Come on Titans !
Cold and windy too hey....it was a bit stormy last night, the park will be a miserable spot.....the stands were likely to be empty today were it not for results falling the Warriors way.
We're well in this.
Half time lead.
Excellent state of affairs.
Gee the game is being reffed from the video box these days depending on which team is set to benefit.
Checking that knock on was laughable.
Yes !
Coal Trains on. Here we go boys
If that wasn't played correctly by Hargreaves that's a turn over......also Maloney got away with a forward pass there.
Wow !!!!
hahaha !!!!
Magic...come on Video Ref give it !
Yes !
James Maloney and Jennings muck up their timing, the ref keeps looking for reasons not to give it ......ruled Try.....IT...
Good start...boys look fired up.
Got me beers in.
Come on Titans smash those Rorters !


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text:
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text:
hello dear kirupa folk
i often need to do alpha / colormatrix transitions on very large, complex movieclips. this has proved to be very heavy on CPU.
i want to create a bitmap photo of an mc, hide the mc, do the transition on the bitmap, change the mc in the background to the target state, and when the transition ended, delete the bitmap and reveal the mc.
i have tried to do this with bitmapdata, but after some time i figured it will take me a couple of days to figure out a clean and efficient way, and i am trying to save myself time. (i hope you dont blame me fot this).
has anyone heard of / can make up in a sec - a class to do this automatically?
very much thanks,


text:
Hello to all Zebra tenants and families.
These melt in the mouth chocolate biscuits are really a chocolate lovers dream.
The biscuits have a very short texture almost like a shortbread, but the whipped chocolate filing is a delicious combination.
We used milk chocolate for the filling but I think dark would have worked well.
Do be careful when melting the chocolate for the filling not to overheat the mixture, if overheated the chocolate and butter will split.
These melting moments are for serious chocoholics, so enjoy.
Happy baking
Guten Appetit
Chocolate Melting moments biscuits (makes approx 15 cookies)
175g butter
50g caster sugar
1 teaspoon vanilla extract
125g plain flour
25g cornflour
25g cocoa powder (sifted)
For filling
100g good quality chocolate
2tablespoons cream
Pre heat the oven to 180c. Line two large baking tray with baking paper


text:
Smoke Signal disables Instagram comment section
June 25, 2020
At the request of district administration, Smoke Signal editors opted to disable the comment section on Monday night's Instagram post in which it announced the district's decision to remove the Indians and Cowboys as the mascots of Pascack Valley and Pascack Hills, respectively. The post had reached 1,855 comments in under 24 hours and while many consisted of debate, there were a number of comments left by anonymous accounts that mocked George Floyd or impersonated Pocahontas or Sacagawea. Other comments targeted individuals' weight or physical appearance.
Administration made the request after receiving guidance from the Hillsdale Police Department, which remains in contact with the Bergen County Prosecutor's Office, according to Interim Principal John Puccio.
"Due to the severity of the situation, we are working with the Hillsdale Police Department to investigate the comments that were posted," Puccio said in an email sent to the student body Thursday afternoon.
The district requested that The Smoke Signal disable further commenting on the Instagram posts due to the possibility that some comments may be considered a bias crime, Interim Principal John Puccio said. A bias or hate crime is a "criminal offense that is committed against a person, property, group of people, or society," motivated "by the offender's bias against a religion, race, disability, sexual orientation, or national origin," according to U.S. Legal.
"[The comments] did not rise to the level of a bias crime. However, that comment section got too inflammatory and it would be possible that it could rise to a bias crime and we don't want to see that take place," said a representative of the Hillsdale Police Department, who asked to remain anonymous due to the matter relating directly with the school rather than the police. "One wrong word can be the difference between really crossing the line these days and when tempers are flared up, unfortunately some of these harsh words can come up very quickly."
The Bergen County Prosecutor's Office gives guidance to the Hillsdale Police Department, which works with administration, according to the police representative. The district encourages students to "to voice their differences," but in a more respectful and productive manner, according to Puccio.
"I was deeply disappointed to read some of the comments that have been made on social media," Puccio said in the email. "I know that those comments are not an accurate representation of our Pascack Valley community as a whole."
Ellie Kim is a junior and is looking forward to her third year as part of The Smoke Signal. She is excited to keep writing and gaining new skills as an...
Spencer Goldstein is a junior and has been a part of The Smoke Signal since his freshman year. He is excited to develop his writing skills as a sports...


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123 Peninsula Drive
Manteo, NC 27954
113 Seabreeze Court
Manteo, NC 27954
The Peninsula subdivision is located in Manteo on the Outer Banks of North Carolina. OuterBanksInvestor.com lists all Peninsula homes for sale in the subdivision including short sales, foreclosures and bank owned properties. Check out the Peninsula market statistics and see all the latest listings to make an informed investment decision about your next Outer Banks home.
There are currently 1 Homes for sale in Peninsula. The average price for Peninsula properties is $399,500which is 0% higher than all Homes within a 20 mile radius. The highest priced property in Peninsula is $399,500 while the lowest priced Home is listed at $399,500. Most of the properties available in Peninsula are Homes . Compared to last month, the average price of real estate in the area has gone up 100%.
Type anything you want here and we will see what we can find in the property descriptions. Maybe a name of a golf course you want to live by or a term like fixer upper.


text:
In the past 12 months, Thanh Hoa has replaced four different coaches, but their results have not improved. This "record" had been set first in 2011 when Dong Tam Long An also replaced four coaches and still ended up getting relegated at the end of the season. … [Read more...] about V. League club fires Italian coach after tepid performance
Quang yen economic zone
Vietnam PM asks for long-term stimulus plan to counter COVID-19 impact
Economist Vo Tri Thanh said the government needs to speed up the execution of the VND62 trillion (US$2.7 billion) stimulus package for coronavirus-stricken individuals and businesses and financial support in the form of more than VND180 trillion ($7.7 billion) worth of delayed tax and land rent collection for companies. … [Read more...] about Vietnam PM asks for long-term stimulus plan to counter COVID-19 impact
Bad refereeing casts shadow over domestic leagues
In the 2019 season, 20 out of 26 referees in V. League 1 made errors during matches (77 percent) and 20 out of 33 assistants got things wrong (61 percent). During V. League 2 last year, 17 out of 23 referees made mistakes (74 percent), while the number for assistants hit 9 out of 25 (36 percent). These numbers proved the poor quality of Vietnamese refereeing. And with the current situation, when the season is still half done, the numbers for 2020 may get much worse. … [Read more...] about Bad refereeing casts shadow over domestic leagues
Local visitors a potential saviour for Sầm Sơn
As many as 200,000 international visitors visited Thanh Hóa last year and many stayed at resorts by the beach. Sầm Sơn remains largely a place for Vietnamese travellers, however, and it will be their staycation this summer who help it move into the future. — VNS … [Read more...] about Local visitors a potential saviour for Sầm Sơn
Hoan Kiem Lake path upgrade nears completion
Hanoi is speeding up replacing pavements around Hoan Kiem Lake with the natural stone and consolidation of the lake bank to mark the 1010th anniversary of Hanoi in October this year. It is expected to be completed before the National Day on September 2. The pavements around Hoan Kiem Lake has deteriorated overtimes. A pilot programme to use natural stone from Binh Dinh and Phu Yen provinces was carried out until June before Hanoi authorities agreed to replace all pavements around the lake. Each stone weighs around 30kg. Three groups have worked day and night since late June in order so that the project will be completed on time. The authorities have also monitored the work to ensure environmental protection and that the construction meets the required standards. The authorities in Hoan Kiem District have started putting cables underground and investing in an automatic watering system and making sure that visitors will have more place to sit and rest. Some photos of the … [Read more...] about Hoan Kiem Lake path upgrade nears completion


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Pooley Wines
Pooley Wines is a five-star rated multi-award winning Tasmanian family winery located in the heart of the Tasmania's famous wine producing region, the Coal River Valley.
The historical property and two separate vineyards in the Coal River Valley have built a reputation for the production of their quality of cool-climate wines such as world class Riesling, Pinot Noir and Chardonnay. Members of the Pooley family have been actively involved in the business for three generations with their longstanding passion and commitment to fine wine being your assurance of uncompromising quality.
If you'd like to explore other wines from this producer, buy directly from them, sign up to their wine club or just have a chat, then please click on the Buy Direct button – we don't mind!


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Left-arm pacer believes the saliva ban will have an adverse effect on the Men in Green pacers
Chief selector-cum-head coach Misbahul Haq will make the final call
Host-cum-singer has launched a project called #ClassicsOnLockdown
This will be the film debut of singer and actor Junaid Khan
Left-arm pacer was charged by on-field umpires Ghaffar and Shamim Ansari
Later on, the same role was played by Aditya Roy Kapoor
Actor revealed him and his family are currently on the hunt for a good script for Junaid Khan's big Bollywood debut
Pacer is expected to join Khulna Tigers in the coming days for the ongoing T20 competition
ICC has issued latest update after the conclusion of Asia Cup 2018
K-P Sports Director General Junaid Khan formally inaugurated the event in which 10 teams will be taking part
Two-day Pakistan intra-squad practice match between ends in a draw
Blackwood stars as West Indies down England in first Test
India's Ganguly confirms Australia tour but seeks shortened quarantine
Pakistan has decided it will not evacuate its citizens from China to counter the threat of the fast-spreading corona virus. Do you agree with the decision?
Polls are non-scientific, reflect only the online audience and can be manipulated.
One can only pray rule of law is enforced rather than another cycle of political point-scoring
Cyber harassment also has severe psychological repercussions
Billion-tree programme
The recent downpour in Karachi has caused much havoc for citizens.
Those who consider taking online classes a piece of cake must do a little research
It has been four years since the death of Abdul Sattar Edhi
Letters will be edited for policy, content and clarity. All letters must have the writer's name and address. You may send your letters to: [email protected]
مویشی منڈیوں کے اوقات کار صبح 6 سے شام 7 بجے تک ہوں گے، اسد عمر
پی آئی اے کے فضائی آپریشن بحال کرنے کیلئے یورپی پارلیمنٹرینزوسول ایوی ایشن حکام کے ساتھ رابطے
پاکستان میں کورونا کے مصدقہ مریضوں کی تعداد 250000 سے تجاوز کرگئی
The writer is an Egyptian researcher with a special interest in Middle Eastern Issues
The writer is an economics and political science graduate, and founder of the educational initiative Averroes
The writer is a psychiatrist. He is a fellow at the department of anesthesiology and critical care management at Johns Hopkins Hospital, Baltimore, Maryland, United States.
This material may not be published, broadcast, rewritten,
redistributed or derived from. Unless otherwise stated, all content is
copyrighted © 2020 The Express Tribune.


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Star Wars Stormtrooper Travel Mug
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Star Wars Stormtrooper Travel Mug
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Whether traveling to a distant galaxy in a starship, or just hopping on the train for the morning commute, home comforts are always important, especially considering the price of coffee on an Imperial Super Star Destroyer these days.
So fill up before you leave and take your favourite drink wherever you go, and if you prefer your coffee on the Dark Side, or your tea a little stronger than the regular footsoldier, you'll find the Stormtrooper Travel Mug is just the thing you need.
Featuring a classic Stormtrooper design, the Travel Mug is completely watertight to avoid spillages, and is insulated to retain the heat of hot drinks. As well as a Stormtrooper helmet, the decal also features a First Order symbol on the reverse of the flask.
Star Wars: The Force Awakens is the seventh film of the record breaking blockbuster franchise, and has introduced the Star Wars Universe to a whole new generation of fans. Appearing prominently throughout the series, the Stormtrooper has become a hugely popular cultural icon, making this the perfect gift for any Star Wars fan, and a great piece of fan memorabilia from the latest trilogy of films. Capacity 16oz. Not suitable for dishwasher use.
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Flat 3Beachside Torcross Kingsbridge
Flat 3Beachside Torcross Kingsbridge
We are delighted to offer a fabulous beach side apartment ideal for a holidayapartment or cosy home with stunning views over the beachfront out to sea over Start Bay. In addition Inland views over the surrounding rural aspects and the renowned freshwater lake and wildlife reserve 'Slapton Ley'.
Located within the village on the seafront promenade and sea wall with easy access onto the splendid pebble Beach. Torcross is an area of outstanding natural beauty within a costal preservation. Situated at the southern end of a three mile pebble beach which is fantastic for families, children and walkers. 'Slapton Ley' nature reserve supports and feeds a wide range of wildlife. Coastal paths connect miles of spectacular coastline.
Village facilities include a public house, cafes & restaurants. Within close proximity to the Village of Chillington with a health centre, post office and convenience store. Easy commute to the highly desired market towns of Kingsbridge and Dartmouth with the bus stop less than 100 yards from the property. This ground floor apartment affords a rear terrace and shed with tandem parking for 2 vehicles and comes highly recommended by sole agents Charles Head. The accommodation briefly affords:
Entering the property through the communal entrance which has been recently refurbished, 3 Beachside Apartments is the first door on the left. The open plan beach hut style living room instantly creates a bright and spacious area to enjoy the stunning sea and coastal views. The fitted bay window is positioned perfectly to enjoy reading a book whilst memorizing over the endless sea views and watching the sunsets in the evenings. With a fitted fireplace it helps create the perfect cosy living space.
At the rear of the living room the kitchen comprises of a modern style' shaker units' with work surfaces incorporating a stainless steel sink and a integral hob with space for a fridge. The added benefit of the kitchen is that it affords the same stunning views from the bay window.
The double bedroom accessed through the hallway offers a fitted double wardrobe and views over the Slapton Ley freshwater lake and wildlife reserve. With the double aspect view from this house, you really do have the perfect position in Torcross. Opposite the bedroom the shower room and toilet is found offering the essential washroom facilities.
At the front, the terrace over looks Torcross beach and provides far reaching sea views perfect for summer evenings enjoying an Al Fresco dinner. At the rear the private terrace with a shed can be found, overlooking the Slapton Ley. There are two tandem parking spaces associated with the property on the gravel driveway shared with the other two apartments, offering plenty of space for your family.
Key Features
- Waterfront location
- Spectacular sea and coastal views
- Two parking spaces
- Views over Slapton Ley
- One Bedroom Beach side apartment
- Perfect for a holiday home
- Less than 20 yards from the beach
- Open plan Living Area and Kitchen
Property Details
|Property Type
|Council Tax Band
|Band C
Room Descriptions
On The Ground Floor
Shower room
Rear terrace
Two Parking Spaces
Council tax band C
We are delighted to offer a fabulous one bedroom studio apartment ideal for a holiday apartment or cosy home with stunning views over the beachfront out to sea over Start Bay.
113 Fore Street
Kingsbridge, Devon
01548 852352
Share This Property
What's my property worth?
Get an accurate indication of the current sale or rental value of your south Devon home. Our property professionals will offer an honest market price from our unrivalled knowledge of the local property scene.


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Purple Revolver Movies ArchiveIf you have a story or a picture you think should be included on the site, please contact us at email@example.com
American: The Bill Hicks Story DVD review
(Wed 29 Sep 2010 12:44)
Some criticised this film for being too sentimental about a man who was not sentimental. Others said it was the most inspiring film of the year, a must see for every fan of Hick's work.
Ben Affleck reduced crime in Boston by employing criminals for The Town
(Mon 27 Sep 2010 16:09)
Movie hard man Ben Affleck is to be more or less knighted by Boston Police for single-handedly reducing crime rates during filming for his bank heist flick The Town.
New Feist doc Look at what the Light did Now to air at Raindance
(Mon 27 Sep 2010 11:04)
Look at What the Light Did Now will have its European premiere at the Raindance Film festival on Sunday 3rdOctober 2010. Further screenings in the UK are to be announced shortly
Legend of the Guardians film review
(Fri 24 Sep 2010 11:33)
Every breed of owl, every color and feathered couture of the species makes an appearance in this film, but these stunning creations do little more than look good, making this into yet another good-guys-versus-bad-guys sa
Jeremy Renner reveals cast of genuine bank robbers for The Town
(Thu 23 Sep 2010 16:25)
Jeremy Renner has revealed how the cast for Ben Affleck's new heist movie The Town consisted primarily of past bank robbers and active criminals, with only a core nucleus of professional actors on set
Everest movie The Wildest Dream premieres at IMAX London
(Tue 21 Sep 2010 11:11)
The Wildest Dream is a stunning documentary of two climbers retracing British explorer George Mallory's final attempt to conquer Mount Everest in 1924 to become the first person to conquer the world's highest peak
This Is War DVD review
(Mon 20 Sep 2010 14:08)
This is war, is a first hand account of life on the front lines as told by a US Marine in Iraq circa 2003.
The Arbour - a Bradford tragedy review
(Mon 20 Sep 2010 12:23)
The Arbor is a documentary about a Yorkshire family torn apart because of a woman's refusal to take care of anyone, including herself.
Buried film review
(Mon 20 Sep 2010 12:04)
Horror can also come in small packages. Even a coffin, as the no frills, literally underground thriller Buried impressively confirms.
The Town film review
(Fri 17 Sep 2010 11:31)
The Town refers to Charlestown, Boston, a place where fathers proudly mentor their sons to follow in their footsteps, which mostly lead to the local jail, but at least the sentiment is there.
Purple Revolver hero of the day: Howard Marks
(Sat 11 Sep 2010 10:48)
Howard Marks has had a colourful life, after studying Physics at Balliol College Oxford he went on to become one of the world's most wanted drug smugglers
Clint Eastwood turned down James Bond role
(Thu 09 Sep 2010 14:17)
Hollywood legend Clint Eastwood turned don the chance to play James Bond it emerged yesterday
Secret Cinema showcases Lawrence of Arabia in Alexandra Palace
(Wed 08 Sep 2010 10:48)
Secret Cinema held it's latest event over the weekend and was perhaps the best kept secret of the year
I Spit On Your Grave collectors DVD boxset set for release
(Wed 08 Sep 2010 00:02)
The controversial rape-revenge horror boxset features new uncut material previously unseen in the UK
Tamara Drewe review - romp and circumstance in the country
(Tue 07 Sep 2010 21:03)
Tamara Drewe is the dream plotline you wish to give the Midsommer Murders episodes that you have to endure with Mum and Grandma on a quiet Sunday evening.
Gemma Arteton banned cow tipping during Tamara Drewe filming
(Tue 07 Sep 2010 19:17)
The statuesque stunner looked glorious in a nude strapless Gucci gown and Bulgari heels, with natural makeup and a simple ponytail, at last night's London premiere
Dorian Gray: The Wayward Lord DVD review
(Mon 06 Sep 2010 11:03)
This story takes a long time to get going, and the mystery of Dorian Gray's portrait is never explained, but there's a real sense of time and place in the movie
My Son, My Son, What Have Ye Done film review
(Mon 06 Sep 2010 10:42)
A title not to be mistaken as being addressed to the filmmaker by his skeptical dad in reaction to this movie, but maybe should be, My Son, My Son, What Have Ye Done manages to come off as both baffling and mystifying at
Al Pacino's 'little friend' in Scarface up for auction
(Sat 04 Sep 2010 15:50)
You can say hello to Al Pacino's 'little friend,' the machine gun from the 1983 film Scarface, which is expected to fetch £100,000 at auction.
Jonah Hex review
(Sat 04 Sep 2010 08:27)
Josh Brolin as the surly supernatural gunslinger is a very different kind of cowboy dodging bullets across the Wild West, or rather Deep South, settling old scores and new ones that may not have even arrived, in some as
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Hi Gabe:
Great sound advice on the Forum about what a shotgun can and cannot do!
I have a stock Mossberg 500 12 shotgun with a modified choke on a 28" vent rib barrel for hunting and this combo gun also included a plain beaded 18" cylinder barrel and plan on using the 18" barrel for self defense inside the home.
While I have a small pistol grip to fit this shotgun, I'm a little apprehensive in using it due to quality of aim and longer followup shots due to not handling the recoil as well as the regular fiberglass stock.
How do you feel about pistol grips?
Answer: I see a great deal of these in central America. They allow the SGN to be meneuvered better and are not much of an issue IF the shots will be taken close range (as is usual). Forget about taking 100 yard slug shots this way, but for CQB at arm's length its not bad.
Also, I have painted the front bead on the 18" cylinder barrel flourescent green, but wonder if there is a better sight arrangement for SD at night inside the home. What do you suggest for sights on a short barrel? Can my local smith install rifle sights if they're available?
Answer: You can get rifle sights, but why bother. It sounds like you have a good set up there.
FYI: I was impressed at the tight shot group (00 Winchester Ranger Low Recoil buckshot) of only 4" at 30 yards with the modified 28" barrel and 2 1/2" - 3" group with the 18" cylinder choke at 10 yards: question - what brand of buckshot do you like and what size? 00, #4?
Answer: My HD SGN has Federal Premium Buckshot #00
What about slugs? I just purchased 10 boxes of Remington Low Recoil LE 1-oz. slugs (from CTD for only $1.86 per box of 5) to practice with.
PMC 00 and #4 buckshot at the same price are also used for range time.
Am I on the right track?
Answer: IMHO, forget #4, stay with #00. As far as LE Low Recoil. Its not that much of an advantage. Think hard about what you will use the SGN for. If I need slugs, I'll go with regular Brenekes, or Foster Slugs (standard power, not reduced)..


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Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.
AssuntosProteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Motores Moleculares/fisiologia , Miosina Tipo V/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Camundongos , Proteínas Motores Moleculares/metabolismo , Miosina Tipo V/metabolismo , Concentração Osmolar , Ligação Proteica
BACKGROUND: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood. RESULTS: From a forward genetic screen, we identified the zebrafish uq 23ks mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the adherens junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co-localization with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. CONCLUSIONS: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284-295, 2019. © 2019 Wiley Periodicals, Inc.
AssuntosCaderinas/metabolismo , Citoesqueleto/ultraestrutura , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Miosina Tipo V/fisiologia , Animais , Forma Celular , Citoesqueleto/metabolismo , Endossomos/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Miosina Tipo V/genética , Miosinas/genética , Miosinas/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.
AssuntosCálcio/química , Cadeias Pesadas de Miosina/química , Miosina Tipo V/química , Miosinas/química , Animais , Biotinilação , DNA/química , Homeostase , Cinesina/química , Cinética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Neurônios/metabolismo , Pontos Quânticos , Ratos , Estresse Mecânico , Potenciais Sinápticos
RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.
AssuntosMembrana Celular/metabolismo , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Transporte Proteico/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Linhagem Celular , Conexina 43/análise , Canal de Potássio ERG1 , Endocitose , Canais de Potássio Éter-A-Go-Go/análise , Junções Comunicantes , Genes Reporter , Sistema de Condução Cardíaco/fisiopatologia , Transporte de Íons , Canal de Potássio Kv1.5/genética , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Miosinas/deficiência , Miosinas/genética , Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function.
AssuntosProteínas do Citoesqueleto/metabolismo , Enterócitos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas rab de Ligação ao GTP/fisiologia , Linhagem Celular Tumoral , Polaridade Celular , Códon sem Sentido , Endossomos/metabolismo , Células HEK293 , Humanos , Isoenzimas/metabolismo , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Microvilosidades/patologia , Mucolipidoses/genética , Fosforilação , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo
The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.
AssuntosTransporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Dimerização , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/metabolismo , Miosina Tipo V/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição
Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA.
AssuntosCadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Transporte de RNA , RNA Mensageiro/metabolismo , Animais , Espinhas Dendríticas/metabolismo , Humanos , Modelos Biológicos , Biossíntese de Proteínas , Estrutura Quaternária de Proteína
Cytoskeletal trafficking systems are becoming more complex at every turn. A new study reports that a yeast myosin V walks on only a select few actin filaments - those that are decorated with tropomyosin.
AssuntosCadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Tropomiosina/fisiologia
Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.
AssuntosAdipócitos/metabolismo , Vesículas Citoplasmáticas/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Insulina/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Células 3T3-L1 , Animais , Membrana Celular/metabolismo , Camundongos , Transporte Proteico/fisiologia
Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to â¼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.
AssuntosCitoesqueleto de Actina/fisiologia , Cinesina/fisiologia , Microtúbulos/fisiologia , Proteínas de Xenopus/fisiologia , Citoesqueleto de Actina/química , Animais , Cinesina/química , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Miosina Tipo V/química , Miosina Tipo V/fisiologia , Conformação Proteica , Coelhos , Xenopus , Proteínas de Xenopus/química
mRNAs encoding polarity and secretion factors (POLs) target the incipient bud site in yeast for localized translation during division. In pheromone-treated cells we now find that these mRNAs are also localized to the yeast-mating projection (shmoo) tip. However, in contrast to the budding program, neither the She2 nor She3 proteins are involved. Instead, the Scp160 RNA-binding protein binds POL and mating pathway mRNAs and regulates their spatial distribution in a Myo4- and cortical ER-dependent fashion. RNA binding by Scp160 is stimulated by activation of Gpa1, the G protein α subunit regulated by the pheromone receptor, and is required for pheromone gradient sensing, as well as subsequent chemotropic growth and cell-cell mating. These effects are incurred independently of obvious changes in translation; thus, mRNA trafficking is required for chemotropism and completion of the mating program. This is, to our knowledge, the first demonstration of ligand-activated RNA targeting in the development of a simple eukaryote.
AssuntosQuimiotaxia/fisiologia , Feromônios/fisiologia , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Tropismo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética
We describe the development of a hierarchic modelling method applied to simulating the processive movement of the myosin-V molecular motor protein along an actin filament track. In the hierarchic model, three different levels of protein structure resolution are represented: secondary structure, domain, and protein, with the level of detail changing according to the degree of interaction among the molecules. The integrity of the system is maintained using a tree of spatially organised bounding volumes and distance constraints. Although applied to an actin-myosin system, the hierarchic framework is general enough so that it may easily be adapted to a number of other large biomolecular systems containing in the order of 100 proteins. We compared the simulation results with biophysical data, and despite the lack of atomic detail in our model, we find good agreement and can even suggest some refinements to the current model of myosin-V motion.
AssuntosModelos Biológicos , Proteínas Motores Moleculares/fisiologia , Miosina Tipo V/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Algoritmos , Animais , Simulação por Computador , Dimerização , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Motores Moleculares/química , Movimento/fisiologia , Miosina Tipo V/química , Ligação Proteica
Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class V myosins were characterized as nonprocessive in vitro, including Myo2p, the essential class V myosin from S. cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or behave processively in the cellular context. Here we show that Myo2p moves processively in vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.
AssuntosCadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Tropomiosina/fisiologia , Actinas/fisiologia , Isoformas de Proteínas
Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (productâ=âMyoB) and a myosin V homolog, myoE (productâ=âMyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape.
AssuntosAspergillus nidulans/crescimento & desenvolvimento , Miosina Tipo II/fisiologia , Miosina Tipo V/fisiologia , Aspergillus nidulans/ultraestrutura , Proteínas Fúngicas , Genoma Fúngico , Imagem com Lapso de Tempo
Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.
AssuntosActinas/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Camundongos , Concentração Osmolar , Ultracentrifugação
Von-Willebrand factor (vWF) is a highly multimerized hemostatic glycoprotein that is stored in endothelial Weibel-Palade bodies (WPB) and secreted upon cell stimulation to act in recruiting platelets to sites of vessel injury. Only fully matured multimeric vWF represents an efficient anchor for platelets, and endothelial cells have developed mechanisms to prevent release of immature vWF. Full maturation of vWF occurs within WPB following their translocation from a perinuclear site of emergence at the trans-Golgi network (TGN) to the cell periphery. The WPB-associated small GTPase Rab27a is involved in restricting immature WPB exocytosis and we searched for links between Rab27a and the actin cytoskeleton that could anchor WPB inside endothelial cells until they are fully matured. We here identify myosin Va as such link. Myosin Va forms a tripartite complex with Rab27a and its effector MyRIP and depletion of or dominant-negative interference with myosin Va leads to an increase in the ratio of perinuclear to more peripheral WPB. Concomitantly, myosin Va depletion results in an elevated secretion of less-oligomeric vWF from histamine-stimulated endothelial cells. These results indicate that a Rab27a/MyRIP/myosin Va complex is involved in linking WPB to the peripheral actin cytoskeleton of endothelial cells to allow full maturation and prevent premature secretion of vWF.
AssuntosCélulas Endoteliais/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Fator de von Willebrand/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Células Endoteliais/fisiologia , Humanos , Imunoprecipitação , Microscopia Confocal , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP
Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.
AssuntosSistema Nervoso Entérico/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Junção Neuromuscular/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Transmissão Sináptica/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microscopia Confocal , Cadeias Leves de Miosina/fisiologia
The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.
AssuntosAxônios/metabolismo , Retículo Endoplasmático/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Miosina Tipo V/fisiologia , Proteínas de Neurofilamentos/metabolismo , Animais , Axônios/fisiologia , Filamentos Intermediários/metabolismo , Filamentos Intermediários/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Distribuição Tecidual
In neuroscience, myosin V motor proteins have attracted attention since they are highly expressed in brain, and absence of myosin Va in man leads to a severe neurological disease called Griscelli syndrome. While in some cells myosin V is described to act as a vesicle transport motor, an additional role in exocytosis has emerged recently. In neurons, myosin V has been linked to exocytosis of secretory vesicles and recycling endosomes. Through these functions, it is implied in regulating important brain functions including the release of neuropeptides by exocytosis of large dense-core vesicles and the insertion of neurotransmitter receptors into post-synaptic membranes. This review focuses on the role of myosin V in (i) axonal transport and stimulated exocytosis of large dense-core vesicles to regulate the secretion of neuroactive substances, (ii) tethering of the endoplasmic reticulum at cerebellar synapses to permit long-term depression, (iii) recycling of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors at hippocampal synapses during long-term potentiation, and (iv) recycling of nicotinic acetylcholine receptors at the neuromuscular junction. Myosin V is thus discussed as an important modulator of synaptic plasticity.