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AWS has released a new whitepaper that has been requested by many AWS customers: AWS Policy Perspectives: Data Residency. Data residency is the requirement that all customer content processed and stored in an IT system must remain within a specific country's borders, and it is one of the foremost concerns of governments that want to use commercial cloud services. General cybersecurity concerns and concerns about government requests for data have contributed to a continued focus on keeping data within countries' borders. In fact, some governments have determined that mandating data residency provides an extra layer of security.
This approach, however, is counterproductive to the data protection objectives and the IT modernization and global economic growth goals that many governments have set as milestones. This new whitepaper addresses the real and perceived security risks expressed by governments when they demand in-country data residency by identifying the most likely and prevalent IT vulnerabilities and security risks, explaining the native security embedded in cloud services, and highlighting the roles and responsibilities of cloud service providers (CSPs), governments, and customers in protecting data.
Large-scale, multinational CSPs, often called hyperscale CSPs, represent a transformational disruption in technology because of how they support their customers with high degrees of efficiency, agility, and innovation as part of world-class security offerings. The whitepaper explains how hyperscale CSPs, such as AWS, that might be located out of country provide their customers the ability to achieve high levels of data protection through safeguards on their own platform and with turnkey tooling for their customers. They do this while at the same time preserving nation-state regulatory sovereignty.
The whitepaper also considers the commercial, public-sector, and economic effects of data residency policies and offers considerations for governments to evaluate before enforcing requirements that can unintentionally limit public-sector digital transformation goals, in turn possibly leading to increased cybersecurity risk.
AWS continues to engage with governments around the world to hear and address their top-of-mind security concerns. We take seriously our commitment to advocate for our customers' interests and enforce security from "ground zero." This means that when customers use AWS, they can have the confidence that their data is protected with a level of assurance that meets, if not exceeds, their needs, regardless of where the data resides.


Early intervention with delinquent borrowers. Servicers must establish or make good faith efforts to establish live contact with borrowers by the 36th day of their delinquency and promptly inform such borrowers, where appropriate, that loss mitigation options may be available. In addition, a servicer must provide a borrower a written notice with information about loss mitigation options by the 45th day of a borrower's delinquency. The rule contains model language servicers may use for the written notice. This section includes an exemption for small servicers as defined above.
READ Louis Says "I Feel Like Killing Myself Because of My Debts"


Sri Lanka Police which was controlled by the Ministry of Law and Order has been moved under the purview of the Minister of Defense.A gazette notification announcing the distribution of subjects for respective ministers notes that 17 government bodies including the 03 armed forces, Civil Security Department, National Cadet Corps, Department of Coast Conservation & National Media Center have been assigned to the Ministry of Defense under President Maithripala Sirisena.
The Central Bank of Sri Lanka, State Banks and it's associated companies, the Public Utilities Commission of Sri Lanka have been moved under the Ministry of Finance and Economic Affairs of which Prime Minister Mahinda Rajapaksa is the subject Minister.
Under the previous government, the Central Bank of Sri Lanka was under the National Policies and Economic Affairs Ministry of which Ranil Wickramasinghe was the Minister.Sri Lankan Airlines & Mihin Lanka which were under the Minister of Public Enterprises are now under Nimal Siripala De Silva who is the Minister of Transport and Civil Aviation.


Stop pharmaceutical companies from advertising on television!!
we need to stop drug companies from polluting commercial time slots, they are very unecessary.
Brian Davis needs your help with "Stop pharmaceutical companies from advertising on television!!". Join Brian and 4 supporters today.


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MODELERSITE.COM > July 2020
Building the Revell 1957 Ford Nascar 1/25 scale
By Andre Pantarotto | 07.21.2020 14:47
I wanted to build the #12 Ralph Moody car. Moody won four races in 1956 for owner Pete DePaolo. He finished eighth in the final points, with 21 Top-10 finishes in 35 races. He raced the first third of 1957, until Ford and others American automobile manufacturers pulled out of racing.
Building the Trumpeter BTM 3 (high speed trench digging vehicle) #09502 1/35 scale
By Pablo Raggi | 07.07.2020 17:09
The BTM is a specialized vehicle of the engineering corps, based on the ATT artillery tractor, which in turn was built over the T-54 hull tank, using the cabin of the ZIS-150 truck. Not only did the BTM derive from this project, but there were at least 4 other designs, all for a specific use, including a version for Antarctic exploration.

redpajama_set_name: RedPajamaCommonCrawl


Scientists claim that when we watch moves, our brain works less than in the process of reading. Books are supposed to turn on our imagination, which broadens the horizons of our thinking skills. However, movies can make you smarter, too. It is only a matter of choosing the right ones. Naturally, detective movies are exactly the kind to make you think: you try to predict the next twist in the plot and guess who the criminal is. That is why our list of top movies to make you smarter for the most part consists of them. But you will also find several unexpected titles here. So, let's explore all of them with our professional writing services.
It this movie together with the characters you will have to investigate mysterious Evan's blackouts. The boy out of the blue has memory losses, after which he finds himself in most ridiculous situations. The movie is based on numerous little jigsaw pieces, which fit together in the end, revealing the final plot twist to you. This movie makes your brain work fast in order not to miss the tiniest details that would eventually lead to the denouement.
By the way, there is a great Easter egg that we cannot but mention here. The movie has more than one ending, so make sure to get familiar with them all. Mixed feelings guaranteed.
Our brain will only benefit from the necessity to juggle past, present and future narratives, as well as actions happening in different realities, that is why we recommend you this non-linear movie. The title Source Code sounds very geeky, but the meaning within touches different spheres of human life, which makes the movie interesting for a wide audience. The main character finds himself in a recursive situation which is bound to repeat until he identifies the criminal. But as the plot develops, we understand that the movie is not only about looking for clever ways to save innocent people from a terrorist, but also about affection and love.
This movie will take you to the world of amnesia and ultimate persistence. After losing his wife, Leonard starts his own search for the murderer. He almost revenged himself once, but got hit in his head. What complicates the things is that the character has developed anterograde amnesia since then that prevents him from remembering new things. Each 15 minutes Leonard's life starts anew, the only lasting memories being those about his wife's death and everything before that. Do not get lost in the loads of memos, Polaroid photos and reminders in the form of tattoos across Leonard's body. You can even benefit from all these devices the character uses: maybe some of them will work for you too as mnemonic techniques.
Will you be able to trace the slightest clues scattered across the movie? Will you get to the core of it before the final scene? Our professional writers must admit that this movie was brilliant, and its ending totally unexpected. Be ready that you will have to watch it at least twice to take in everything the script writers implied. This movie is a masterpiece by Christopher Nolan that will surely affect you to the deep of your heart.
As you can see, Christopher Nolan tops our list of intellectual movies producers. This is his second brainchild we recommend you in this essay, and now you will get to know why.
Interstellar combines human knowledge of physics with wanderlust: but in this case people travel not because they want to but because they need to. A terrible catastrophe impends over the human race, and the only thing to deal with it is to gather all the skills and knowledge in order to conquer space. This movie is not purely fantastic, as we already mentioned. It is an example of sci-fi, which relies on real science in many instances. If you want to learn more about it, we suggest for you the book by Kip Thorne entitled The Science of Interstellar.
Anyway, what we are talking about here is the movie. Watch Interstellar, if you have not yet done so, and be sure that you will not consider those 3 hours spent on the movie wasted.
Another movie from 2011 that your brain will definitely like. It tells us a story about the life of Dr John Forbes Nash Jr, a famous mathematician. Have you heard about the Nash Equilibrium before? If not, you will find a lot of interesting things about it after watching the movie. This theory is a cornerstone of A Beautiful Mind, but there are various discussions on the Internet whether it was explained right in the movie. Well, if you want to contribute to it, watch the movie as soon as possible, and you will be able to join online disputes and put in your two cents' worth.
A Beautiful Mind also touches upon the issue of psychiatric illnesses, if these are among your range of interests. What is more, the movie reveals the process of struggling with them. For the most part, the movie is biographic, but some private details of the mathematician's life were deliberately concealed from the audience; probably, they would only distract us from the main idea.
From our previous books vs movies essay you already know a lot about this common dilemma in the modern world, but we hope we could solve it for you in this post. There is no need to deprive yourself of the pleasure of spending several hours in front of the screen if what you watch will make you a more educated person or improve your thinking skills. Stick with our recommendations and at your earliest convenience watch the movies from our list that you have not seen before. Please note that you will have to re-watch some of them, maybe even several times, because of the intricate plots that are hard to grasp straight away. This should provide you with a brilliant pastime for the next couple of weeks!


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Royal ladies and the stories behind their tiaras
Priceless family heirlooms
By Samantha Stewart|11:05pm Dec 27, 2020
1 of 11Attribution: Getty
The British royal family – Queen Elizabeth II in particular – has an enviable collection of tiaras.
Each of the bejewelled headpieces have histories so rich, they could rival their price tags. Click through to discover the fascinating histories behind these tiaras.
The Queen's George IV State Diadem is one of the most iconic pieces in her vast collection of tiaras.
Although the piece is exclusively worn by Queens, it was initially made for King George IV, who wore it on the day of his coronation in 1821.
Over time it has been worn by Queen Victoria, Alexandra, Mary, and Elizabeth II.
The Queen's Alexandra's Kokoshnik Tiara is classic design that has a Romanov-related backstory.
The Kokoshnik Tiara was presented to Alexandra, Princess of Wales, as a 25th wedding anniversary gift in 1888 by Lady Salisbury. Alexandra wanted a tiara in the style of a kokoshnik (Russian for "cock's comb"), a traditional Russian folk headdress, a style she had seen worn by her sister, Marie Feodorovna, the Empress of Russia.
The sparkling Cambridge Lover's Knot tiara was one of Diana's most iconic pieces and over time has become Kate's go-to, no doubt for its sentimental value. The stunning piece was created for Queen Mary in 1914 from pearls and diamonds from her family's collection.
On her wedding day, Princess Eugenie chose an unexpected family heirloom; the Greville Emerald Kokoshnik tiara. The diamond and emerald headpiece made by French jeweler Boucheron in 1919, belonged to Dame Margaret Greville, a famous society hostess who, upon her death, left her jewels to the Queen Mother.
The Meander Tiara is a Greek design and was believed to have been a gift to Princess Andrew – born Princess Alice of Battenberg – after marrying Prince Andrew of Greece and Denmark in 1903. Alice owned the tiara until 1947, when she gave it to her new daughter-in-law, Princess Elizabeth, as a wedding gift. In recent years the tiara was worn at the wedding of Princess Anne's daughter, Zara Phillips.
The tiara worn by the Duchess of Sussex on her wedding day was made for Queen Mary in 1919, and currently belongs to Queen Elizabeth II. The tiara features a diamond centre-stone filigree design and was worn atop of Meghan's 16-foot-veil.
Not all tiaras are from the Queen's jewellery vault. One of Princess Diana's favourites – the Spencer Tiara – belongs to her family. Diana wore the diamond headpiece on her wedding day, as did Lady Sarah Spencer; Jane, Baroness Fellowes, and Victoria Lockwood.
The Queen Mother received the Greville Tiara in 1942 when Mrs. Greville had given her all of her jewellery. The diamond and platinum tiara was made for Mrs. Greville in 1921 by Lucien Hirtz, the chief designer for Boucheron in Paris. The Queen has never worn this tiara in public, the Duchess of Cornwall did however when she married Prince Charles in 2005.
10 of 11Attribution: Getty
The Cartier Halo crown has nearly 1000 diamonds in total and was purchased by King George VI in 1936 as an anniversary gift to his wife. Eight years later it was regifted to Queen Elizabeth II who lent the beautiful headpiece to Kate on her wedding day.
The Grand Duchess Vladimir Tiara originally belonged to Russian Grand Duchess Vladimir, before it was purchased by Queen Mary. She eventually passed it on to her granddaughter Queen Elizabeth II.
-- no more galleries --


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author: Adam Zagoria
2020 Long Island Lutheran forward Zed Key has three official visits set, working on last two
By ADAM ZAGORIA
GLEN HEAD, N.Y. — A slew of college coaches came out to Long Island Lutheran on Tuesday afternoon to watch 2020 power forward Zed Key and 2020 point guard Andre Curbelo.
Marquette's entire staff including head coach Steve Wojciechowski met with Key Tuesday, as did Florida coach Mike White and his staff. Illinois head coach Brad Underwood and two assistants were on hand. St. John's head coach Mike Anderson and assistant Van Macon and Rutgers assistant Steve Hayn were also at the open gym.
"It's just a blessing to see just them watching me and Andre and a couple other kids on the team," the 6-foot-8, 215-pound Key said. "Just to see where all the hard work we put in starts paying off now. High D-1 coaches are in attendance at every open gym."
2020 @ZedBasketball14 of @LuHiBasketball
will take officials to Marquette 9/21 And Florida 9/28. Just visited Ohio State and has 2 more. Story coming. pic.twitter.com/HqFwyUmmoS
— Adam Zagoria (@AdamZagoria) September 10, 2019
Key, who helped lead Long Island Lutheran to a New York State Federation Class AA title last season, is narrowing down his list of schools and closing in on a decision this fall.
Key took an official visit to Ohio State this past weekend and will take officials to Marquette (Sept. 21) and Florida (Sept. 28). He will have two officials remaining and may take some unofficials as well.
As for the Ohio State visit, he said, "It was a really good visit. When I think about Ohio State, I think about a huge spread-out campus, it's not like that. Everything's right in the center of campus that you need. All the classes are right there. It's a really nice campus.
"[Their message] was that nothing's promised and just to work. Me playing the four spot, they're expecting a couple people at my position to go to the NBA. So just come in, work hard and be ready to play." Junior forward Kaleb Wesson could go pro after next season.
Key met Tuesday with the whole Marquette staff.
"They were just talking about the campus and how would I fit in as a player," he said. "They were talking about my character and how it would fit in right with the team. They talked about the basketball resources of the program. "
Key speaks regularly with Marquette assistant Dwayne Killings.
"Yes, we talk about the campus, we're just building a relationship," he said.
Marquette is also among the final four for Archbishop Stepinac point guard R.J. Davis, who had a home visit with the staff Tuesday night. Key and Davis are friendly.
"Yes, that's my bro," Key said. "We haven't talked about [playing together], we just talk about basketball."
Key also met Tuesday with Florida's White and the staff ahead of his official there.
"We were talking about the same thing, character and how my character would fit into the team," he said. "Just talked about my visit and what they have to offer me academically as well."
Illinois is also strongly in play for both Key and Curbelo, but Key has yet to set up an official there.
"I got three set up, I just have to figure out the other two and I think that Illinois is going to be one," he said.
Key has also visited St. John's unofficially.
"They're going to have a really good team this year," he said. "And they like the way I play as a post-presence and to be able to shoot the mid-range and the three-pointer. So just be who I am and I'll fit in with the team really well."
Rutgers, St. John's, Providence and Illinois remain in the mix for the other two official visits, although he could take unofficials to the locals.
"I just got to sit down with my family and see which one I really want to visit," he said.
As for Rutgers, Key is impressed with the new RWJBarnabas Health Athletic Performance Center which houses a practice court and the new basketball facilities.
"They have no fours in my class, they just had somebody commit who's like a five [Dean Reiber] but everyone's leaving, everyone's a senior, so I'll come in and play right away," he said. "And then also the coaching staff, the new practice gym, it's exclusive."
Key models himself after a mix of Tobias Harris and Joel Embiid "just to be able to handle the ball, and pick-and-pop
"He's just worked so hard on his body this offseason," LuHi coach John Buck said. "He's come such a long way in six months that if he keeps working at it, there's a reason why so many high-major kids are now on him after this summer. I think he can play with anyone in the country if he continues to work."
Key hopes to decide by this fall.
"Yes, yes, I'll sit down with my parents, see what they have to say and figure out which college fits me the best," he said.
Follow Adam Zagoria on Twitter
Written by Adam Zagoria
Adam Zagoria is a Basketball Insider who covers basketball at all levels. A contributor to The New York Times and SportsNet New York (SNY), he is also the author of two books and is an award-winning journalist and filmmaker. His articles have appeared in ESPN The Magazine, SLAM, Sheridan Hoops, Basketball Times and in newspapers nationwide. He also won an Emmy award for his work on the SNY mini-documentary on Syracuse guard Tyus Battle. A veteran Ultimate Frisbee player, he has competed in numerous National and World Championships and, perhaps more importantly, his teams won the Westchester Summer League (WSL) championships in 2011 and 2013. He lives in Manhattan with his wife and children.
Shot consistency has propelled 2020 guard Norance Berry
DeMarr Langford cuts to three schools


What method do you use for cleaning ?
All Fresh Carpet Cleaners use a method that most "carpet manufacturers recommend." That method is hot water extraction. Natural fibers are cleaned with hot water extraction but at a lower temperature to prevent shrinkage. With the hot water extraction method, a cleaning solution is typically applied first to break up and loosen soil. Then hot water is sprayed onto the fibers and immediately extracted, flushing the fibers clean. According to the top leaders in the carpet cleaning industry, this is the most effective cleaning method.
Do I need to vacuum before the crew arrives ?
Yes, we highly recommend that you vacuum at least one day before or the day that the crew will arrive for the cleaning. Vacuum cleaning your home or business regularly is essential for good health; the ultimate goal of vacuuming is to safeguard your health. Did you know that humans shed over 1 million skin cells per hour? These dead skin cells mingle with airborne dust (soil particles) and accumulate in carpets, rugs, and furniture. They provide a great developing environment for dust mites and other dangerous microorganisms. So when you do not vacuum regularly, the tiny microbes are constantly floating around from you walking on the soiled carpet or sitting on your soiled furniture, which can cause a lot of serious problems for humans, especially small babies because they are closer to the floor, people with asthma and inhalant-related allergies, and also the elderly.
What do you consider as an area ?
How long will it take to clean my carpets ?
Do you move furniture ?
Yes. There is no charge to move furniture. We are professionally trained to move your furniture. After the cleaning is completed, we will return it to its rightful place on protective padding as needed. We do suggest you remove items off of your furniture like electronics, plants, lamps and knick-knacks. We will move most furniture that can be safely moved. There are several items we will not move, including: pianos, electronics, china cabinets, beds, dressers with mirrors, pool tables, etc.
How long will it take the carpet to dry ?
I have heard that cleaning my carpet makes it soil faster. Is this true ?
I have had my carpet cleaned, but the spots have reappeared. What should I do ?
Although most spots that are removed from carpet do not reappear, some substances in carpet backing may wick to the surface after cleaning. If this happens, please give us a call as soon as possible.
Do you guarantee removal of all spots or stains in the carpet ?
We cannot guarantee that we will remove all spots or stains. There are substances that can permanently discolor the carpet fibers. The age of the carpet and the type of carpet fibers also plays a factor in this. The technician will try to determine if this is the case prior to cleaning, but they are not always able to determine this before trying to remove the spots.
After my carpet was cleaned, it had bubbles or ripples in the carpet. What caused this ?
As the carpet dries, these bubbles or ripples should start to lay flat again. Once it is dry, they should be virtually unnoticeable. This is usually a sign that the carpet was in need of re-stretching prior to the cleaning. If these ripples do not go away after it is completely dry, please contact us as soon as possible.
Will carpet cleaning get rid of fleas in my carpet ?
We cannot guarantee that the cleaning will get rid of all the fleas. We usually recommend that the customer kill the adult fleas first by whichever method they prefer. This is something that a professional exterminator might be able to help with. Please keep in mind that the carpet is not the only place fleas will lay eggs. You might consider having your upholstery treated and cleaned as well as any area rugs or pet beds since fleas can invade these areas too.
Why do your prices vary from other carpet cleaners ?
When comparing prices, make sure you ask a lot of questions, and please keep a few things in mind. Our price includes treating for spots, moving furniture, and a 10-day satisfaction policy. There are no hidden fees or additional charges. Also, we never sub-contact, so our technicians are always background checked, drug tested and properly trained employees. This is what allows us to stand behind the quality of all of our work.
How do you guarantee customer satisfaction ?
We are happy to work with you to correct any situation that may arise. In some cases, this can mean spot cleaning specific areas of concern or even completely re-cleaning the area in question. In the end, we're not satisfied until you are !


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Prada Warehouse by AMO
15/01/2018. FW18 Mens Fashion Show [2 VIDEOS]
metalocus, INÉS LALUETA
The new Prada showsapce project by AMO has access through plastic curtains, into colourfully lit space was transformed into an oneiric and surreal archive, organized into sectors each corresponding to a specific theme.
FW18 Mens Fashion Show. Prada Warehouse by AMO. Image courtesy of PRADA
In a venue, adjoining its fondazione on the south-eastern outskirts of Milan, piled with pallets of boxes in plywood and foil, AMO (OMA's research and design studio) completes the setting for the Prada Mens Fall Winter 2018 spring/summer presentation.
For the first time, Prada invited four renowned creatives to each work on an item of clothing.
"Boxes, crates and all the other objects that colonize the warehouse are marked with a series of ambiguous symbols that express the multiplicity of the prada identity. Classical themes like idealized accessories, gizmos, mascots and recurring motifs of the collections are transformed into emblems that hint at the content of the crates," explains Prada.
Description of project by AMO
For the Prada FW 2018 Men's and Women's fashion show, AMO converts the depositories of Viale Ortles, Milan, into an oneiric and surreal archive, where the multifaceted essence of Prada comes to life, turning the warehouse into a venue that both speaks of Prada's accumulated identities and its place within the contemporary.
The building is organized in sectors that each correspond to a specific theme. Boxes, crates and all the other objects that colonize the warehouse are marked with a series of ambiguous symbols that express the multiplicity of the Prada identity. Classical themes like idealized accessories, gizmos, mascots and recurring motifs of the collections are transformed into emblems that hint at the content of the crates.
Showspace by AMO.
OMA. Office for Metropolitan Architecture
Office for Metropolitan Architecture (OMA) is a leading international partnership practicing architecture, urbanism, and cultural analysis. OMA's buildings and masterplans around the world insist on intelligent forms while inventing new possibilities for content and everyday use. OMA is led by ten partners – Rem Koolhaas, Ellen van Loon, Reinier de Graaf, Shohei Shigematsu, Iyad Alsaka, David Gianotten, Chris van Duijn, Ippolito Pestellini Laparelli, Jason Long and Michael Kokora – and maintains offices in Rotterdam, New York, Beijing, Hong Kong, Doha and Dubai.
OMA-designed buildings currently under construction include Taipei Performing Arts Centre, Qatar National Library, Qatar Foundation Headquarters, Bibliothèque Multimédia à Vocation Régionale in Caen, Fondation d'Entreprise Galeries Lafayette in Paris, Bryghusprojektet in Copenhagen, Musée National des Beaux-Arts du Québec, and Faena Arts Center in Miami.
OMA's recently completed projects include Garage Museum of Contemporary Art in Moscow (2015); Fondazione Prada in Milan (2015); G-Star Headquarters in Amsterdam (2014); Shenzhen Stock Exchange (2013); De Rotterdam, a large mixed-use tower in the Netherlands (2013); CCTV Headquarters in Beijing (2012); New Court, the headquarters for Rothschild Bank in London (2011); Milstein Hall at Cornell University in Ithaca, New York (2011); and Maggie's Centre, a cancer care centre in Glasgow (2011). Earlier buildings include Casa da Música in Porto (2005), Seattle Central Library (2004), and Netherlands Embassy in Berlin (2003).
Total Space by OMA-AMO
04/07/2016 - 2017 SS Prada Men's and Women's show. [MIL] Italy - metalocus, PALOMA DE LA QUINTANA
2016 FW Prada Men's and Women's Show by OMA-AMO
20/01/2016 - Prada [MIL] Italy - metalocus, JOSÉ JUAN BARBA
AMO designed 90s-style Nightclub in Paris for Miu Miu
07/07/2015 - [PAR] France - metalocus, INÉS LALUETA
OMA releases the 2016 SS Prada Real Fantasies [VIDEO] by AMO
05/02/2016 - [VIDEO] from OMA - metalocus, JOSÉ JUAN BARBA
PANDA, a Project by OMA and Bengler
13/09/2016 - A digital platform to disrupt the sharing economy. 2016 Oslo Architecture Triennale [Oslo] Norway - metalocus, JOSÉ JUAN BARBA


We find that the peripheral ER in Saccharomyces cerevisiae forms a dynamic network of interconnecting membrane tubules throughout the cell cycle, similar to the ER in higher eukaryotes. Maintenance of this network does not require microtubule or actin filaments, but its dynamic behavior is largely dependent on the actin cytoskeleton. We isolated three conditional mutants that disrupt peripheral ER structure. One has a mutation in a component of the COPI coat complex, which is required for vesicle budding. This mutant has a partial defect in ER segregation into daughter cells and disorganized ER in mother cells. A similar phenotype was found in other mutants with defects in vesicular trafficking between ER and Golgi complex, but not in mutants blocked at later steps in the secretory pathway. The other two mutants found in the screen have defects in the signal recognition particle (SRP) receptor. This receptor, along with SRP, targets ribosome–nascent chain complexes to the ER membrane for protein translocation. A conditional mutation in SRP also disrupts ER structure, but other mutants with translocation defects do not. We also demonstrate that, both in wild-type and mutant cells, the ER and mitochondria partially coalign, and that mutations that disrupt ER structure also affect mitochondrial structure. Our data suggest that both trafficking between the ER and Golgi complex and ribosome targeting are important for maintaining ER structure, and that proper ER structure may be required to maintain mitochondrial structure.
The ER usually consists of a polygonal meshwork of membrane tubules and sheet-like cisternae (Palade 1956). These structures are connected with one another and with the continuous membrane of the outer nuclear envelope. Much of the ER, called RER, is involved in protein translocation. It is studded with ribosomes carrying nascent chains that are transferred into or across the ER membrane as they are being synthesized. In some cells, smooth regions of the ER are found that lack ribosomes and have a different structure and function. Little is known about how the complex morphology of the ER is formed and maintained, or what role the structure plays in functions of the ER.
In mammalian cells, the ER tubule network is highly dynamic and is continually being extended to the periphery of the cell (Lee and Chen 1988; Waterman-Storer and Salmon 1998). It is rearranged by tubules sliding along one another and by new tubules emerging from preexisting tubules and fusing with others. The formation and maintenance of the ER network is thought to require microtubules (Terasaki et al. 1986; Terasaki 1990). Depolymerization of microtubules with drugs like nocodazole causes a gradual and reversible contraction of the ER to the center of the cell (Lee et al. 1989). Newly forming tubules are thought to be pulled out of existing tubules by motor proteins migrating along microtubules and/or by being dragged by the tip of polymerizing microtubules. In vitro, microtubules have also been proposed to be required for the formation of ER-like networks of membrane tubules (Dabora and Sheetz 1988). However, the in vitro formation of ER-like tubule networks in the absence of microtubules has recently been reported (Dreier and Rapoport 2000). In addition, maintenance of ER structure does not solely rely on a microtubule network, since ER tubules frequently do not coalign with it (Terasaki et al. 1986; Lee et al. 1989). In plant cells, actin filaments, rather than microtubules, are thought to be important for ER formation and maintenance (Terasaki 1990; Liebe and Menzel 1995).
Little is known about the structure of ER in Saccharomyces cerevisiae. Indirect immunofluorescence and EM have shown that the yeast ER consists primarily of perinuclear and cortical domains, with a few tubules connecting them (Matile et al. 1969; van Rijn et al. 1975; Novick et al. 1980; Rose et al. 1989; Preuss et al. 1991). Structural details of the cortical ER, which is closely apposed to the plasma membrane, remain to be elucidated. In addition, it is unclear which mechanisms or molecules are involved in keeping the ER at the cell periphery, and whether the structure of the peripheral ER remains intact during the cell cycle. Here, we show that the peripheral ER forms a dynamic tubular network throughout the cell cycle and have isolated mutants that dramatically affect ER structure.
The strains used are listed in Table 1. Media were prepared according to standard methods (Sherman 1991). The plasmid pJK59 encodes Sec63-GFP, a fusion of the S65T V163A mutant of GFP to the COOH terminus of Sec63p under the SEC63 promoter. It was constructed by amplifying SEC63 and ∼400 bp of its 5′ promoter from pPS599 (from R. Schekman, University of California, Berkeley, CA) with primers 5′-GGTCTAGACTTCAAGTTCCCAGCGAG-3′ and 5′-GGCTCGAGCTTCTGGTGATTCATCATCTTC-3′. The resulting product was cloned between the XbaI and XhoI sites of the CEN/URA3 plasmid, pJK50 (Kahana et al. 1998). The plasmid pWP1055 encodes ss-GFP-HDEL under the MET25 promoter. The DNA encoding green fluorescent protein (GFP) was amplified from pJK59 using oligonucleotides that added sequences encoding the signal sequence of carboxypeptidase Y at the NH2 terminus (5′-AGATACTCTAGAATGAAAGCATTCACCAGTTTACTATGTGG-ACTAGGCCTGTCCACTACACTCGCTAAGCCGCTAGCAAAGGAGAAGAACTC-3′) and HDEL at the COOH terminus of GFP (5′-GATATACGAATCCCAATTCGTCGTGGCAGCCGGATCCTTTGTATAGTTC-3′). The product was then cloned into the CEN/URA3 plasmid p416MET25 (Mumberg et al. 1994) between the XbaI and EcoRI sites.
A collection of 1,016 mutants with growth defects at 37°C was obtained from C. Cole (Dartmouth Medical School, Hanover, NH; Amberg et al. 1992) and transformed with pJK59. This plasmid could not be introduced into 24 of the mutants. The mutants containing pJK59 were grown at 25°C, were shifted to 37°C for 3 h, and were then examined live in a ZEISS Axioplan II fluorescence microscope using a ZEISS 100× Plan Apochromat oil immersion objective. No fluorescent signal could be detected in 27 of the mutants after the shift to nonpermissive temperature.
Cells expressing GFP were mounted in growth medium and examined live. For time course experiments, coverslips were sealed with Valap. Images were acquired using a DeltaVision microscope (Applied Precision Instruments) built around a ZEISS Axiovert microscope with a PXL CCD camera (Photometrics) and a ZEISS 100× Plan Apochromat oil immersion objective. All images shown were deconvolved and scaled using Delta Vision software.
To stain the actin cytoskeleton with Alexa 594 phalloidin (Molecular Probes), cells were fixed in 4% formaldehyde, 50 mM KPO4, pH 6.5, and 1 mM MgCl2 for 2 h at room temperature. They were then washed once with PBS pH 7.4 and resuspended in PBS at 10 OD600 /mL. 92.4 μL of the cell suspension was mixed with 7.6 μL of Alexa 594 phalloidin (6.6 μM stock in methanol) and incubated for 1 h at room temperature in the dark. The cells were washed three times with cold (4°C) PBS and viewed immediately.
Immunofluorescence with antitubulin antibody and rhodamine-conjugated secondary antibody (Molecular Probes) was performed as described in Pringle et al. 1991.
For Fig. 6 B, the cells were grown for 1 h in medium supplemented with 0.1 μg/ml DAPI to visualize nuclear DNA.
Mitochondria were stained with TMR-CH2-Cl (Mitotracker, Molecular Probes) as described in Nunnari et al. 1997.
Cells were fixed in 1.5% KMnO4 for 20 min, poststained with uranylacetate for 4 h, and were dehydrated by incubation with 50, 70, 96, and 100% ethanol for 15 min each. The cells were then incubated in fresh 100% ethanol for 30 min and embedded in Epon 812. Ultrathin sections were cut with a diamond knife and examined in a Philips EM 400. The relative surface area of the ER was calculated with the point counting technique (Weibel and Bolender 1976). The final magnification of the micrographs was 6,000 and the point spacing of the test system was 1.5 cm.
Pulse-labeling and immunoprecipitation were done essentially as described in Stirling et al. 1992, except that 5 OD600 units of cells were labeled for 7 min with 250 μCi of 35S protein-labeling mix (NEN Life Science Products).
For the experiments with cycloheximide, cultures were grown logarithmically at 25°C, 1 μM cycloheximide (Sigma-Aldrich) was added to half of each culture, and the cultures were then grown at 37°C for 6 h. This amount of cycloheximide decreased the growth rate of wild-type cells about fivefold and did not significantly affect the growth rate of the srp101-47 and srp102-510 mutants. A portion of the cultures was pulse-labeled and Kar2p was immunoprecipitated as described above. To depolymerize microtubules, cells were grown with 20 μg/mL nocodazole (1 mg/mL stock in DMSO; Sigma-Aldrich) for 2.5 h (Jacobs et al. 1988). Latrunculin-A (Molecular Probes) was added to cultures at 200 μM from a 10 mM stock in DMSO (Ayscough et al. 1997), and cultures were grown for the indicated amount of time.
The fraction of Sec61p bound to ribosomes was determined basically as described in Panzner et al. 1995. Cultures were grown in YPD to an OD600 of 0.5 at 25°C, and then half of each culture was shifted to 37°C for 3 h while the rest continued to grow at 25°C. NaN3 was then added to 10 mM and the cultures cooled in an ice-water bath. The cells were pelleted and resuspended with an equal volume of buffer containing 50 mM potassium acetate, 10 mM magnesium acetate, 20% glycerol, and 100 mM Hepes, pH 7.8, and lysed in a Biospec bead beater. The lysates were spun at 4,000 g to remove unbroken cells and debris, and then at 12,000 g to remove mitochondria and nuclei. Microsomes were pelleted by spinning at 500,000 g for 30 min. The membranes were washed in buffer containing 800 mM potassium acetate, 10 mM magnesium acetate, 10% glycerol, and 50 mM Hepes, pH 7.8, and resuspended in this buffer. Microsomes were solubilized by adding digitonin to 2.5% and incubating at 4°C for 30 min. Ribosomes were pelleted by spinning at 500,000 g for 30 min. To release nascent chains from the microsomes, 2 mM puromycin (Fluka) and 1 mM GTP were added before solubilization with digitonin and the microsomes were incubated for 1 h on ice and 30 min at 30°C (Gorlich and Rapoport 1993). The amount of Sec61p in the fractions was determined by immunoblotting using an 125I-labeled secondary antibody (NEN Life Science Products). All samples were run in triplicate and quantitated using a Fujix PhosphorImager. Samples were run at various dilutions to ensure that the quantification was performed within the linear range of the PhosphorImager.
Previous immunofluorescence microscopy with fixed S. cerevisiae cells only allowed the visualization of the peripheral ER as a bright ring close to the plasma membrane (Rose et al. 1989; Preuss et al. 1991). To obtain a more detailed picture of the ER, we used a fusion of the ER protein Sec63p to GFP (Sec63-GFP) and employed high-resolution fluorescence microscopy with live cells. A stack of images with focal planes 0.2-μm apart were obtained with a wide-field optical sectioning microscope and deconvolved. Two of the images from each stack are shown in Fig. 1, with focus planes close to the center and periphery of the cell. With the improved resolution, we were able to demonstrate that the peripheral ER forms a network of interconnected tubules (Fig. 1, bottom left). This network could be seen on either side of the cell in only one or two image planes close to the plasma membrane. The nuclear ER, on the other hand, forms a continuous membrane (Fig. 1, top left). To confirm that the peripheral network is ER, we used a GFP-fusion protein targeted to the ER lumen. We expressed GFP with an NH2-terminal signal sequence and a COOH-terminal ER retention signal (HDEL; Pelham et al. 1988). This fusion protein, called ss-GFP-HDEL, shows a localization pattern similar to Sec63-GFP (Fig. 1, right). Thus, the peripheral ER in yeast looks strikingly similar to the ER network in mammalian cells. The network was seen at all stages of the cell cycle and appeared in the daughter cell as soon as the bud started to form. The ER network is also thought to be maintained during mitosis in higher eukaryotes (Ellenberg et al. 1997).
The peripheral ER forms a network of interconnected tubules. Fluorescence microscopy of wild-type cells expressing either Sec63-GFP or ss-GFP-HDEL. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm.
We wondered if the yeast cortical ER is dynamic as it is in higher eukaryotes. Cells expressing Sec63-GFP were visualized in the fluorescence microscope at three-minute intervals (Fig. 2 A). The peripheral ER in the daughter cell (above the mother cell) is highly dynamic and is completely rearranged in three minutes. To facilitate comparison between the frames, stars have been placed at identical locations in the daughter cell in each image. The structures near the stars are qualitatively quite different at each time point. The ER in the mother cell was somewhat more stable than in the daughter cell, with some regions showing little change over the course of the experiment. The star in the mother cell indicates a region that remains relatively unchanged during the course of the experiment. This qualitative difference in the extent of ER structural alteration in daughter and mother cells was seen consistently in many experiments (not shown).
Actin is required for the dynamics of the peripheral ER. Time-lapse images of cortical ER in wild-type cells expressing Sec63-GFP. A, Images of a cell taken at 3-min intervals (mother cell on bottom). Bar, 5 μm. To facilitate comparisons between frames, stars have been placed in identical places in each frame. B, Images of a portion of a cell taken at 5-s intervals. Indicated are an instance of ring closure (a) and formation of a new ER tubule (b). Bar, 1 μm. C, Images of a cell taken at 3-min intervals 10 min after 200 μM latrunculin-A was added to the medium (mother cell on bottom). Bar, 5 μm.
Video microscopy of mammalian cells revealed three types of movement of the ER: tubule branching, ring closure (the contraction of a loop of ER to a single tubule), and tubule sliding (the movement of a junction of ER tubules along a tubule; Lee and Chen 1988). Similar movements occur in S. cerevisiae. Fig. 2 B shows time-lapse microscopy of a portion of a mother cell with pictures taken every five seconds. An example of ring closure occurs between 5 and 20 s (Fig. 2, arrow a points to ring). An instance of tubule branching occurs between 25 and 30 s (Fig. 2, arrow b points to the new tube). We find that tubule branching and ring closure occur at approximately the same rate as in mammals (Lee and Chen 1988). Therefore, ER tubule movements appear to occur similarly in yeast and mammalian cells.
To examine the role of microtubules in ER formation, we incubated yeast in medium containing nocodazole. After two hours, microtubules could not be detected by immunofluorescence with antitubulin antibodies (not shown; Jacobs et al. 1988). Neither the structure of the ER (Fig. 3 A) nor ER dynamics (not shown) were affected by the disruption of microtubules. These results indicate that, unlike the situation in mammals, microtubules play no role in the maintenance of ER structure.
Neither actin filaments nor microtubules are required to maintain cortical ER structure. A, Images of the ER in wild-type cells expressing Sec63-GFP were taken after 2.5 h of growth in medium with either 20 μg/ml nocodazole or 200 μM latrunculin-A. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm. B, Wild-type cells expressing Sec63-GFP were fixed and stained with Alexa 594 phalloidin to visualize actin. Images were acquired while focusing on the cell cortex. Arrows point to regions in which actin filaments and ER tubules align. Bars, 5 μm.
We next tested the role of actin filaments and patches in the formation and maintenance of the ER. We first assessed the alignment of tubules of the peripheral ER with cortical actin filaments in fixed cells (Fig. 3 B). Although fixation slightly distorts the ER, there are clearly a few regions where ER tubules and actin filaments align (indicated by arrows). However, most ER tubules do not align with any actin filament. Next, we treated yeast cells with latrunculin-A, which results in the disruption of actin structures within minutes, as seen by staining with rhodamine-phalloidin (not shown; Ayscough et al. 1997). However, even after growth for two hours in medium with latrunculin-A, the ER structure was not disrupted, although there appeared to be some minor proliferation of ER tubules (Fig. 3 A). Strikingly, minutes after addition of latrunculin-A, the dynamics of the ER was greatly reduced (Fig. 2, compare C with A, daughter cell on top in both). To facilitate comparison between the frames, stars were placed at identical locations in the daughter cell in each frame. To quantitatively compare the changes of ER structure seen in Fig. 2A and Fig. C, we determined the pixel values at different time points in the same portion of each image of the daughter cell, and calculated an average correlation coefficient. The coefficient for the drug-treated cell (0.779) was clearly higher than for the control cell (0.293), and may have been underestimated because of a slight drift of the sample or Brownian movement. Taken together, our results suggest that the cytoskeleton is not required for maintenance of the ER structure, but actin filaments play a role in ER dynamics.
We used a genetic screen to identify proteins that are required for maintenance of the structure of the cortical ER. Since maintaining ER structure may be essential for cell viability, we screened a collection of temperature-sensitive mutants (Amberg et al. 1992) for those that had altered cortical ER structure or defects in segregating the peripheral ER into the daughter cell. Approximately 1,000 mutants were transformed with the plasmid encoding Sec63-GFP. They were grown at permissive temperature (25°C), shifted to nonpermissive temperature (37°C) for three hours, and examined in a fluorescence microscope. Mutants which displayed ER throughout the cell were numerous and were discarded since it is well known that ER proliferation can be caused by overexpression of membrane proteins or disrupting ER to Golgi complex transport. Three mutants that had disrupted ER structure were identified (WPY152, WPY153, and WPY154).
In all three mutants, the mutations that cause the ER structure defects were recessive, segregated 2:2 as determined by back-crosses with a wild-type strain, and were linked to the temperature-sensitive growth phenotype. We isolated plasmids from genomic libraries that complemented the growth defect of each of the mutants at 37°C. Using a combination of DNA sequencing, subcloning, and integrative mapping, we determined that the mutations responsible for the altered ER structure in the mutants are in SEC27 (WPY153), SRP101 (WPY154), and SRP102 (WPY152). We named the altered alleles of these genes sec27-95, srp101-47, and srp102-510. SEC27 encodes a component of COPI, a coat-forming complex involved in vesicle budding (Duden et al. 1994). SRP101 and SRP102 encode the α and β subunits of the signal recognition particle (SRP) receptor, a heterodimeric complex of GTPases in the ER membrane (Gilmore et al. 1982a,Gilmore et al. 1982b; Meyer et al. 1982; Ogg et al. 1992, Ogg et al. 1998). SRP and SRP receptor target ribosome–nascent polypeptide complexes to the ER membrane for cotranslational protein translocation. We sequenced srp101-47 and srp102-510. The alteration in Srp101p (L422P) affects a residue that is not conserved between species, but is close to two regions likely to be involved in GTP hydrolysis (Ogg et al. 1992; Montoya et al. 1997). The change in Srp102p (L58P) is in a region of unknown function.
In the sec27-95 mutant found in the screen, the peripheral ER in mother cells was disorganized and appeared to be converted from tubules to sheets. In ∼50% of the cells, the ER in daughter cells was dramatically reduced and disorganized (Fig. 4 A). The phenotype was not simply the result of mislocalization of Sec63-GFP, since ss-GFP-HDEL showed a similar distribution (not shown). In thin-section EM, there was a reduction in the amount of peripheral ER at nonpermissive temperature in both mother and daughter cells. Only small areas of the peripheral ER were seen, in contrast to the situation in wild-type cells in which most of the plasma membrane has ER tubules near it (Fig. 5). To quantitate these differences, we determined the surface area of the ER (both perinuclear and cortical) relative to total cell volume. There was a significant decrease in the relative surface area in sec27-95 cells compared with wild-type cells which became more pronounced at nonpermissive temperature (Table 2).
Mutants with conditional defects in vesicular transport between the ER and Golgi complex have altered ER structure at nonpermissive temperature. Images of cells expressing Sec63-GFP acquired while focusing on either the center or periphery of the cells. Bars, 5 μm. Arrows point to daughter cells in some cells. A, Images of sec27-95 cells grown either at 25°C or 3 h after shift to nonpermissive temperature (37°C). B, Images of sec21-1, sec23-1, and ret1-3 cells 3 h after shift to 37°C. C, Images of ufe1-1 cells 2 h after shift to 37°C and cdc48-3 cells 4 h after shift to 37°C.
sec27-95 cells have less peripheral ER at nonpermissive temperature. Thin-section EM of wild-type (A and B) and sec27-95 (C and D) cells grown at 25°C (A and C) or 3 h after shift to 37°C (B and D). L, Lipid droplet; M, mitochondrium; N, nucleus; PM, plasma membrane; V, vacuole; ER, endoplasmic reticulum. Bars, 1 μm.
A similar phenotype was shared by mutants with conditional defects in other components of COPI (sec21-1 and ret1-3), a mutant with a defect in the functionally related coat complex COPII (sec23-1), and Ufe1p, an ER-localized vesicle soluble N-ethylmaleimide–protein attachment protein receptor (v-SNARE; ufe1-1; Fig. 4B and Fig. C). Mutants with defects in other components of COPI or COPII (sec28Δ::HIS3, sec27-1, sec13-1, and sec16-2) had disorganized ER structure at nonpermissive temperature, but did not have the dramatic reduction in the amount of ER in daughter cells seen in the other mutants (not shown). It is unclear why this phenotype is seen in some mutants with defects in ER–Golgi complex traffic and not in others. Perhaps the rate and extent of inactivation of the mutant proteins differs in these two groups. This might also explain why this phenotype is seen in the sec27-95 mutant we isolated, but not the previously identified sec27-1 mutant. Previous EM studies on sec mutants with ER–Golgi apparatus trafficking defects have also shown that ER structure becomes disorganized at nonpermissive temperature (Novick et al. 1980; Duden et al. 1994). In contrast to these mutants that affect traffic between the ER and Golgi complex, mutants with conditional defects in later steps of the secretory pathway (sec1-1, sec2-41, sec 3-2, sec4-8, sec6-4, sec 8-9, sec9-4, and sec15-1) did not show changes in ER morphology after three hours at nonpermissive temperature (not shown).
Besides its role in vesicular traffic from the Golgi apparatus to the ER (Lewis et al. 1997), Ufe1p has also been implicated in homotypic ER fusion (Patel et al. 1998). Since homotypic ER fusion may well be required to maintain ER structure, we looked at a temperature-sensitive mutant in Cdc48p, another protein implicated in the process (Latterich et al. 1995). Surprisingly, even after four hours at nonpermissive temperature, ER structure remained normal in a cdc48-3 mutant (Fig. 4 C).
In the SRP receptor mutants the ER structure was also most disturbed in daughter cells, which often contained one bright fluorescent spot, and in some cases did not contain any detectable ER (Fig. 6 A). However, most mother cells also lacked detectable tubular regions of the ER, and instead contained numerous bright fluorescent spots. Abnormal ER structure was first observed one hour after shift to elevated temperature, and became pronounced after two hours. The structure of the perinuclear ER was not disrupted in the mutants; a ring of Sec63p-GFP was seen around the nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI; Fig. 6 B). Both SRP receptor mutants have normal ER structure at the permissive temperature (Fig. 6 A). We ruled out that the mutants only cause the mislocalization of Sec63-GFP, rather than actually disrupting ER structure, because ss-GFP-HDEL also has an altered distribution in the mutants at the nonpermissive temperature (not shown). Analysis of the mutants by thin-section EM revealed a reduction in the amount of ER at the periphery of the cells, particularly at nonpermissive temperature (Fig. 7 and Table 2). In addition, large sheets of membrane near the nucleus can be seen that are not present in wild-type cells (Fig. 5). These structures are likely to be responsible for the brightly fluorescing spots seen in the fluorescence microscope.
Mutants with altered ER morphology at the nonpermissive temperature. A, Images of srp101-47 and srp102-510 cells expressing Sec63-GFP grown at either 25°C or 2 h after shift to nonpermissive temperature (37°C). Arrows point to the daughter cells in some images. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm. B, srp101-47 cells grown for 2 h at 37°C and stained with DAPI to visualize nuclear DNA (blue). Bar, 5 μm.
Mutants with conditional defects in SRP receptor have altered peripheral ER morphology at nonpermissive temperature. Thin-section EM of srp102-510 (A and B) and srp101-47 (C and D) cells grown at 25°C (A and C) or 3 h after shift to 37°C (B and D). L, Lipid droplet; M, mitochondrium; N, nucleus; PM, plasma membrane; V, vacuole. Bars, 1 μm.
A conditional mutation in SRP itself also affects ER structure. Yeast cells bearing a mutation in Sec65p, the homologue of the mammalian SRP19 subunit (Stirling and Hewitt 1992), have abnormal ER morphology at the nonpermissive temperature, similar to that seen in the srp101-47 and srp102-510 mutants isolated in the screen (Fig. 8 A).
Phenotypes of various translocation mutants. A, Images of sec65-1, sec61-3, sec61-2, and sec61-2 Δssh1 cells expressing Sec63-GFP were taken 3 h after shift to nonpermissive temperature (37°C). Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm. B, Translocation of Kar2p was tested 1 h after shift to the nonpermissive temperature (37°C). The cells were pulse-labeled for 7 min and Kar2p was immunoprecipitated. The positions of mature Kar2p (m) and of Kar2p that has not undergone signal sequence cleavage (p) are indicated.
Like other mutants with defects in SRP and SRP receptor, srp101-47 and srp102-510 mutants have a protein translocation defect; both mutants accumulate precursor of the ER lumenal protein, Kar2p (Fig. 8 B). However, the translocation defect per se may not be responsible for the altered ER morphology. We tested a number of mutants with temperature-sensitive or cold-sensitive defects in other components of the protein translocation machinery and none of them had abnormal ER structure. Specifically, temperature-sensitive mutations in the major component of the translocation channel, Sec61p (mutants sec61-3 and sec61-2), have only a few minor changes in ER structure three hours after shift to the nonpermissive temperature (Fig. 8 A). These mutants develop a translocation defect less than one hour after shift to elevated temperature (Fig. 8 B; and Rose et al. 1989; Stirling et al. 1992), but continue to grow for a few hours (not shown). We ruled out that differences in strain background account for the difference between the sec61 mutants and the SRP pathway mutants, by moving the sec61 alleles into the same strain background as the SRP receptor mutants we isolated (data not shown). ER structure is also not significantly disrupted in cold-sensitive sec61-11, sec61-23, sec61-24, or sec61-32 mutants after they were grown at nonpermissive temperature for up to six hours (not shown). A sec61-2 mutant that also lacks the Sec61p homologue Ssh1p (Δssh1 sec61-2) has a strong protein translocation defect (Finke et al. 1996), but no ER morphology defect (Fig. 8 A). We also looked at two mutants with temperature-sensitive defects in proteins required for posttranslational translocation of proteins into the ER, a process that does not require the SRP pathway (Panzner et al. 1995). Both mutants (kar2-159 and sec62-1) had only slight ER morphology alterations similar to those seen in the sec61 mutants (not shown). Thus, it seems that the altered ER morphology is not a consequence of a general translocation defect.
Neither SRP101 nor SRP102 are essential genes, but cells lacking them grow approximately five times slower than wild-type cells (Ogg et al. 1992, Ogg et al. 1998). The srp101-47 and srp102-510 mutants we isolated were also able to grow slowly at the nonpermissive temperature. After prolonged growth at 37°C (>24 h) the ER morphology defect was no longer observed (not shown). Similarly, cells with deletions of SRP101, SRP102, or SRP54 (which encodes a component of SRP), showed no ER morphology defect (not shown).
One way the SRP pathway might affect ER structure is via ribosome targeting. We reasoned that slowing translation might restore ribosome binding in mutants with a defect in the SRP pathway by giving ribosome–nascent chain complexes more time to bind to the ER membrane. Sublethal doses of the translation elongation inhibitor cycloheximide were added to cultures of wild-type, srp101-47, and srp102-510 strains immediately before shifting them to the elevated temperature. After six hours, the mutants grown without cycloheximide had ER morphology defects (not shown), whereas those grown in the presence of the drug had almost normal ER (Fig. 9 A). In addition, as described for other translocation mutants (Lee and Beckwith 1986; Ogg and Walter 1995), cycloheximide suppressed or reduced the translocation defect of the mutants (Fig. 9 B). Thus, the role of the SRP pathway in maintaining ER morphology is likely related to its known function in targeting ribosomes to the ER membrane.
Cycloheximide suppresses both the ER morphology and translocation defects of SRP receptor mutants. Strains expressing Sec63-GFP were grown at 25°C and then for 6 h at 37°C with or without 1 μM cycloheximide. A, Images of the cells grown with cycloheximide. They were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm. B, A portion of the cultures were pulse-labeled for 7 min and lysates were immunoprecipitated for Kar2p. m, Mature Kar2p; p, precursor of Kar2p.
To determine ribosome binding to the ER, we measured the amount of Sec61p that is associated with ribosomes after solubilization of ER membranes in detergent. ER membranes from wild-type, sec61-3, and srp101-47 cells were washed with high salt to remove loosely bound ribosomes not engaged in translocation (Gorlich and Rapoport 1993), and solubilized in digitonin. The ribosomes were pelleted, and the amount of cosedimenting Sec61p was determined by quantitative immunoblotting (Fig. 10). At permissive temperature, ∼30% of the Sec61p was bound to ribosomes in all three strains (Fig. 10, bars 1, 5, and 9). Release of the nascent chains from the ribosomes with puromycin greatly decreased the amount of Sec61p pelleting with the ribosomes (Fig. 10, bar 2; and data not shown), indicating that the association requires the presence of a translocating polypeptide. When the strains were grown for three hours at nonpermissive temperature, the fraction of Sec61p bound to ribosomes reproducibly decreased to ∼20% in the srp101-47 strain (Fig. 10, bar 6), whereas it remained about the same in the sec61-3 (Fig. 10, bar 10) and wild-type strains (Fig. 10, bar 3). The amount of Sec61p per cell in these strains was about the same when they were grown at permissive temperature or after three hours at nonpermissive temperature (data not shown; Biederer et al. 1996). When the srp101-47 mutant was grown at 37°C for prolonged periods, conditions that restore ER morphology, the amounts of Sec61p bound to ribosomes increased to normal levels (Fig. 10, bar 8 vs. 6). A similar correlation between restoration of ER morphology and ribosome binding was seen after treatment of the mutant with cycloheximide (Fig. 10, bar 7 vs. 6). A small increase of ribosome-associated Sec61p was observed when wild-type cells were grown with cycloheximide (Fig. 10, bar 4 vs. 3), which might be expected when the rate of translation is slowed. Taken together, these results suggest that the SRP pathway influences ER morphology through its effect on ribosome binding.
Ribosome binding to Sec61p. Wild-type, sec61-3, and srp101-47 cells were grown at 25°C and then shifted to 37°C for the indicated number of hours. In some cases, cycloheximide was added to the medium when the cultures were shifted to elevated temperature. ER membranes were isolated, washed with high salt, and solubilized with digitonin. The ribosomes were pelleted and the amount of Sec61p in the pellet determined by quantitative immunoblotting. Each bar is the average of at least three independent determinations. Some samples were treated with puromycin before solubilization to release nascent polypeptides (column 2). The differences between columns 5 and 6 and columns 3 and 4 are statistically significant (P < 0.01).
We found that in the SRP receptor mutants, not only peripheral ER, but also mitochondrial structure, is disrupted. In wild-type cells, mitochondria form branched reticular structures near the cell cortex (Hermann and Shaw 1998; Fig. 11 A). In the SRP receptor mutants we isolated, mitochondria appear to fragment; at nonpermissive temperature, a number of bright spots were seen throughout the cytoplasm (Fig. 11 B). Mitochondrial structure is normal when the mutants are grown at permissive temperature (not shown). Interestingly, when the ER and mitochondria were visualized in the same cell, fragments of the disrupted ER were frequently localized next to fragments of mitochondria (Fig. 11 B). Mutants with defects in ER–Golgi complex trafficking that affect ER structure (e.g., ret1-3), also have altered mitochondrial structure. As with the SRP mutants, the disrupted ER and mitochondrial structures are often near one another (Fig. 11 C). Even in wild-type cells, the tubules of the peripheral ER sometimes coaligned with mitochondrial tubules (Fig. 11 A). These data suggest that the membranes of the ER and mitochondria are linked at certain sites and that the ER may have an effect on the structure of mitochondria. The reverse may not be true. Conditional mutations in two mitochondrial outer membrane proteins, Mmm1p and Fzo1p, cause mitochondria to form large spheres (mmm1-1) or fragment (fzo1-1) at nonpermissive temperature (Burgess et al. 1994; Hermann et al. 1998; Rapaport et al. 1998). ER structure is unaffected in these mutants (Fig. 11D and Fig. E).
The SRP receptor mutants also disrupt mitochondrial structure. Cells expressing Sec63-GFP were labeled with TMR-CH2Cl to visualize mitochondria and viewed live. Wild-type cells were grown at 30°C. The other strains were grown at 25°C and then shifted to 37°C for 2 h (srp101-47), 2.5 h (ret1-3), 4 h (mmm1-1), or 30 min (fzo1-1). Arrows point to regions of alignment of mitochondria and ER tubules in wild-type cells. Images were acquired while focusing on either the center or periphery of the cells. Bars, 5 μm.
A major result of this work is the demonstration that the peripheral ER in yeast, previously viewed simply as a bright ring by immunofluorescence (Rose et al. 1989; Preuss et al. 1991), is a tubular network, similar in structure to the ER in higher eukaryotes. The tubules have a relatively constant diameter and undergo the same kinds of dynamic changes that have been described in mammalian cells. As in mammals, the ER network is maintained during mitosis. Yeast cells can therefore serve as an excellent model system in which to study the mechanisms underlying the formation and maintenance of ER networks.
Our data indicate that mechanisms proposed for maintaining the reticular structure of the ER in mammalian cells are probably not correct for yeast. We found that microtubules are not required to maintain the structure of the peripheral ER, in contrast to their function in mammalian cells. This is perhaps not too surprising, since there are few cytoplasmic microtubules in yeast. While actin does form an extensive cortical network, it is also not required for the maintenance of ER structure. Since there is also relatively little coalignment between actin structures and the ER network, ER tubules are probably stabilized by mechanisms other than association with an underlying cytoskeleton scaffold. The actin cytoskeleton does appear to be required for ER dynamics, which are drastically reduced minutes after addition of latrunculin-A to the medium. However, it is difficult to rule out that the effect may be indirect. Actin filaments are also required for the dynamics of mitochondria in yeast (Simon et al. 1995), but again the mechanism remains to be elucidated.
Given that the nuclear and peripheral ER are structurally distinct and that the cytoskeleton does not play a role in maintaining ER structure in yeast, we decided to use genetics to search for other components that do. In our visual screen, we looked for mutants that affect either ER inheritance or the maintenance of the ER. Only three out of the thousand mutants we screened had such defects, indicating that a general perturbance of cell physiology does not readily lead to these phenotypes. It is clear that the screen has not been saturated, and indeed we have found additional mutants affecting ER structure that were not identified in the screen.
One class of mutants we isolated affects ER structure and the propagation of the ER from the mother to the daughter cell by disrupting vesicular transport between the ER and Golgi complex. Defects later in the secretory pathway did not affect ER structure or inheritance. How exactly vesicular transport between the ER and Golgi complex is required for ER structure is unclear. Resident ER proteins are known to be transported to the Golgi apparatus at some low rate (Teasdale and Jackson 1996), and it therefore seems possible that retrograde transport from the Golgi apparatus to the ER may be required to recycle ER proteins needed to maintain proper structure.
Surprisingly, we find that ER structure is not affected in a temperature-sensitive mutant with a defect in a protein (Cdc48p) required for homotypic ER fusion in vitro (Latterich et al. 1995). Since yeast ER is dynamic, it seems unlikely that ER structure could be maintained in the absence of homotypic ER fusion. It may be that, in vivo, other proteins are able to substitute for the role of Cdc48. Alternatively, it may be that Cdc48p plays a role in the ER–ER fusion required for nuclear division, but is not essential for fusion events in peripheral ER.
Our data show that the SRP pathway plays an important role in maintaining the reticular structure of the ER. The linkage could be direct. Only mutants with defects in the α and β subunits of the SRP receptor and in SRP itself had the ER disruption phenotype; other mutants with translocation defects did not show this effect. In fact, some of the mutations in Sec61p, a component required for transport of all substrates, have stronger translocation defects than the srp101-47 and srp102-510 mutants we isolated (Stirling et al. 1992; Finke et al. 1996; Pilon et al. 1998). In addition, the Sec61p mutants can continue to grow at the nonpermissive temperature for a few hours after onset of the translocation defect, and their ER remains normal. Despite this evidence, it is hard to exclude that the SRP pathway affects ER structure by blocking the translocation of a particular protein. However, we consider it more likely that the SRP pathway affects ER structure via ribosome targeting to the ER membrane. We find a small, but reproducible, reduction in ribosome binding to the ER in a SRP receptor mutant, but not a Sec61p mutant or wild-type strain, when they are grown at elevated temperature. In addition, under conditions where ER morphology is restored, ribosome binding is increased back to wild-type levels. The experiments with cycloheximide are consistent with this interpretation. Slowing translation with sublethal amounts of cycloheximide restored ribosome binding and suppressed both the ER morphology and translocation defects of the SRP receptor mutants. These data show that both phenotypes are related to ribosome targeting and argue against an additional function of the SRP pathway. It is unclear how ribosome binding to the ER might affect its structure. In mammals, membrane-bound ribosomes have been proposed to be connected with each other by a protein network (Kriebich et al. 1983; Amar-Costesec et al. 1984; Marcantonio et al. 1984). Perhaps, ribosome binding to the Sec61p or Ssh1p complexes affects this network. A role for ribosome binding in determining ER structure might also explain the morphological differences between the rough and smooth portions of the ER that have been noted since the organelle was first described (Palade 1956).
The SRP pathway is clearly not essential for ER formation and maintenance since cells with deletions of SRP pathway components have normal ER structure. In addition, both the translocation and ER morphology defects in the SRP receptor mutants we isolated are partially ameliorated during prolonged growth without this pathway. Adaptation of SRP pathway mutants has been noted before and was not found to be the result of suppressing mutations (Hann and Walter 1991; Ogg et al. 1992). While it is possible that suppressing mutations may occur in our mutants when they are grown at high temperature, it is more likely that they also undergo adaptation. Since we found that ribosome binding is restored under these conditions, our data suggest that it is SRP-independent ribosome targeting that restores ER structure. SRP-independent ribosome targeting may explain both the small changes in ribosome binding to the ER in SRP pathway mutants when they are shifted to nonpermissive temperature and the fact that the SRP pathway is not essential for viability (Hann and Walter 1991; Ogg et al. 1992, Ogg et al. 1998). Indeed, SRP-independent, direct binding of ribosome–nascent chain complexes to ER membranes has been demonstrated in vitro (Jungnickel and Rapoport 1995).
Finally, our data point to an interesting connection between the structures of the ER and mitochondria. Both organelles form reticular networks close to the plasma membrane and are often closely apposed to one another. A close association between these two organelles has also been observed in mammalian cells (Rizzuto et al. 1998). In SRP receptor mutants, both structures are disrupted, but maintain their association, indicating that they are physically linked at a number of sites. It is possible that the ER is required for maintaining the mitochondrial reticulum. Interestingly, the reverse may not be true since mutations that disrupt mitochondrial structure leave the ER intact. A structural connection between the ER and mitochondria is consistent with known functional interactions between the two organelles. For example, it has been proposed that direct contact between the ER and mitochondria is required for phospholipid transport between them (Shiao et al. 1998; Achleitner et al. 1999), and Ca2+ released from one organelle can act locally on the other (Rizzuto et al. 1998).
We thank R. Schekman, F. Winston, M. Latterich, J. Nunnari, R. Jensen, P. Walter, P. Novick, C. Shamu, D. Botstein, and T. Sommer for strains; C. Shamu, K. Matlack, and L. Dreier for critically reading the manuscript; A. Kram and J. Zagers for help with EM; M. Rolls and J. White for help with quantification of images; and B. Glick for helpful advice.
T.A. Rapoport is a Howard Hughes investigator and W.A. Prinz is supported by a postdoctoral fellowship from the American Cancer Society.
Jason A. Kahana's current address is Ludwig Institute for Cancer Research, CMM-East, Rm 3080, 9500 Gilman Dr., La Jolla, CA 92093-0660.
Abbreviations used in this paper: GFP, green fluorescent protein; SRP, signal recognition particle.
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Abstract: [First paragraph] Fredric Jameson's pointed question1 from his 1997 essay is persistently examined and explored throughout David Simon's Treme (HBO, 2010-). Indeed, Treme asks how space is political and in response incorporates multiple figurations of negotiations of public spaces in post-Katrina New Orleans. Like Simon's The Wire (HBO, 2002-2008) before it, Treme functions as a sustained critique of the ways in which city spaces are used by both disenfranchised individuals as well as political and corporate elites. For Simon, the local is the primary point of departure for systemic political critique. While his New Orleans narrative/s may be flawed at times and are, perhaps, primarily based on learned knowledge rather than acquired through experience (as in The Wire), Treme nonetheless provides a sustained spatial critique at the level of the local. Storylines turn on the ways in which central characters struggle to reclaim their place in the city, whether through finding a new place to live, locating exiled friends and family, re-building a business or simply dodging disaster tourists. As in real life, individual strategies for surviving in the city after the storm are predicated in Treme on the degree of privilege prior to it; the ways in which social advantages, or the lack thereof, inform everyday life after the hurricane is traced and contrasted in overlapping character narratives.
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Home > Uttar Pradesh > Degree > Chandpur > Maa Saraswati Mahila Mahavidyalayay
Maa Saraswati Mahila Mahavidyalayay, Chandpur, Uttar Pradesh
Chandpur (District Varanassi)
Uttar Pradesh, India
Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh is a recognised institute / college. Status: Private Non-Aided . Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh was established on / in 2003.
Principal of Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh is Smt. Manorma Srivastava.
Maa Saraswati Mahila Mahavidyalayay is situated in Chandpur of Uttar Pradesh state (Province) in India. This data has been provided by www.punjabcolleges.com. Mobile No(s) of concerned persons at Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh are Sri Rajeshwar Patel (Manager), 9415353579.
Website of Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh is www.maasaraswatimmv.org.
Contact Details of Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh are : 0542-2372382
Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh runs course(s) in Degree stream(s).
Approval details: Maa Saraswati Mahila Mahavidyalayay is affiliated with Veer Bahadur Singh Purvanchal University (VBSPU), Jaunpur (Uttar Pradesh)
Media coverage of Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh, Uttar Pradesh
NCTE appeal order for Maa Saraswati Mahila Mahavidyalayay Chandpur
F.No.89 - 92-2010 - Appeal
NATIONAL COUNCIL FOR TEACHER EDUCATION
Hans Bhawan, Wing II, 1, Bahadurshah Zafar Marg, New Delhi - 110002
O R D E R
WHEREAS the appeal of Maa Saraswati Mahila Mahavidyalaya, Varanasi, Uttar Pradesh dated 08-02-2010 is against the Order No. F.NRC-NCTE-F-3-UP-2663-155th Meeting-2010-15639 dated 29-01-2010 of the Northern Regional Committee, refusing recognition for conducting B.Ed course on the grounds
1) The land area is only 2507.66 sq.m and the college is running BA and MA programmes.
2) The built up area is only 2000 sq.m for BA, MA and proposed B.Ed programmes which is not as per NCTE norms for B.Ed".
AND WHEREAS the Correspondent, Maa Saraswati Mahila Mahavidyalaya (hereinafter referred to as the appellant), preferred an appeal dated 08-02-2010 to the National Council for Teacher Education, New Delhi (hereinafter referred to as the Council) under Section 18 of the NCTE Act, 1993 against the said Order.
AND WHEREAS Shri. Rajeshwar PD Singh, Manager and Dr. Ravindra Kumar Shrivastava, Principal, Maa Saraswati Mahila Mahavidyalaya, Varanasi, Uttar Pradesh presented the case of the appellant institution on 16.3.2010. In the appeal and during personal presentation it was submitted that the ground of refusal of recognition to the institution was against the factual position on the spot as well as document records, map, the replies of the appellant as well as the other relevant material which were submitted alongwith the application.
AND WHEREAS the Council noted that in the application submitted by the institution, the available build up area indicated was 1950 sq.mtrs. As against this, in an affidavit dated 1.10.2007 submitted, the available land and built up area indicated were 2507.62 sq.mtrs and 2000 sq mtrs. respectively. The building plan submitted by the institution showed that the size of the plot was 748.50 sq.mtrs. and the built-up area shown was 541 sq.mtrs. Thus, the institution made contradicting claims regarding availability of the land and built up area in various documents submitted from time to time. The Council also noted that even if it was accepted that the institution was having 2507.66 sq.mtrs. of land and built up area of 2000 sq.mtrs. it was not sufficient as per the NCTE norms as the institution was running B.A. and M.A. programmes also in the same premises and, as per the NCTE norms for running B.Ed. course alone the requirement of land and built up area are 2500 sq.mtrs. and 1500 sq.mtrs. respectively.
The Council was therefore, of the view that recognition was refused to the institution by NRC for valid reasons and there was no ground to accept the appeal.
AND WHEREAS after perusal of documents, memorandum of appeal, affidavit and after considering oral arguments advanced during hearing, the Council reached the conclusion that there was no ground to accept the appeal and hence it should be rejected. Accordingly, the appeal was rejected and NRC's order dated 29-01-10 was confirmed.
NOW THEREFORE, the Council hereby confirms the Order appealed against.
(Hasib Ahmad)
Member Secretary
1. The Manager, Maa Saraswati Mahila Mahavidyalaya, Chandpur, Industrial Estate, District, Varanasi -, Uttar Pradesh
2. The Secretary, Ministry of Human Resource Development, Department of School Education and Literacy, Shastri Bhawan, New Delhi.
3. Regional Director, Northern Regional Committee, A-46, Shantipath, Tilak Nagar, Jaipur - 302004, Rajasthan.
4. PS to Chairperson
5. The Secretary, Education (looking after Teacher Education) Government of Uttar Pradesh, Lucknow.
The Manager, Maa Saraswati Mahila Mahavidyalaya, Chandpur, Industrial, Estate Varanasi, Varanasi - , Uttar Pradesh
F.No.89-215-2009-Appeal
Hans Bhawan, Wing II, 1, Bahadurshah Zafar Marg, New Delhi - 110 002
WHEREAS the appeal of Maa Saraswati Mahila Mahavidyalaya, Varanasi, Uttar Pradesh dated 16-03-2009 is against the Order No.
F.NRC-NCTE-F-7-UP-2663-2008 dated 27-01-2009 of the Northern Regional Committee, Refusing recognition for conducting B.Ed course on the grounds that The NRC noted that the State Govt. recommendation letter no. 2006-Sattar -6 -2008 -2(323)2008 dated 27.08.2008 is negative. The NRC decided that the appeal for B.Ed. course is hereby rejected.
AND WHEREAS the Correspondent, Maa Saraswati Mahila Mahavidyalaya (hereinafter referred to as the appellant), preferred an
appeal dated 16-03-2009 to the National Council for Teacher Education, New Delhi (hereinafter referred to as the Council) under Section 18 of the NCTE Act, 1993 against the said Order.
AND WHEREAS Rajeshwar Prasad Singh, Mamber and Shri Anil Singh, Lipik, Maa Saraswati Mahila Mahavidyalaya, Varanasi, Uttar Pradesh presented the case of the appellant institution on 29-05-2009. In the appeal and during personal presentation it was submitted that the appellant was having adequate infrastructural and instructional facilities as per norms and as per Regulations 2007, NOC from State Govt. was not mandatory and hence refusal of the recognition by NRC in reference to the State Govt. recommendations was not reasonable and logical.
AND WHEREAS the Council noted from the U.P. Govt. G.O. dt. 11-02-05 that institutions offering undergraduate courses atleast in any two disciplines of Science-Arts-Commerce were only eligible to conduct teacher training programmes and the same Govt.. vide its G.O. dt. 01-06-09 had withdrawn its earlier G.O. The Council also noted that an application for recognition of teacher education course has to be considered in accordance with the provisions in the NCTE Act and Regulations and that there was no such condition in the NCTE Act-Regulations that an applicant institution should have been running two degree courses. The Council was therefore of the view that the refusal of recognition to the institution on said ground was not justified. The Council, therefore, came to a conclusion that there was enough justification in accepting the appeal and remanding back the case to NRC to consider the case on merit and there-after passing appropriate orders.
AND WHEREAS after perusal of documents, memorandum of appeal, affidavit, VT Report, and after hearing oral arguments advanced during the hearing, the Council reached the conclusion that there was adequate justification for accepting the appeal and remanding back the case to NRC for consideration of the case on merits, as per the provisions in the NCTE Act.- regulation. Accordingly the appeal was accepted and the case was remanded back to NRC for further examination on merit.
NOW THEREFORE, the Council hereby remands back the case of Maa Saraswati Mahila Mahavidyalaya, Varanasi, Uttar Pradesh to the NRC, NCTE, for necessary action as indicated above.
1. The Manager, Maa Saraswati Mahila Mahavidyalaya, Chandpur, Industrial, Estate Varanasi, Varanasi - , Uttar Pradesh
Summary: Maa Saraswati Mahila Mahavidyalayay, Chandpur Uttar Pradesh website, mobile, contact address and approval / recognition details.