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(Ideally all nine readings should be proclaimed tonight. But for reasons of space, I can only comment on four.)
Dennis Weaver's recent death reminded me of something he said in an old radio interview. When asked about his years as Matt Dillon's sidekick in the Gunsmoke series, he pointed out, "All the old movie and TV western heroes have sidekicks. It's the only way the audience can find out what the hero's thinking." Such characters are as much a dramatic device in westerns as soliloquies are in Shakespeare's plays. Without them, we'd never know what's going on in someone's mind.
Fr. Raymond Brown was convinced that our sacred authors use angels in a similar way - as literary devices giving readers the meaning behind specific events. In tonight's gospel, for instance, Mary Magdalene, Mary the mother of James, and Salome simply come upon an empty tomb. The absence of Jesus' body can be interpreted in different ways: someone could have taken it, or they might have gone to the wrong tomb. We need Mark's "young man" to give the definitive Christian interpretation: "You are looking for Jesus of Nazareth, the one who was crucified. He has been raised up; he is not here."
As comforting as these words are to the women, there's one problem: none of us normally go around with sidekicks, or break into a soliloquy during significant times of our life, nor do angels suddenly appear to provide us with the meaning behind our experiences. We live a life overflowing with unknowns.
It's our responsibility to surface meaning behind the people, events and situations which otherwise pass through our lives in a meaningless procession.
Paul, in tonight's eighth reading, supplies us with the meaning of our first formal step in the faith: baptism. Ignoring highfalutin theological language, the Apostle states that when people are immersed in the water of baptism, they're committing themselves to be immersed just as deeply into this death-dealing world as the historical Jesus was. Yet, when they're lifted up out of the water, they're proclaiming that they're now experiencing the same life which the Father gave Jesus.
Most Scripture scholars believe the Exodus account of the crossing of the sea has been greatly exaggerated both by several hundred years of oral tradition and the sacred author's attempt to show the significance of the event. If it happened exactly the way our passage describes, how could anyone ever gripe and complain about Yahweh bringing them out of Egypt as some of them do later in the wilderness? The author has undoubtedly integrated his or her interpretation into the narrative, so that we, the readers, can instantly see something in the crossing which probably took the original participants years to understand.
That's why it's good to listen carefully to what Yahweh proclaims in our Deutero-Isaiah passage. "Though the mountains leave this place and the hills be shaken, my love will never leave you; nor my covenant of peace be shaken, says Yahweh who has mercy on you."
Perhaps the only way we can understand what God thinks about us or surface the meaning of God's actions in our lives is to return to something the prophet presumed all followers of God would presume: God loves us and will never desert us, even during those moments when we feel unloved and deserted.
Especially on this night, our readings remind us that we live a life of faith. Yet when we want to convey our experience of faith to others, we often have to fall back on as many "devices" as our sacred authors did.


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The Buffalo Bills and New York Jets will make some history when they kick off on Thursday night.
Not on the field, but on your phone.
The game between the AFC East rivals will be streamed live on Twitter, meaning millions around the world can watch the live game action from their smartphones, tablets and other mobile devices.
The NFL had a similar venture with Yahoo for a game last season, but the league signed a deal with Twitter in April to deliver 10 Thursday night games through the mobile app.
Twitter also announced on Wednesday that its app will be available on Xbox One, Apple TV and Amazon Fire TV, just in time for the first NFL game.
Here is a look at the details and what it means for fans around the world:
Streaming live television is becoming more of a trend in recent years, with some consumers preferring to cut their cable television subscriptions in favor of viewing their favorite programs through the internet. This occasionally takes away the option of watching the program live as it airs, but not in the case of Twitter's agreement with the NFL.
To access the stream, you don't even need a Twitter account. Simply open Twitter's mobile app or go to twitter.com, click on the ''moments'' tab and it will show up on the screen . Viewers will see the same broadcast production - same announcers, camera angles, etc. - as those who tune in to CBS on television to watch the game.
For those who would rather stream the game on a larger screen than their handheld phones or tablets, Xbox One, Apple TV and Amazon Fire TV are now equipped with the Twitter app to use on television screens.
For those who want to watch the old fashioned way, the game is still available on TV via CBS and on the NFL Network.
While the production quality and most other components of the broadcast itself will be identical to the one viewed on CBS, those watching through Twitter will see curated tweets related to the game running alongside the video.
Twitter has long billed itself as the ''second screen'' for the TV viewer, with users having a live, running conversation and commentary about what they are watching on their phones as a companion to the content they are viewing on TV.
This stream will marry the two screens, allowing users to tweet away to their heart's content while never having to take their eyes off the ball.
Twitter paid more than $10 million for the chance to stream the 10 games. In contrast, CBS and NBC are paying $45 million per game for the Thursday night package and Yahoo reportedly paid nearly double that just for the Jaguars-Bills game in London last year.
The NFL is not known for offering discounts, but Twitter's user base of more than 313 million people worldwide may have helped the company secure the bid. All of the advertising during the streamed version will be the same as those ads aired on television, save for a few that Twitter was able to sell itself.
While more and more people are leaving cable television for streaming services, the audiences are still small in comparison. The Yahoo game last year in London averaged a worldwide audience of about 2.4 million viewers, but that game was blacked out on television for any viewers outside the local markets of Buffalo and Jacksonville.
To compare, a Jets-Dolphins game from London last year televised on CBS drew 9.9 million viewers in the U.S. alone.
But the NFL is banking on Twitter's international reach to help draw in audiences from around the world that the league has spent years trying to cultivate.
The short-message service has had difficulty monetizing its popularity. The company has yet to turn a profit despite the hundreds of millions of people who use their product, so hitching itself to the all-powerful NFL is a leverage play to increase the eyeballs and advertisers.
AP NFL websites: http://pro32.ap.org and http://twitter.com/AP-NFL

subset: commoncrawl


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London Flagship
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Tel +44 (0) 20 7493 6767
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The store, one of the largest luxury goods stores in the world, is the perfect showcase for the extensive range of Asprey products including jewellery and silverware, timepieces, leather goods and silk, china and crystal.
The team of Asprey craftsmen, experts in making silver, jewellery, leathergoods and engraving are based on the top floor of the building, allowing Asprey to continue the bespoke service for which it is renowned.
Pop Up At The Dorchester Hotel
The Dorchester Hotel
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Tel +44 (0) 20 7319 7138
Opening Times:
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Sunday: 12pm to 5pm


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General information
EMDR (Eye Movement Desensitization and Reprocessing) is a powerful and effective psychotherapy that enables people to heal from the symptoms and emotional distress that are the result of disturbing life experiences. EMDR relies on the brain's self-healing capacity. It helps to activate your brain's natural emotional healing system and remove improperly stored images, thoughts or feelings that get in the way.
The goal of EMDR is to process completely the experiences that are causing problems, and to include new ones that are needed for full health. By alternately stimulating the right and left sides of the brain, EMDR processing allows experiences that are causing problems to be "digested" and stored appropriately in your brain, thereby allowing room for the potential for new positive experience.
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For More Information
Call 847-529-8300 x 0 or email at email@example.com


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Level: Beginner Sessions: 1 Hours: 3 hours
Project OverviewLearn how to solder mixed metals without a mess. Sweat soldering will reduce bleeding of solder onto mixed metals. Additionally, you learn how to cut, shape and forge sterling silver. Put all the pieces together to create these distinctive earrings.
Bench Block (steel or anodized aluminum) - Used as a hammering surface for metal stamping or working with metal.
Bezel Shears – Used to cut metal sheet. There are many available on the market, any of which will work, however they should NOT be serrated. (Tin snips or airplane shears will also work, however, they may be large and cumbersome.) If the project has curves in the design and you own a pair, bring curved bezel shears. This is an optional item for most classes. Shears will be provided for use during classes.
Disc Cutter – Used to punch discs out of metals. One set will be provided to share in every class.
Basic hand tools – Any quality is appropriate for my classes. These do not need to be high end or expense.
- Round nose - Typically it is preferred that the round nose be a fine tip. Especially for classes with detailed wire work.
- Chain/needle nose – Tapered jaws for general metal and wire work. Non-serrated are requested for all of my classes. Typically requested for classes to be used for opening and closing jump rings; in which case, 2 pairs are requested or a combination of chain nose and flat nose is suitable.
- Flat nose – Similar to chain nose but the jaws are not tapered. Non-serrated are requested for all of my classes.
Magnifier or Optivisor – For almost all classes this is listed as an "As Needed" tool. If you have sight challenges it is recommended that you are properly prepared. For advance stone setting classes this is required! If you decide to use reading glasses consider better than 2.5x.
Needle File Set – Used for filing smaller pieces of metal and tight places. When required, either diamond coated or fine tooth is preferred. Some students may prefer to use smaller files as opposed to the student files for their projects.
Nylon nose pliers – Flat nose pliers with PVC grips o the heads. Mostly used for straightening wire. In my classes they are used to prevent marring when bending metal. Only required for some classes, but this is mostly an optional item.
Ott Light or Task Lamp - This is optional item that is listed for every class. It is up to you to determine whether or not you will need to have one depending on your vision.
Rawhide/Leather Mallet – Shape and flatten metal without marring it. #2 mallet is a standard size. Instructor will provide for every class needed. A yellow nylon mallet may be used in its place, however a rubber one may not.
Side Cutters - Also known as wire cutters or snippers.
For many of my classes I prefer Ultra Flush side cutters because it eases the workload. Ultra flush is the most flush you can buy as opposed to just flush cutters. If you don't own a pair of Ultra Flush cutters, you may want to invest in a good pair either Lindstrom or Swanstrom. There is usually not a different in price between ultra and just flush cutters so why not just buy the best?
The image shows just a side cutter (top) and ultra flush (bottom).
Optional Soldering Kit
Items in this Soldering Kit will be provided for use during the class. If you are interested in ordering a kit please click on the image. Kit includes: Butane torch, butane, 1 tube solder paste, fire tweezers, copper tweezers, Sparex, tripod with mesh, flux, soldering board. (Note: kit will be delivered at class. If you require the kit prior to class please contact us to make arrangements.
Ring Mandrel, Steel – Used for shaping and forming metal into rings. A plastic one will not work in its place. Instructor will provide to share (1 for every 2 people) for every class needed.
Student Files – Standard size is 2/o. For filing metal. . Instructor will provide to share for every class needed.
Super Grip Gloves – Optional for all classes, but most have found these very helpful for drilling class and classes that require a lot filing sanding. Not only will they protect your hands, but the grip will allow you to hold onto the stones you are working with.


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Tonight I am chairing an event with my friend and former PhD supervisor Professor Guy Claxton, for the second of our series of public events on Spirituality, Tools of the Mind, and the Social Brain. The event is booked out with a large waiting list, but you can watch or listen to the event live, download after the event, or follow (and give) comments at the hashtag #RSASpirituality.
For now, I offer the following extracts as an appetiser. I am sure Guy's thinking has evolved since his inaugural address at Bristol University in 2002 around the time we first met, but I'm equally sure the quality of thinking and expression will be just as good, if not better. Guy's main argument tonight will be similar in spirit, enriched by some of his recent thinking on embodied cognition. The following extract follows from the claim that spiritual experiences tend to bring four shifts in the quality of our experience: aliveness, belonging, mystery and peace of mind, which Guy examines as follows:
"Underlying all these four shifts in the quality of experience seems to be an expansion in the sense of identity, so that instead of feeling like an anxious bubble, in constant danger of being jostled or pricked, one feels more union or wholeness, both within and without, and this brings with it more kinship and more trust. It is no coincidence that the descriptions are couched is such glowing language, for those who report them overwhelmingly appraise them as positive and valuable. While from the outside it is possible – as Freud and others have done – to interpret accounts of the Common Experience sceptically or pathologically, from the inside, there is little doubt that something precious, even momentous, has occurred.
I think that experiences like that are small gifts, little tastes of spirituality. And those tastes are attractive, and often leave, when they fade, as they mostly do, a thirst for more of what they have betokened. So here is my definition of spirituality. First, it involves the feeling of being drawn towards such qualities and experiences, and of wanting to increase their likelihood, frequency or stability. The urge is not to seek them piecemeal, however, but to develop the quality of being which underpins them. Second, spirituality may involve a strengthening desire to seek the company of people who seem to possess these qualities more strongly: to find what Buddhists call a sangha. And third, and less comfortably, the spiritual impulse may involve a feeling of heightened dissatisfaction with the absences or the opposites of such qualities. A taste of intensified aliveness may make normal energy levels and perceptions feel grey and dull. That blast of 'belonging' can make that orphaned feeling all the more intolerable. A surge of 'mystery' may make adventure seem more attractive than conventional security. And even a moment of deeply felt inner peace can painfully accentuate a more familiar feeling of self-consciousness or confusion."


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As the time for the premier for The Force Awakens draws near, excitement is brewing for those new and familiar with the series. For decades many fans have speculated on the comings and goings of the characters from the original Star Wars Trilogy. With the canon now being rewritten, The Force Awakens hopes to take us to places in the galaxy far far away that have never been seen before. Screen shots from the movie trailers hint at the return of many familiar characters. Of course this just leads to more questions, but one question that has been left unanswered for decades deals with an object rather than a character. That object is the lightsaber wielded by Luke and Anakin Skywalker. Yes, fans might want to know where that lightsaber is, but for that matter, who actually owns the lightsaber? For that answer the lightsaber must be analyzed through the intricate lense that is property law.
As you might recall this was the second lightsaber Anakin built. His first lightsaber was destroyed on Geonosis during the events of Attack of the Clones. From the Clone Wars series there is no depiction of him making the new lightsaber, but there was an episode showing younglings crafting their own Jedi weapons. For the sake of this analysis, we will assume that Anakin made his second lightsaber in a similar matter. However, this does not mean that the Jedi Order may claim ownership over the weapon (think crafts that you bring home from summer camp, but more dangerous). This would mean the lightsaber is Anakin's personal property. This means that Anakin has the true legal title to the lightsaber.
After Anakin and Obi-Wan's battle on Mustafar, Obi-Wan took the lightsaber with him before entering into a self-imposed exile on Tatooine. At no point does Anakin give the lightsaber to Obi-Wan, therefore Anakin still retained rightful title in the lightsaber. It is not clear if Obi-Wan meant to take it for himself or if he knew that he would give it to either Luke or Leia. Either way, this was arguably still an act of larceny. Connecticut law defines larceny as:
A person commits larceny when, with intent to deprive another of property or to appropriate the same to himself or a third person, he wrongfully takes, obtains or withholds such property from an owner. Conn. Gen. Stat. § 53a-119 (2015).
Since Obi-Wan cut off Anakin's legs and arm immediately before taking the lightsaber, it seems a likely conclusion that Obi-Wan intended to deprive Anakin of the lightsaber by taking it and leaving Anakin to smolder in the lava of Mustafar.
Meanwhile, Obi-Wan has been exerting dominion and control over the lightsaber. He kept it with him on Tatooine during his exile, and nineteen years after the events of Revenge of the Sith, Obi-Wan presented the lightsaber to Luke Skywalker, saying that it belonged to his father. At this point, Luke was under the impression that his father was already dead. To his knowledge then, there was no other owner of the lightsaber still living. He took the lightsaber from Obi-Wan as a gift, not knowing that the true title of the lightsaber still resided with his living father. Connecticut defines a gift as follows:
A gift is the transfer of property without consideration. It requires two things: a delivery of the possession of the property to the donee, and an intent that the title thereto shall pass immediately to him. Coppola v. Farina, 50 Conn. Sup. 11, 13, 910 A.2d 1011 (2006).
There are two elements that must be satisfied. First, that the possession of the lightsaber transfer to Luke, and second, the intent that the title immediately transfer to Luke. While the first element was satisfied, the second could not be fulfilled as Obi-Wan never had the title to the lightsaber. As such the gift fails, however, Luke is still in (unlawful) possession of the lightsaber.
Fast forward to the events of the Empire Strikes Back in which Luke has his hand cut off by Darth Vader (Anakin Skywalker) and is deprived of the lightsaber. However, shortly after this he learns that Darth Vader is his father. Upon this revelation, he is aware that his father, the true owner of the lightsaber, is alive. Darth Vader has a valid claim against Luke for the return of the lightsaber. In terms of criminal conduct though, it is unlikely that Luke could be charged with receipt of stolen property. In the state of Connecticut "a person is guilty of larceny by receiving stolen property if (he/she) receives stolen property knowing that it has probably been stolen or believing that it has probably been stolen." Conn. Gen. Stat.§ 53a-119(8). In this instance, Luke had no reason to think that the lightsaber had been stolen since he was convinced that his father was dead. Granted Luke probably should have asked a few more questions regarding the provenance of the lightsaber, but this does not make him liable. As a result, though Darth Vader would have had a claim, there would likely not have been a criminal claim against Luke.
Regardless of the claims against him, Luke lost the lightsaber in an undisclosed location on Cloud City. If he was still exerting dominion and control over the lightsaber, he would be on notice to return it to its true owner. Luke, however, was not in possession of the lightsaber after learning that his father was still alive. As such, he was not necessarily responsible for returning it at that point.
Moving then to the events of Return of the Jedi. After the death of Darth Vader, Connecticut intestacy law suggest that his personal property would then belong to his living heirs. In this case, the two living heirs would be Luke and Leia Skywalker (since Padme, their mother, is also deceased). See Conn. Gen. Stat.§ 45a-438. However, there is an argument to be made that Luke contributed to the death of Darth Vader by weakening him before his confrontation with Emperor Palpatine. Albeit Darth Vader acting on his own to save his son, this does not change the fact that Luke intentionally acted to harm his father. But for Luke's lightsaber duel, Darth Vader may have been able to fend off Emperor Palpatine. More importantly, after the exchange Luke drags his father's body to a hangar in an effort to leave the second Death Star. In the end, Darth Vader asks Luke to help him take off his mask. Luke acknowledges that this will kill Darth Vader. Although Darth Vader claims that this is inevitable, Luke still takes off the mask knowing that it would lead to Darth Vader's death. While some jurisdictions recognize "right-to-die" legislation, Connecticut is not one of them. As a result, Luke may be charged with murder. In Connecticut, "A person is guilty of murder when, with intent to cause the death of another person, he causes the death of such person or of a third person or causes a suicide by force, duress or deception . . . ." Conn. Gen. Stat.§ Sec. 53a-54a.
The reason Luke's actions are relevant to the lightsaber ownership is due to the fact that Connecticut has a slayer law. A slayer law prevents certain individuals from inheriting property if they caused the death of the person leaving the estate. In Connecticut, if Luke is found guilty of murdering his father (or being an accessory to his father's murder) then he is prohibited from inheriting or receiving part of the estate from the victim (i.e. his father). Conn. Gen. Stat.§ Sec. 45a-447(c)(1). According to Connecticut law, the residual estate would then pass to Anakin's other heirs. Conn. Gen. Stat.§ Sec. 45a-447. This means that the Connecticut Superior Court could determine he is guilty of murder, but a conviction would not bar him from inheriting the lightsaber. Only when he exhausts all of his appeals would he be barred from inheriting. Sulser v. Winnick, Conn. Super. LEXIS 2137 (2007).
If Luke were to be found responsible for Darth Vader's death, then the statute would leave Leia Skywalker, Darth Vader's only other living heir, to inherit their father's entire estate (which may consist of shares in a now imploded Death Star). Whether or not Leia is aware of this, the true title to Darth Vader's lightsaber would go to Leia. The unique nature of the lightsaber may prohibit Leia from wielding it like her brother or father. Certain laws may require the lightsaber to be registered for Leia to possess it, or the law could require Leia to have a permit for the lightsaber. This is likely not the case as it would equate lightsabers to handguns under Connecticut law. However, Connecticut still finds a knife with an automatic spring release from the handle with a blade longer than one and one-half inches to be a dangerous weapon. See Conn. Gen. Stat.§ Sec. 29-38. A lightsaber surely has a similar release function with a saber length of longer than one and one-half inches. While this law does not make mere possession of the lightsaber illegal, carrying the lightsaber in a vehicle or on one's person would certainly be illegal with few exceptions. Although Leia may have title to the lightsaber, she could be prevented from taking it with her for weekend getaways in the Millennium Falcon.
This leads us to Star Wars VII. While images show Finn (John Boyega) holding what looks to be the lightsaber in question this has not been definitively confirmed. How he received or acquired the new lightsaber will impact the necessary legal analysis. In the hands of Finn, we see the memorable blue glow of the lightsaber that has not been seen (in the film timeline) since Luke learned of his parentage in The Empire Strikes Back. While the lightsaber's current location is yet to be seen, its fate will likely play some role in the plot of The Force Awakens. Leia has the legal title to that fabled piece of property, but she may not even realize it. In the end, no one can legally obtain ownership of the lightsaber in question without Leia transferring title.


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From The Missouri Republican, August 20, 1862.
This article was republished in the September 2007 issue of The Shrapnel .
WARNING: This is a verbatim reprint of a period humor article and contains language that may be offensive to some.
The Humor of the War-Dashes at Military Life with a Free Pencil.
Mr. Newell, whom it is no breach of faith to name as the writer of the "Orpheus C. Kerr Papers,"* has struck an original vein of humor, and works it with decided effect. The war has given birth to no more amusing book than this. It is thoroughly good humored and loyal but exposes the weak side of our military policy in a way which shows the absurdities of many measures which have hitherto controlled the campaign against the rebels; the loose administration of the oath of allegiance, for instance, furnishing our author with an inexhaustible fund for raillery. It is now well known that the rebels who have taken the oath have openly violated it, returning to the ranks of their fellow-traitors so soon as interest or inclination dictated. Others errors of our past military management are relentlessly ridiculed in these "Papers"-sometimes unjustly, but generally with excellent reason.
Originally appearing as contributions to a Sunday journal in this city, the "Papers" have been widely copied and read. There runs through the handsome volume in which the first series is now collected, a dry humor that explains the popularity of the writer during his year of hebdominal appearance before the public. New chapters of autobiographical character have been added to the published volume, and the originals have been pruned or touched up, so that in their amended form the "Papers" may be regarded as a notable contribution to the humorous literature of the country. Occasionally the humor degenerates into coarseness, and a severer pruning of the coming volumes of the series will improve it-yet, wise things are said in a funny way, and the book is a good one to laugh over.
Under date of Washington, April, 1861, ""Orpheus" gives his opinion of ther rebels in this
The chivalrous South, my boy, has taken Fort Sumter, and only wants to be "let alone." Some things of a Southern sort I like, my boy; Southdown mutton is fit for the gods, and Southside particular is liquid sunshine for the heart; but the whole country was growing tired of new South wails before this, and my present comprehensive estimate of all there is of Dixie may be summed up in twelve straight lines, under the general heading of
'Neath a ragged palmetto a Southerner at rest,
A-twisting the band of his Panama hat,
And trying to lighten his mind of a load
By humming the words of the following ode:
"Oh! For a nigger, and oh! for a whip;
Oh! for a cocktail, and oh! for a nip!
Oh! for a shot at old Greeley and Beecher;
Oh! for a crack at a Yankee school-teacher;
Oh! for a captain, and oh! for a ship;
Oh! for a cargo of niggers each trip."
And so he kept oh-ing for what he had not,
Not contented with owing for all that he'd got.
These "twelve straight lines" are worthy of Hood.
Here is a fair hit at the way in which the recruiting service was conducted in the early stages of the war:
The Calcium Light regiment was recruited at great expense in New York, and went into camp on Riker's Island, until Secretary Cameron ordered his Colonel to bring him on immediately for the defense of Washington. The regiment has three officers, and will elect the others as soon as his voice is strong enough. He says that he is a regiment of 1,000 men; he says that 1,000 is simply the figure 1 and three ciphers, and that he represents the 1, and his three officers the three ciphers.
I believe him, my boy!
Villiam Brown of Regiment 5, Mackeral Brigade, asked his Colonel last week for leave to go to New York on recruiting service, and got it. He came back to day, and says the Colonel to him:
"Where's your recruits?"
Villiam smiled sweetly, and remarked that he didn't see it.
"Why, you went to New York on recruiting service, didn't you?" exclaimed the Colonel.
"Yes," says Villiam. "I went to recruit my health."
The Colonel immediately administered the oath to him. The oath, my boy, tastes well with lemon in it.
The General of the Mackeral Brigade is no friend to England. He is reported to have made this strong speech:
"We have borne with Great Britain a great while, my boy; but it is now time for us to take Canada, and wipe every vestige of British tyranny from the face of the globe. The American eagle, my boy, flaps his dark wings over the redhead of battle, and as his scarlet eyes rest for a moment on the English Customhouse, he softly whispers-he simply remarks-he merely ejaculates-Gore!
"Americans! fellow-citizens! foreigners! and people of Boston! shall we longer allow the bloated British aristocracy to blight us with base Abolition proclivities, while Mr. Seward is capable of holding a pen?
"'Hail blood and thunder! welcome, gentle Gore!
Let the loud hewgag shatter every shore!
High to the zenith let our eagle fly,
Ten thousand battles blazing in his eye!
Nail our proud standard to the Northern Pole,
Plant patent earthquakes in each foreign hole!
Shout havoc, murder, victory and spoils,
Till all creation crouches in our toils!
Then, when the world to our behest is bent,
And takes the Herald for its punishment,
We'll pin our banner to a comet's tail
And shake the Heavens with a big "All Hail!'"
"That's the spirit of America, my boy, taken with nutmeg on top and a hollow straw. Very good for invalids."
The following are amusing bits:
Finding himself master of the situation, Capt. Villiam Brown called the solitary chivalry to him and issued the following proclamation:
"Citizens of Accomac! I come among you not as a incendiary and assassin, but to heal your wounds and be your long-lost father. Several of the happiest months in my life were not spent in Accomac, and your affecting hospitality will make me more than jealously watchful of your liberties and the pursuit of happiness. (See the Constitution.)
"Citizens of Accomac! These brave men of whom I am a spectator are not your enemies; they are your brothers, and desire to embrace you in fraternal bonds. They wish to be considered your guests, and respectfully invite you to observe their banner of our common forefathers. In proof whereof I establish the following orders:
"I. If any nigger come within the lines of the United States army to give information, whatsomever, of the movements of the enemy, the aforesaid shall have his head knocked off, and be returned to his lawful owner, according to the groceries and provisions of the fugitive slave ack. (See the Constitution.)
II. If any chicken or other defenseless object belonging to the South be brought within the lines of the United States army, by any nigger, his heirs, administrators and assigns, the aforesaid shall have his tail cut off, and be sent back to his rightful owner at the expense of the Treasury Department.
III. Any soldier found guilty of shooting the Southern Confederacy, or bothering him in any manner whatsomever, the same shall be deemed guilty of disorderly conduct, and be pronounced an accursed Abolitionist.
VILLIAM BROWN, Eskevire.
Captain Conic Section Mackeral Brigade, Commanding Accomac."
The mud at present inclosing the Mackeral Brigade is unpleasant to the personal feelings of the corps, but the effect at a distance is unique. "As you survey that expanse of mud from Arlington Heights," continued Captain Bob Shorty, "with the veterans of the Mackeral Brigade wading about in it up to their chins, you are forcibly reminded of a limitless plum-pudding, well stocked with animated raisins."
"My friend," says I, "the comparison is apt, and reminds me of Shakespeare's happier efforts. But tell me, my Pylades, has the dredging for those missing regiments near Alexandria proved successful?"
Captain Bob Shorty shook the mire from his ears, and then, says he:
"Two brigades were excavated this morning, and are at present building a raft to go down to Washington after some soap. Let us not utter complaints against the mud," continued Captain Bob Shorty, reflectively, for it has served to develop the genius of New England. We dug out a Yankee regiment from Boston first, and the moment those wooden nutmeg chaps got their breath, they went to work at the mud that had almost suffocated them, mixed up some spoiled flour with it, and are now making their eternal fortunes by peddling it out for patent cement."
It pleases me greatly to announce, my boy, that the General of the Mackeral Brigade believes in McClellan, and gorgeously defends him against the attacks of that portion of the depraved Press which has friends dying of old age in the Army of the Potomac.
"Thunder!" says he to Captain Bob Shorty stirring the oath in his tumbler with a toothbrush, "the way Little Mac is devoting himself to the military squelching of this here unnatural rebellion is actually outraging his physical nature. He reviews his staff twice a day, goes over the river every five minutes, studies international law six hours before dinner, takes soundings of the mud every time the dew falls, and takes so little sleep that there's two inches of dust on one of his eye-balls. Would you believe it," says the General, placing the tumbler over his nose to keep off a fly, "his devotion is such that his hair is turning gray and will probably dye!"
Captain Bob Shorty whistled. I do not mean to say that he intended to be musically satirical, by boy; but if I should hear such a canary bird remark, after I'd told a story, some one would go home with his eye done up in rainbows.
* * * "Read that ere Napoleonic docky-ment," says William, handing me a scroll. It was as follows:
"EDICK-Having noticed that the press of the United States of America is making a ass of itself, by giving information to the enemy concerning the best methods of carrying on the strategy of war, I do hereby assume control of all special correspondents, forbidding them to transact anything but private business; neither they nor their wives, nor their children, to the third and fourth generation.
"I. It is ordered that all advice from editors to the War Department, to the General Commanding, or the Generals Commanding the armies in the field, be absolutely forbidden; as such advice is calculated to make the United States of America a idiot.
"II. Any newspaper publishing any news whatever, however obtained, shall be excluded from all railroads and steamboats, in order that country journals, which receive the same news during the following year, may not be injured in cirkylation.
"III. This control of special correspondents does not include the correspondent of the London Times, who wouldn't be believed if he published all the news of the next Christian era. By order of
VILLIAM BROWN, Eskevire,
"Captain Conic Section Brigade."
On reaching Accomac, my boy, we found Captain Villiam Brown at the head of the Conic Section of the Mackeral Brigade, dressed principally in a large sword and brass buttons, and taking the attitude of the sun with a glass instrument operated by means of a bottle.
"Ah!" says Villiam, "You are just in time to hear my speech to the sons of Mars, previous to the capture of Manassas by the United States of America!"
Hereupon Villiam mounted a demijohn laid lengthwise, and says he:
"Fellow-Anacondas: Having been informed by a gentleman who has spent two weeks at Manassas, that the Southern Confederacy has gone south for its health, I have concluded that it is time to be offensive. The great Anaconda, having eluded Barnum, is about to move on the enemy's rear:
"'Rear aloft your peaks, ye mountings,
Rear aloft your waves, O sea!
Rear your sparklings crests, ye fountings,
For my love's come back to me!'
The day of inaction is past, and now the United States of America is about to swoop down like an exasperated eagle on the chickens left by the hawk. Are you ready, my sagacious reptiles, to spill a drop or so for your soaking country? Are you ready to rose up as one man--
"'The rose is red,
The willets blue,
Sugar is sweet, and
Bully for you.'
"Ages to come will look down on this day and say: "They died young."The Present will reply: 'I don't see it;' but the present is just the last thing for us to think about. Richmond is before us, and there let it remain. We shall take it in a few years:
"'It may be for years and it may be forever,
Then why art thou silent, O pride of me heart,'
which is poickry. I hereby divide this splendid army into one corps dammee, and take command of it."
At the conclusion of this thrilling oration, my boy, the corps dammee formed itself into a hollow square, in the centre of which appeared a mail-clad ambulance.
Onward moved the magnificent pageant toward the plains of Manassas, the Anatomical Cavalry being in advance and the Mackeral Brigade following closely after.
Arriving on the noted battle field, we found nothing but a scene of desolation; the rebels gone; the masked batteries gone; and nothing left but a solitary daughter of the sunny South, who cursed us for invading the peaceful homes of Virginia, and then tried to sell us stale milk at six shillings a quart.
When Captain Villiam Brown surveyed this spectacle, my boy, his brows knit with portentous anger, and says he:
"So much for wasting so much time. Ah!" says Villiam, clutching convulsively at his canteen, "we have met the enemy, and they are hours-ahead of us."
Like four and twenty thunder storms the howitzers roared together, and had not the Orange county veterans forgotten to put in any balls, there is reason to believe that some windows would have been broken. Another discharge, however, was more successful, as it knocked the top off the chimney.
The Southern Confederacy appeared at the window again, and says he:
"If you fellows don't quit that racket down there you'll irritate me pretty soon."
* * * "Mr. Davis," says Samyule to the Confederacy at the window, "if we do not irritate you, will you consent to be reconstructed?"
"Reconstructed!" says the Confederacy, thoughtfully: "reconstructed! Ah!" says he, "you mean will I consent to be born again?"
"Yes," says Samyule, metaphysically; "will you consent to be borne again, as we have borne with you heretofore?"
The Confederacy thought awhile, and then says he:
"Consider me reconstructed."
As that was all the Constitution asked, of course there was no more to be done, and the Orange County Howitzers returned to their original position in the mire-the English gentlemen remarking that the appearance and discipline of our troops were satisfactory to Albion.
Fighting according to the Constitution, my boy, is such an admirable way of preventing carnage that some doctor ought to take out a patent for it as a cheap medicine.
"Orpheus" does not confine himself to war topics. His views of the National Anthem business, of the "Jane Eyre" school of novels, and of the American experience of Garibaldi are very droll.
Volume one of The Orpheus C. Kerr Papers can be found online here.


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I substituted beef fat when looking up how much lye to use. Hopefully I calculated it correctly. It's something you really don't want to mess around with. If your lye to fat ratio is wrong then you might end up with a chemical burn like Ed Norton in Fight Club. I had the balsamic vinegar standing by just in case.
The recipe I created was:
- 1 cup rendered bison fat
- 60.8 grams lye
- 1/3 cup water
- paprika added generously for color
- ~ 1 teaspoon vanilla essential oil for scent
I weighed the lye crystals on the coffee filter carefully using the electronic scale. Then I mixed them into the bowl with 1/3 cup of water on the right. The water and lye reacted chemically and heated up.
Meanwhile I melted the bison fat and started monitoring the temperature of both the fat and the lye mixture.
It took some jockeying to get both temperatures to drop to 98 degrees at the same time. I ended up chilling and reheating the fat, but eventually I zeroed in on the target temperature. Once the temperature of the fat and the lye were both around 98 degrees I poured the lye into the fat and mixed.
I stirred for about 10-15 minutes to fully mix the lye with the fat. As it cooled it started to thicken. Once a drip from the spoon lingered on the surface of the mixture I knew it was time to pour.
I quickly mixed in the paprika and vanilla oil and then poured it into a plastic mold.
I'm kind of surprised at how much the ingredients made. Now I will let these set up for 3-4 days. Then I will knock them out and let them cure for 3 weeks. Hopefully the final products will not burn me.


text:
We'll be dropping musical classic throughout the night for you to sing your hearts out too and back by popular demand.... We will be opening the BASEMENT for 2 rooms of dancing delights!
That's right kids... It's a new year and I'm getting older so why not throw the biggest, dirtiest, most outragous a house party to celebrate!
The BBC is to air the moment Lord Mandelson was outed by columnist Matthew Parris for the first time since it happened more than a decade ago.
The recording, to be played during an interview with the former Business Secretary on Radio 4, has been banished from the airwaves since 1998, when the slip occurred during an edition of BBC2's Newsnight.
Now radio bosses gave the go-ahead for its broadcast during an Archive On 4 programme in which the Labour politician looks back at his career.
In Meeting Myself Coming Back, Lord Mandelson talks openly about the incident for the first time, and says he was "upset" his sexuality was seen to have any political importance and the aggressive way he was pursued by journalists over the matter was "unacceptable".
Parris let slip about the politician's homosexuality when he told Newsnight host Jeremy Paxman there were "two gay members of the cabinet at least". He went on: "Chris Smith is openly gay and Peter Mandelson is certainly gay."
Paxman responded: "Ah, I think we'll just move on from there, I'm not quite sure where he is on that."
His sexuality was the subject of a story in the News Of The World more than a decade earlier but was largely forgotten due to his lower profile at the time.
Hours after the broadcast, an internal memo was issued to producers at the BBC effectively barring them from mentioning the issue. In his autobiography, The Third Man, Lord Mandelson says the BBC imposed a "ban" on any reference to the exchange.
In the new Radio 4 programme he is played the recording before giving his thoughts. He tells presenter John Wilson: "What upset me first of all was that they should be debating this as though it was some sort of question that hadn't been answered or that it was of some political importance.
"I had been outed by the News of the World some 10 years before in 1987 and had long since got over it and got through it. What I didn't accept very easily was this had suddenly become the subject of a political discussion on Newsnight, when in my view it had absolutely nothing whatsoever to do with my life as a politician or what I was doing as a minister."
Source: PA
© Copyright 2009 Pinkwire, Talent Media.
Designd & powerd by ENTWURF.


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Posted by Redazione on May 26, 2017
Meet the unique development tool IoT Starter Kit with which the development of your wireless Internet applications will be a matter of play. The necessary software and services are ready for you, there are lots of tutorials for installation and configuration. Additional HW extensions can be attached to the kit as a variety of sensors and actuators.
For wireless connectivity,IQRFhas been chosen as the world's number one in reliable wireless mesh network technologies for more than 14 years. In this basic development kit, you will find an IQRF part that includes a sensory and relay kit that allows you to collect basic sensor data (temperature, light intensity, voltage) or control connected devices.
Communication with these devices is accomplished using DPA commands implemented in custom programs – Custom DPA Handlers.
At the borderline between the IQRF network and the computer network, there is a popular UP developer board from AAEON. This gateway boasts excellent equipment (2GB + 32GB eMMC memory, Intel® Atom ™ x5 Z8350 Processor 64 bit-up to 1.92GHz) and high resilience.
The IQRF Daemon has been developed for this gateway. It provides communication with the IQRF network and sends the data in the required format via selected communication channels to local and remote services. In particular, the MQTT protocol and the JSON message format are used. Regular reading of values from the IQRF network provides a prepared scheduler.
For installation and configuration of the operating system and services, see https://github.com/iqrfsdk/iot-starter-kit.
Data are sent from the IQRF network via the MQTT Channel into the Microsoft Azure IoT Hub, from where it can be further processed. You can take advantage of a simple Power BI visualization or a more sophisticated solution with other robust tools offered by Microsoft Azure.
Check out a short videoon how to set up the Microsoft Azure Gateway and IoT Hub to get a basic dashboard with visualization of the data you are getting.
Where can I get the IoT Starter Kit?
The IoT Starter Kit can be purchased at IQRF Alliance e-shop or with the help of distributors. The participants of the IQRF Summit 2017, which is held in Prague from 7th to 8th June, receive a significant discount on the eshop. In addition, they can attend a free workshop for this product and other extensions. The workshop will be held during the summit.
IQRF® technology is a wireless technology for data transmission in mesh networks with extremely low power consumption and high reliability. The transmission is bi-directional and fast enough for both data collection and control of electronic devices. It has been developed by the Czech company MICRORISC and in 2014 it was awarded a scientific award the Czech Head.
The latest operating system IQRF OS 4.0 ensures uncompromised security, whether in the secure addition of new devices to the network, in network data transmission or in additional user encryption of the transmitted data, all based on the AES-128 standard.


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Clathrin‐mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin‐coated pit (CCP) dynamics led us to propose the existence of a rate‐limiting, regulatory step(s) that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin‐1, which was thought to be neuron specific, is activated by the Akt/GSK3β signaling cascade in non‐neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin‐1 in RPE cells by inhibition of GSK3β accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR‐Cas9n‐mediated knockout and reconstitution studies establish that dynamin‐1 is activated by Akt/GSK3β signaling in H1299 non‐small lung cancer cells. These findings provide direct evidence for an isoform‐specific role for dynamin in regulating CME and reveal a feed‐forward pathway that could link signaling from cell surface receptors to the regulation of CME.
Dynamin‐1, previously thought to be neuron specific, is activated by an Akt/GSK3β signaling cascade in non‐neuronal cells. Dynamin‐1 activation alters the rate and regulation of clathrin‐mediated endocytosis, providing a feed‐forward pathway between endocytosis and signaling.
An endocytic checkpoint monitors the fidelity of clathrin‐coated vesicle (CCV) formation.
Dysregulated clathrin‐mediated endocytosis (CME) alters cell signaling and proliferation.
Dynamin‐1 and dynamin‐2 differentially regulate early stages of CME.
Endogenous dynamin‐1 is activated by an Akt/GSK3β signaling cascade, illustrating a first non‐neuronal role of dynamin‐1.
Clathrin‐mediated endocytosis (CME) constitutes the major entry pathway for signaling receptors, transporters and adhesion molecules into eukaryotic cells (Traub, 2009; McMahon & Boucrot, 2011; Ferguson & De Camilli, 2012). CME is a multi‐step process involving the formation, stabilization and maturation of clathrin‐coated pits (CCPs), leading to the incorporation of surface receptors and their ligands into clathrin‐coated vesicles (CCVs). During CCP maturation, the CME core machinery consisting of clathrin, dynamin and AP2 interacts with a myriad of endocytic accessory proteins (EAPs), many of which are recruited to CCPs via binding to the appendage domain (AD) of the α‐adaptin subunit of AP2 (Schmid & McMahon, 2007). Following maturation, membrane scission is catalyzed by the GTPase dynamin, leading to the formation of cargo‐containing vesicles.
Although the endocytic machinery is well understood, many questions remain as to how CME is regulated. Analyses of CCP dynamics by live cell imaging revealed striking differences in their assembly and maturation kinetics, including the existence of short‐lived, abortive CCPs (Ehrlich et al, 2004; Loerke et al, 2009; Taylor et al, 2012) and early rate‐limiting step(s) required to monitor the fidelity of CCP maturation (Aguet et al, 2013). The manipulation of cargo concentration (Loerke et al, 2009), the induction of receptor clustering (Liu et al, 2010), as well as small interfering RNA (siRNA) knockdown of a subset of EAPs (Mettlen et al, 2009b) shifted the relative proportions of abortive vs. productive CCPs and/or altered the lifetimes of abortive CCPs. In addition to establishing a role for these factors in CCP maturation, these findings led to the hypothesis that transitions leading to the formation of mature CCPs are gated by a checkpoint mechanism(s) that monitors coat assembly, cargo recruitment and membrane curvature (Mettlen et al, 2009a). While still controversial (McMahon & Boucrot, 2011; Ferguson & De Camilli, 2012), the strongest evidence for the existence of an endocytic checkpoint derives from recent observations of the rapid and active turnover of aberrant flat clathrin lattices that formed in cells expressing a truncated α‐adaptin subunit lacking the AD (ΔαAD) (Aguet et al, 2013). Despite their severe defect in early stages of CCP maturation, the rate of TfnR uptake in these cells is unimpaired due to two compensatory mechanisms. First, the rate of CCP initiation doubles, and second, those CCPs able to bypass the endocytic checkpoint undergo a more rapid and dysregulated maturation. Thus, these cells represent a powerful tool to study the regulation of CME.
Clathrin‐mediated endocytosis regulation is important because cell surface signaling is governed by endocytic trafficking, either by the rapid clearance of surface receptors leading to the attenuation of incoming signals or by the formation of endosome‐specific signal transduction complexes (Schenck et al, 2008; Sorkin & von Zastrow, 2009; Zoncu et al, 2009; Platta & Stenmark, 2011; Palfy et al, 2012). Indeed, a recently generated hierarchical map of endocytic membrane trafficking has revealed close interactions between signaling and endocytic pathways (Liberali et al, 2014), which remain unexplored.
The GTPase dynamin, which assembles into a fission apparatus at the necks of deeply invaginated CCPs, has been proposed to also function as a regulator of early, rate‐limiting steps in CCV formation (Sever et al, 1999; Loerke et al, 2009; Aguet et al, 2013). There are three major mammalian isoforms: dynamin‐1 (Dyn1), which is presumed to be neuron specific; dynamin‐2 (Dyn2), which is ubiquitously expressed; and dynamin‐3 (Dyn3), mainly expressed in testis, brain and lung. Although sharing significant sequence identity, Dyn1 and Dyn2 differ in their curvature‐generating/sensing properties (Liu et al, 2011). It has been proposed that Dyn1, which has more potent curvature‐generating properties, is suited for rapid, compensatory endocytosis at the synapse, whereas the curvature‐sensing properties of Dyn2, which requires a narrow neck to trigger its assembly, are suited to monitor and regulate early stages of CCP maturation (Liu et al, 2011). Indeed, there is evidence for isoform‐specific functions of Dyn1 and Dyn2 in CME both at the synapse (Ferguson et al, 2007) and in non‐neuronal cells (Liu et al, 2008), respectively. However, the basis for these differences and their functional significance remains poorly understood.
Through mechanistic studies of endocytosis in cells defective in early stages of CCP maturation, we have discovered an endosome‐associated signaling pathway that activates endogenously expressed but normally quiescent dynamin‐1 in non‐neuronal cells. This signaling pathway exists in normal RPE cells and is activated in the non‐small cell lung cancer cell line, H1299. Acute activation of dynamin‐1 increases the rate of CCP initiation, alters CCP dynamics and triggers rapid, dysregulated CME. These data establish an isoform‐specific role for dynamin in regulating early stages of CME and point to the existence of a two‐way crosstalk between signaling and CME.
Dysregulated CME produces nascent CCVs defective in endosomal trafficking and maturation
Despite being severely defective in the early stages of CCP maturation (Aguet et al, 2013), the rate of Tfn uptake in ΔαAD cells is unaffected (Motley et al, 2006; Aguet et al, 2013). We first confirmed that the compensatory endocytosis leading to efficient TfnR uptake was clathrin dependent (Supplementary Fig S1A) and that the steady‐state levels of surface TfnR remained unaffected in control and ΔαAD cells (Supplementary Fig S1B). Thus, although an operational checkpoint in these cells rapidly turns over the majority of aberrant CCPs, we proposed that a subset of productive CCPs mature in a more rapid and dysregulated manner, as a result of bypassing the endocytic checkpoint (Aguet et al, 2013). Although best understood in the context of cell division, by definition checkpoints function as fidelity monitors to ensure the accuracy of the molecular events being monitored. If a subset of CCVs formed in ΔαAD cells had indeed bypassed this regulatory step(s), then they should exhibit defects that would have functional consequences for their downstream processing. We tested this hypothesis by comparing early endosomal trafficking in control cells reconstituted with full‐length α‐adaptin (FL cells) and ΔαAD cells.
Newly formed nascent endocytic vesicles undergo multiple rounds of homotypic and heterotypic fusion at the cell periphery, driving the maturation of larger early endosomes that then move toward the perinuclear region (Gorvel et al, 1991; Rink et al, 2005). Early endosomes undergo acidification and mediate the sorting of selective cargos for recycling and/or degradation (Mellman, 1996; Maxfield & McGraw, 2004; Grant & Donaldson, 2009). We first examined the trafficking of labeled transferrin (Tfn) through early endosomes by incubating both FL and ΔαAD cells with a brief (2‐min) pulse of Tfn‐AlexaFluor®647 (TfnAF647), followed by a second 2‐min pulse with Tfn‐AlexaFluor®568 (TfnAF568). As expected (Fig 1A, top panel), rapid homo‐ and heterotypic fusion events in FL cells resulted in significant mixing between the first and second pulses of Tfn and their colocalization in endosomal compartments both at the periphery and in perinuclear regions of the cell (Fig 1A, see arrows). In contrast, in mutant cells (Fig 1A, bottom panel) there was very little mixing between the two pulses of Tfn, with the second pulse of TfnAF568 remaining largely at the cell periphery, suggesting a delay in homotypic fusion and defects in nascent endocytic vesicle maturation and trafficking.
As early endosomes mature, their lumenal content becomes increasingly acidic through the action of V‐ATPases (Forgac, 2007). To measure the rate and extent of acidification of Tfn‐containing nascent endosomes, we incubated FL and ΔαAD cells with Tfn conjugated to the pH‐sensitive probe pHrodo Red and followed the increase in fluorescence emission as an indicator of increased acidification of Tfn in early endosomes. In both cell lines, the fluorescence of internalized pHrodo Red‐Tfn increased, indicating acidification, and reached a plateau within 5 min before slowly decreasing as Tfn moved into a less acidic recycling compartment (Fig 1B). However, the degree of acidification was significantly lower in mutant as compared to control cells, again indicating that early endosomal maturation is defective in these cells.
Homotypic and heterotypic fusion events during early endosome maturation aid the formation of sorting endosomes, from which Tfn recycles back to the cell surface via perinuclear recycling endosomes. Thus, we expected to see a defect in the rate of Tfn recycling, which we next determined by loading cells with a pulse of biotinylated‐Tfn (BSS‐Tfn) for different time points, followed by the recycling of BSS‐Tfn back to the plasma membrane. Surprisingly, we observed that a significant portion of internalized BSS‐Tfn pre‐loaded with short pulses (≤ 20 min) recycled with faster kinetics in ΔαAD cells (Fig 1C and Supplementary Fig S2A and B). Thus, a large fraction of nascent endocytic vesicles formed during dysregulated CME fail to mature and instead rapidly return to the plasma membrane. When perinuclear recycling endosomes were loaded with a longer pulse of BSS‐Tfn (60 min), the rates of recycling were indistinguishable, indicating that these later stages of endocytic trafficking were unaffected (Supplementary Fig S2C).
During maturation, early endosomes sequentially acquire the Rab5 effectors APPL1 and EEA1 that function as scaffolding molecules on endosomal surfaces (Rink et al, 2005). Immunofluorescence of EEA1‐ (Fig 1D, Supplementary Fig S3A) and APPL1‐positive (Fig 1D, Supplementary Fig S3B) early endosomes revealed their substantial accumulation in ΔαAD when compared to FL cells. Furthermore, APPL1‐ and EEA1‐positive early endosomes displayed a significantly more peripheral localization in ΔαAD cells (Fig 1E). Given that there was no corresponding increase in the protein levels of either APPL1 or EEA1 (data not shown), we interpret the accumulation and localization of these intermediates as a delay in maturation. Together, these data establish that nascent endocytic vesicles formed in ΔαAD cells have profound defects in subsequent vesicular trafficking and maturation.
Dysregulated CME alters cell signaling
CME plays an important role in the regulation of multiple signaling pathways (Le Roy & Wrana, 2005; Traub, 2009). Besides dysregulated CME and aberrant trafficking of nascent CCVs, the ΔαAD cells also exhibited enhanced cell proliferation (Fig 2A). The kinase Akt is known to orchestrate diverse fundamental processes, including cell proliferation. Consistent with this, we detected a significant increase in the steady‐state activation/phosphorylation of Akt in ΔαAD cells (Fig 2B). The scaffold protein APPL1 has been shown to play an important role in the regulation of Akt (Schenck et al, 2008). Therefore, we next investigated whether the accumulation of APPL1‐positive early endosomes contributed to the enhanced Akt activity measured in these cells. The siRNA‐mediated knockdown of APPL1 in ΔαAD cells significantly reduced Akt phosphorylation, back to the levels seen in FL cells (Fig 2C and D).
Given the role of CME in regulating signaling, we next examined the effects of dysregulated CME on downstream signaling from activated EGF receptors. Stimulation of serum‐starved cells with 20 ng/ml of EGF induced Akt activation (pT308 and pS473) to a much greater extent in ΔαAD cells compared to control cells (Fig 3A, quantification in B). We also observed increased ERK activation in response to EGF (Supplementary Fig S4A and B), but no significant increase under steady‐state conditions (data not shown). Akt can activate multiple downstream pathways depending, in part, on its cellular localization (Palfy et al, 2012). The APPL1‐dependent recruitment of Akt to early endosomes has been shown to regulate phosphorylation of GSK3β kinase but not TSC2, which is activated in the cytosol by Akt upon growth factor stimulation (Schenck et al, 2008) (Fig 3C). We therefore tested for differences in growth factor stimulation of these two proteins. Consistent with enhanced signaling from accumulated APPL1‐positive endosomes, EGF‐stimulated phosphorylation of GSK3β but not TSC2 was strongly enhanced in ΔαAD cells, relative to FL cells (Fig 3D and E). Taken together, these results indicate that dysregulated CME leads to the accumulation of early endosome intermediates that prolong and enhance specific downstream signaling pathways.
Dynamin‐1 is specifically enlisted for rapid, compensatory endocytosis
The GTPase dynamin, which assembles into a fission apparatus at the necks of mature, deeply invaginated CCPs, is also recruited at early stages of CCP maturation (Damke et al, 1994; Ehrlich et al, 2004; Taylor et al, 2012) and has been proposed to function as a regulator of early, rate‐limiting steps in CCV formation (Mettlen et al, 2009a; Aguet et al, 2013). Although Dyn1 is highly expressed in neuronal tissues and enriched at the synapse, transcriptional analyses reveal that mRNA levels for the Dyn1 and Dyn2 isoforms are similar in most tissues (dynamin‐1: http://biogps.org/gene/1759/; dynamin‐2: http://biogps.org/gene/1785/). Consistent with this, Dyn1 expression has been detected by Western blotting in many non‐neuronal cell lines, including the RPE cells used here (Supplementary Fig S5A and B). Given their potential isoform‐specific differences (Liu et al, 2011, 2012), we next investigated the effects of Dyn1 and Dyn2 siRNA‐mediated knockdown on CME in control and ΔαAD cells.
As expected, the siRNA‐mediated knockdown of Dyn2 potently inhibited CME in both FL and ΔαAD cells (Fig 4A and B). Strikingly, the siRNA‐mediated knockdown of Dyn1 potently inhibited TfnR uptake in ΔαAD (Fig 4B), but had no significant effect on CME in FL cells (Fig 4A). Importantly, we did not detect differences in Dyn1 or Dyn2 expression levels between FL and ΔαAD cells, nor were there compensatory changes in expression upon knockdown of either Dyn1 or Dyn2 (Supplementary Fig S5). From these data, we conclude that Dyn1 is expressed but normally quiescent in these non‐neuronal cells and can be activated to mediate rapid compensatory CME.
GSK3β directly regulates dynamin‐1‐dependent CME
We next investigated the mechanism by which Dyn1 is activated. At the synapse, Dyn1 activity is highly regulated by cycles of phosphorylation/dephosphorylation. For example, GSK3β‐mediated phosphorylation of Ser774, which is not conserved in Dyn2, regulates Dyn1 activity (Clayton et al, 2010; Smillie & Cousin, 2011). Intriguingly, Akt is known to phosphorylate GSK3β and inhibit its activity (Schenck et al, 2008; Palfy et al, 2012), which might then allow for the consequent activation of Dyn1. Given that phosphorylation of both Akt and GSK3β is enhanced in ΔαAD cells (Figs 2 and 3), we wondered whether this signal transduction cascade could be responsible for activation of Dyn1 and induction of more rapid, compensatory CME. We first used a Ser774 phospho‐specific antibody and confirmed that Dyn1 is phosphorylated in FL cells and that the steady‐state levels of Dyn1 (pSer774) were indeed significantly decreased in ΔαAD cells (Fig 5A).
We then directly tested the role of this signaling cascade in the differential regulation of CME in FL and ΔαAD cells using several inhibitors of either Akt or GSK3β (Fig 5B). Uptake assays performed in the presence of the GSK3β inhibitors Bio (Supplementary Fig S6A and B) or CHIR‐99021 (Supplementary Fig S6C) led to an increase in the rate of CME in control cells, while having no effect on ΔαAD cells (Fig 5C and D). This effect was due to activation of Dyn1, as it was abrogated by siRNA‐mediated knockdown of Dyn1, but still evident upon knockdown of Dyn2 (Fig 5C). Correspondingly, treatment with the PI3K/Akt inhibitor LY294002 (Supplementary Fig S6A and B) or the more specific Akt inhibitor X (Supplementary Fig S6C) showed no effect on CME in FL cells, but reduced the rate of CME in ΔαAD cells to that seen upon siRNA‐mediated knockdown of Dyn1 (Fig 5F).
Given the altered distribution of APPL1 endosomes seen in ΔαAD cells and their impact on Akt activation, we next examined the effect of APPL1 depletion on CME rates. As expected, and similar to the effect of Akt inhibition, the siRNA‐mediated knockdown of APPL1 resulted in a significant decrease in the rate of CME in these cells, but not in control (FL) cells (Fig 6A). The generation of peripheral APPL1 early endosomes has been shown to be partially dependent on CME (Zoncu et al, 2009). Consistent with this and our biochemical studies, inhibition of Akt in ΔαAD cells led to a reduction in the amount of peripheral APPL1 early endosomes (Fig 6B). In contrast, in control cells the inhibition of GSK3β, which in turn activates Dyn1 and triggers dysregulated CME, leads to the accumulation of APPL1 early endocytic intermediates (Fig 6C).
To exclude the possibility that these effects are specific to ΔαAD mutant cells, we next asked whether Dyn1 could be activated via this signaling pathway in the parental ARPE‐19 cells. As seen in FL cells, CME in untreated ARPE‐19 cells was strongly dependent on Dyn2 but not Dyn1, and it was insensitive to Akt inhibitors (Fig 7A). Strikingly, the inhibition of GSK3β in ARPE‐19 cells resulted in an increased rate of TfnR uptake, which was abrogated upon siRNA knockdown of Dyn1, but not Dyn2 (Fig 7B). Taken together, these results establish that Dyn1 can be directly activated through an Akt/GSK3β kinase cascade to increase the rate of CME.
The crosstalk between signaling and dynamins alters CCP dynamics and dysregulates CME
We previously showed that CCPs undergo a complex, multistep maturation process that is reflected in their broad lifetime distribution (Loerke et al, 2009; Aguet et al, 2013). We have also shown that CCPs that fail to recruit Dyn2 exhibit an exponentially decaying lifetime distribution, indicative of an unregulated process, and suggesting that dynamin might regulate early stages of CCP maturation (Loerke et al, 2009; Aguet et al, 2013). Finally, those few CCPs that are able to mature in ΔαAD cells also exhibit a near‐exponential lifetime distribution reflective of an unregulated maturation process (Loerke et al, 2009; Aguet et al, 2013). Thus, we wondered whether the activation of Dyn1 might be sufficient to accelerate CCP maturation and alter its regulation. CCPs in ARPE‐19 cells stably expressing EGFP‐CLCa exhibit the expected broad lifetime distribution (Fig 7C, red curve) reflective of a multi‐step regulated maturation process. Strikingly, upon acute treatment with the GSK3β inhibitor CHIR‐99021, the curve shifts to a quasi‐exponentially decaying lifetime distribution, indicative of a less regulated process (Fig 7C, green curve). Moreover, the median lifetime of CCPs decreases from ~53 s in control cells to ~35 s in GSK3β‐inhibited cells, in agreement with the more rapid Tfn uptake measured biochemically. Consistent with the biochemical studies shown in Figs 4 and 5, the siRNA‐mediated knockdown of Dyn1 alone had no effect on CCP dynamics in ARPE‐19 cells (Fig 7C, red and blue curves); however, Dyn1‐depleted cells no longer responded to inhibition of GSK3β (Fig 7C, purple curve). From these data, we conclude that the effects of GSK3β inhibition on CME are through phosphorylation of Dyn1 and that its activation alters the rate and regulation of early stages in CCP maturation. Unexpectedly, we also detected a dramatic increase in the rate of CCP initiation upon inhibition of GSK3β, which was again completely dependent on Dyn1 (Fig 7D). These results indicate that signaling through Akt/GSK3β modulates the activity of Dyn1, leading to enhanced rates of CCP initiation and maturation, as well as altered regulation of CME in normal cells.
Akt signaling activates dynamin‐1 in H1299 lung cancer cells
The Akt/GSK3β signaling pathway is highly activated in many cancers, and it has been suggested that CME can be dysregulated in cancer (Mosesson et al, 2008; Mellman & Yarden, 2013). Therefore, we next tested whether Dyn1 might function in CME in cancer cells displaying high levels of Akt phosphorylation, such as H1299, non‐small cell lung cancer (NSCLC) adenocarcinoma cells (Hernandez et al, 2013). Indeed, we found by siRNA‐mediated knockdown of Dyn1 and Dyn2 that H1299 cells are partially dependent on Dyn1 for efficient TfnR uptake (Fig 8A), while also relying on Dyn2. Consistent with activated Dyn1 in H1299 cells, CME is only slightly accelerated in the presence of GSK3β inhibitors, but markedly reduced by inhibition of Akt (Fig 8B). To further explore Dyn1 function in cancer cells, we generated a Dyn1 knockout (KO) H1299 cell line using CRISPR‐Cas9n and then reconstituted these cells with either WT‐Dyn1‐EGFP or S774A‐Dyn1‐EGFP, a mutant that cannot be phosphorylated by GSK3β (Fig 8C). Consistent with the effects of Dyn1 siRNA treatment of H1299 cells, knockout of Dyn1 in H1299 (Fig 8D, white bars) resulted in an ~40% decrease in the rate of TfnR uptake, to approximately the same level as inhibition of Akt in parent cells (Fig 8D, black bars). Importantly, Akt inhibition did not further reduce TfnR uptake in Dyn1 KO cells (Fig 8D, white bars), suggesting that the effects of Akt inhibition on CME are indeed dependent on Dyn1. Reconstitution of Dyn1 KO H1299 cells with either WT‐Dyn1‐EGFP (Fig 8D, light gray bars) or S774A‐Dyn1‐EGFP (Fig 8D, dark gray bars) fully restored the rate of TfnR uptake back to control levels, indicating that Dyn1‐EGFP is fully active in these cells. That expression of S774A‐Dyn1‐EGFP did not increase the rate of CME suggests that other phosphorylation sites, for example S778, which are required for GSK3β recognition (Clayton et al, 2010) might also regulate dynamin‐1 activity. Importantly, whereas WT‐Dyn1‐EGFP‐expressing cells still showed sensitivity to Akt inhibition, S774A‐Dyn1‐EGFP‐expressing cells were no longer responsive to Akt inhibition. These results establish that the effects of Akt inhibition on CME are primarily mediated by Dyn1 activation through phosphorylation of S774 and that enhanced Akt signaling in cancer cells can indeed lead to activation of Dyn1 and dysregulated CME.
The findings presented here provide direct evidence for the role of dynamins as key regulators of early steps of CCP formation and demonstrate that alterations in the regulation of CME can profoundly influence downstream endosomal maturation and trafficking. We establish the existence of an Akt/GSK3β‐dependent signaling cascade in non‐neuronal cells that directly links activation of dynamin‐1 to alterations in the early stages in CCP initiation and maturation. Together, these results suggest the intriguing possibility that signaling receptors could generate a feed‐forward signal through Akt and GSK3β to activate dynamin‐1, dysregulate CME and enhance signaling properties.
Live cell imaging and quantitative image analysis of CME had revealed the existence of a subpopulation of short‐lived, abortive CCPs (Ehrlich et al, 2004; Loerke et al, 2009; Taylor et al, 2012; Aguet et al, 2013). Our previous studies reporting that the proportion of abortive vs. productive CCPs is dependent on cargo loading (Loerke et al, 2009; Liu et al, 2010) and affected by siRNA knockdown of a subset of EAPs (Mettlen et al, 2009b) led us to propose the existence of an endocytic checkpoint gating CCP maturation. In further support of this model, here we show that the products of CME (i.e., nascent endocytic vesicles) formed in ΔαAD cells by bypassing this regulation are functionally altered: Homotypic fusion and subsequent early endosomal maturation stages are significantly delayed, endosome acidification is reduced, and the portion of TfnRs undergoing rapid recycling is increased. Together, these data further support the existence and significance of an endocytic checkpoint that monitors the fidelity of CME. Not addressed here, and in need of further study, are the molecular mechanisms involved in sensing defects in CCP formation and in actively turning over aberrant CCPs. It will also be important to identify the precise molecular alterations in nascent CCVs that underlie the dramatic changes seen in their downstream maturation. Identification of these differences would provide important insight into what aspects of CCP maturation are monitored by the endocytic checkpoint. While beyond the scope of this study, candidates include defects in the packaging of SNARE proteins or the V‐ATPase, both of which might be required for subsequent targeting and fusion events.
Here we have focused on identifying the compensatory mechanism by which CME is fully restored in mutant cells despite a severe defect in early stages of CCP maturation. We show that the accumulation of APPL‐1 endosomes leads to the constitutive activation of Akt and phosphorylation of GSK3β to suppress its inhibitory phosphorylation of endogenous Dyn1. Activation of Dyn1 drives rapid, compensatory endocytosis in these non‐neuronal cells. Most studies have focused on the burst of dynamin recruitment to promote membrane fission at the end of CCP maturation (Doyon et al, 2011; McMahon & Boucrot, 2011; Ferguson & De Camilli, 2012); however, there exists growing evidence that dynamin also functions in controlling early, rate‐limiting stages of CCP maturation (Sever et al, 1999, 2000; Loerke et al, 2009; Taylor et al, 2012; Aguet et al, 2013). The data presented here strongly supports that dynamins can function, in an isoform‐specific manner, as master regulators of CME.
Although Dyn1 is highly expressed in neurons and is generally considered to be a neuron‐specific isoform, mRNA levels of Dyn1 and Dyn2 are actually comparable in most tissues. Consistent with this, the signaling pathway we have identified is also operable in parental ARPE‐19 cells and other non‐neuronal cells. Acute inhibition of GSK3β accelerates CME in a Dyn1‐dependent, but Dyn2‐independent manner. Strikingly, CCP dynamics in ARPE‐19 cells acutely treated with GSK3β inhibitors fully mirror those of productive, curved CCPs in ΔαAD cells. Specifically, the rate of CCP initiation approximately doubles and productive CCPs mature in a more rapid and unregulated manner (compare Fig 6A and B this report with Figs 6E and 7E in Aguet et al, 2013). Thus, we conclude that Dyn1 alters the regulation of early stages of CME including CCP initiation and maturation. A role for Dyn1 in CCP assembly has also been suggested by a recent study in which acute photoablation of Dyn1 in Drosophila prevented AP2 and clathrin assembly at the synapse (Kasprowicz et al, 2014).
We previously showed that Dyn1 and Dyn2 have distinct biochemical properties (Liu et al, 2011) that correlate with their differential effects on CME. Dyn1 is a powerful curvature generator that could compensate, in part, for the defect in curvature generation of nascent CCPs in ΔαAD cells. In contrast, Dyn2 is highly curvature sensitive, yet is targeted more efficiently to CCPs by virtue of its divergent Pro/Arg‐rich domain (Liu et al, 2011). While associated with CCPs, Dyn2 could monitor early stages of maturation. Formation of a narrow neck at the base of fully mature and deeply invaginated CCPs could trigger Dyn2 assembly and fission. Evolutionary history also supports a regulatory role for dynamin (Liu et al, 2012) in that it appears much later in evolution than the core CME machinery, which dates back to the earliest common eukaryotic ancestor (Field et al, 2007). Further work will be needed to understand the isoform‐specific differences behind Dyn1 and Dyn2 regulation of CME and to identify specific up‐ and downstream effectors of this complex multi‐step process. We note that CME in ΔαAD cells depends on both Dyn1 and Dyn2. Whether they work together at single CCPs or independently at subsets of CCPs remains to be determined.
Although endocytosis and cellular signaling have been previously viewed as separate processes, endocytosis is now recognizably linked to many aspects of cellular signaling (Sorkin & von Zastrow, 2009; Di Fiore & von Zastrow, 2014). Recently, stimulation of cells with EGF has been shown to modulate the number of early endosomes (Villasenor et al, 2015), in a mechanism that can in turn potentially regulate signaling. Moreover, a recently generated map of endocytic membrane trafficking revealed new potential links between signaling and endocytic pathways (Liberali et al, 2014). Strikingly, whereas Akt2 inhibition/knockdown severely reduced Tfn uptake, inhibition of GSK3β strongly enhanced CME in HeLa cells (Liberali et al, 2014). These results independently confirm our findings, support the importance of the Akt/GSK3β axis in regulating CME and further suggest that our results are likely valid in other cell types. Here we establish that this kinase cascade operates through the activation of Dyn1 leading to rapid and dysregulated endocytosis (Fig 9). Whether Dyn1 can be activated in a cargo‐selective manner to alter the dynamic behavior and trafficking of a specific subset of CCPs and CCVs is an intriguing possibility worthy of further study.
The crosstalk between aberrant signaling and the regulation of dynamins, which we have shown can lead to enhanced proliferation, may partially explain the impact of dysregulated CME in several diseases, including cancer. Given that Akt is overactive in numerous tumor cells, the activation of this signaling cascade could in turn induce significant differences in the dynamics of CME, lead to the accumulation of early endosomal intermediates and rapid receptor recycling and thus serve as a potent generator of survival signals that sustain high proliferative activity. In this way, activating Dyn1 might function as a feed‐forward mechanism to enhance proliferative signals. Indeed, our analysis of the roles of Dyn1 and Akt in regulating CME in H1299 NSCLC cells supports this view. Interestingly, Dyn1 was found to be overexpressed in certain cancers, including leukemia, lung and colon adenocarcinomas (Haferlach et al, 2010; Hong et al, 2010); hence, the overexpression and/or potential Akt‐driven activation of Dyn1 may have profound implications for the role of dysregulated CME in cancer.
Materials and Methods
Cell culture
ARPE‐19 cells reconstituted with full‐length (FL) or ΔAD α‐adaptin were derived as previously described (Aguet et al, 2013). cDNA encoding the full‐length (FL) or truncated ΔAD α‐adaptin was kindly provided by M.S. Robinson (Cambridge Institute for Medical Research). Expression of α‐adaptins within each stable cell cohort was determined by Western blotting using the anti‐AP2α (#AC1‐M11, Pierce); the cohort with the expression level closest to endogenous α‐adaptin was chosen for further experiments. All cell lines were grown under 5% CO2 at 37°C in DMEM high glucose medium (Life Technologies) supplemented with 20 mM HEPES, 10 mg/ml streptomycin, 66 μg/ml penicillin and 10% (v/v) fetal calf serum (FCS, HyClone).
Transferrin receptor internalization and recycling from endosomal compartments
Transferrin (Tfn) internalization and recycling experiments were performed using anti‐TfnR mAb (HTR‐D65) (Schmid & Smythe, 1991) or biotinylated‐Tfn (BSS‐Tfn), respectively. ARPE‐19 cells were grown overnight in 96‐well plates at a density of 2.0 × 104 cells/well and pre‐incubated with 4 μg/ml of D65 in Tfn assay buffer (PBS4+: PBS supplemented with 1 mM MgCl2, 1 mM CaCl2, 5 mM glucose and 0.2% bovine serum albumin) at 4°C for 30 min. Cells were washed (3× PBS4+) to remove unbound ligand and further incubated in the presence (continuous uptake) of additional ligand at 37°C for the indicated time points. Cells were then immediately cooled down (4°C) to arrest internalization. Following a washing step to remove unbound ligand (3× PBS4+), the remaining surface‐bound ligand was removed from the cells by an acid wash step (5 × 2 min 0.2 M acetic acid, 0.2 M NaCl, pH 2.5). Cells were washed with PBS and then fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) in PBS for 20 min and further permeabilized with 0.1% Triton X‐100/PBS for 10 min. Internalized D65 ligand was assessed using a goat anti‐mouse HRP‐conjugated antibody (Life Technologies), further developed with OPD (P1536, Sigma‐Aldrich), and the reaction was stopped by using 5 M H2SO4. The absorbance was read at 490 nm (Biotek Synergy H1 Hybrid Reader). Internalized ligand was expressed as the percentage of the total surface‐bound ligand at 4°C (i.e., without acid wash step), measured in parallel.
For Tfn internalization in FL and ΔαAD cells using the GSK3β inhibitors Bio (Sigma) or CHIR‐99021 (Sigma), and the PI3K/Akt inhibitor LY294002 (Calbiochem) or the Akt inhibitor X (Calbiochem), cells were initially pre‐incubated in the absence (i.e., control) or presence of the indicated inhibitor (10 μM) for 30 min at 37°C, followed by pre‐incubation with 4 μg/ml of D65 in Tfn assay buffer at 4°C for 30 min, in the continued absence or presence of the inhibitor. Tfn internalization assays were then performed as described above. Percentage of Tfn uptake was calculated relative to the initial total surface‐bound ligand at 4°C for all the assays.
For the recycling of Tfn from endosomal compartments, cells were pulsed for different time points (5‐60 min) with 8 μg/ml of BSS‐Tfn at 37°C. Following internalization, plates containing cells were transferred to ice, washed with cold PBS and acid‐washed to remove surface‐bound ligand. Cells were then incubated in PBS4+ containing 2 mg/ml of unlabeled Tfn at 37°C for the indicated time points. Remaining BSS‐Tfn that recycled to the surface was again removed by acid wash, and the cells were further processed as described for the internalization assay, but this time using streptavidin‐POD (Roche). The decrease in intracellular Tfn (recycling) was calculated relative to the total internal pool of ligand internalized.
siRNA transfection
ARPE‐19 cells were treated with a previously established siRNA sequence using RNAiMAX (Life Technologies, Carlsbad, CA, USA) to silence endogenous proteins, following the manufacturer's instructions. Briefly, 110 pmol of the indicated siRNA and 6.5 μl of RNAiMAX reagent were added in 2 ml of OptiMEM (Life Technologies) in each well of a 6‐well plate containing cells for 4 h. Transfection was performed at day 1 and day 3 after plating, and experiments were performed at day 5. The exogenously expressed FL and ΔAD α‐adaptins harbor mutations that confer resistance to siRNA silencing. siRNA oligos used to target the following proteins were endogenous α‐adaptin (5′‐GAGCAUGUGCACGCUGGCCA‐3′), dynamin‐1 (5′‐GGCUUACAUGAACACCAACCACGAA‐3′), dynamin‐2 (Dyn2_1: 5′‐CCGAAUCAAUCGCAUCUUCUU‐3′ and Dyn2_2: 5′‐GACAUGAUCCUGCAGUUCAUU‐3′), as well as CHC as previously described (Huang et al, 2004). The AllStars Negative siRNA non‐targeting sequence was purchased from Qiagen.
Microscopy/fluorescence‐based assays
Immunofluorescence: ARPE‐19 FL and ΔαAD cells (1.5 × 105 cells per well in a 6‐well plate) grown O/N on glass cover slips were washed with PBS and fixed in 4% PFA in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X‐100 for 10 min and further blocked with Q‐PBS (0.01% saponin, 2% BSA, 0.1% lysine, pH 7.4) for 30 min. After three washes with PBS, cells were incubated with a mouse anti‐EEA1 antibody (#610457, BD Transduction Labs) or a rabbit anti‐APPL1 (#3858, Cell Signaling) in Q‐PBS for 1 h using the recommended dilution. Cells were further washed three times with PBS and further incubated with suitable AlexaFluor®‐labeled secondary antibodies (Life Technologies) for 45 min. After three additional washes with PBS, samples were mounted on Fluoromount G (Electron Microscopy Sciences) on glass slides and examined using a 100 × 1.49 NA objective (Nikon) mounted on an epifluorescence Ti‐Eclipse inverted microscope.
Tfn pulse/chase: TfnAF568 and TfnAF647 were obtained from Life Technologies. For dual‐pulse experiments using fluorescent Tfn, FL and ΔαAD cells were initially pulsed with 4 μg/ml of TfnAF647 for 2 min. Following a cold washing step to remove unbound ligand (3× PBS; 4°C), the remaining surface‐bound ligand was removed by an acid wash step (3 × 2 min 0.2 M acetic acid, 0.2 M NaCl, pH 2.5). Cells were washed with PBS and then pulsed with 4 μg/ml of TfnAF568 for 2 min, washed with cold PBS (4°C), acid‐washed again and fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) in PBS for 20 min. After three additional washes with PBS, samples were mounted on Fluoromount G (Electron Microscopy Sciences) on glass slides and examined using a 100 × 1.49 NA objective (Nikon) mounted on a Ti‐Eclipse inverted microscope. EEA1‐ and APPL1‐immunostained cells were imaged with an exposure time of 200 ms for both channels using a CoolSNAP HQ2 monochrome CCD camera with 6.45 × 6.45 μm2 pixels (Photometrics, Tucson, AZ, USA).
Endosomal acidification: Time‐lapse of Tfn internalization and acidification in FL and ΔαAD cells was performed by incubating cells with pHRodo‐Tfn and recording the fluorescence emission (585 nm) every 60 s for 20 min using a Synergy H1 Hybrid Multi‐Mode Microplate Reader.
EEA1 and APPL1 staining and analysis by TIRFM
Detection of fluorescence spot signals by TIRFM.
Cells expressing EGFP‐CLCa were immunostained for APPL1 (#3858, Cell Signaling) and EAA1 (#610457, BD Transduction Labs), followed by labeled secondary antibodies (Life Technologies). Fluorescent signals, illuminated using an incident angle set to visualize 250 nm from the coverslip, were detected using the spot detection algorithm as previously described (Aguet et al, 2013). Clathrin‐coated structures and early endosomes were assumed to be diffraction‐limited, producing a signal that is well approximated by a 2D Gaussian function in TIRF microscopy. Fluorescence signals were identified by numerically fitting a 2D Gaussian at candidate locations, and statistically testing the amplitude against the residual background noise estimated at each location. The detection was performed independently for the three fluorescence channels.
Identification of cell boundaries.
Cell boundaries were automatically identified from the cytosolic background of EGFP‐CLCa. The raw images were first smoothened using a Gaussian filter (S.D. = 5 pixels). The intensity distribution of the smoothened image was always bimodal, with the two modes representing background signal outside cells and fluorescence inside cells. A threshold was automatically selected as the local minimum between the two modes.
Object density relative to cell boundary.
Object densities relative to the cell boundary were calculated from all objects that were closer to a cell boundary than the image border. The densities were normalized by the area of the band of pixels equidistant from the cell edge corresponding to each bin of the density histogram.
Western blotting
Cells were washed three times with PBS and harvested/resuspended in 100–150 μl of reducing Laemmli sample buffer. The cell lysate was boiled for 10 min and 30 μg of cell lysate was loaded onto an SDS gel. After transferring to a PVDF membrane, membranes were probed with antibodies against the following proteins: APPL1 (#3858, Cell Signaling), phospho‐Akt S473 (#4060, Cell Signaling), phospho‐Akt T308 (#13038, Cell Signaling), Akt (#9272, Cell Signaling), phospho‐ERK (#4370, Cell Signaling), ERK (sc‐93, Santa Cruz Biotechnology), phospho‐TSC2 (#3611, Cell Signaling), TSC2 (sc‐893, Santa Cruz Biotechnology), phospho‐GSK3β (#5558, Cell Signaling), GSK3β (#610201, BD Transduction Laboratories), dynamin‐1 (ab52852, Abcam), dynamin‐1 phospho‐S774 (ab55324, Abcam) and dynamin‐2 (sc‐6400, Santa Cruz Biotechnology), according to the manufacturers' instructions.
Cell proliferation and EGF‐dependent signaling
ARPE‐19 FL and ΔαAD cells (1.0 × 106 cells) were seeded in a 6‐cm dish containing DMEM with 10% FBS. Eight hours after seeding, cells were washed three times with PBS and starved in DMEM containing 0.2% FBS for 16 h. One dish was left untreated (i.e., control). The remaining dishes were treated with 20 ng/ml of EGF for the indicated time points (0–180 min). At the end of each time point, cells were washed three times with PBS and harvested/resuspended in 100–150 μl of reducing Laemmli sample buffer. The cell lysate was boiled for 10 min, and 30 μg of cell lysate was subjected to SDS–PAGE. After transferring to a PVDF membrane, the membrane was used to probe with several EGF/EGFR downstream effectors as described above. Cell proliferation was assessed by MTS assay (Promega), according to the manufacturer's instructions.
TIRF microscopy
Total internal reflection fluorescence (TIRF) microscopy was performed as previously described (Loerke et al, 2009). Briefly, RPE cells expressing EGFP‐CLCa were imaged using a 100 × 1.49 NA Apo TIRF objective (Nikon) mounted on a Ti‐Eclipse inverted microscope equipped with the Perfect Focus System (Nikon). During imaging, cells were maintained in DMEM lacking phenol red and supplemented with 2.5% fetal calf serum. Time‐lapse image sequences from different cells were acquired at a frame rate of 1 frame/s and exposure time of 150 ms using a CoolSNAP HQ2 monochrome CCD camera with 6.45 × 6.45 μm2 pixels (Photometrics).
Image and data analysis
All image and data analyses were carried out in Matlab (MathWorks, Natick, MA, USA), using custom‐written software. Up‐to‐date versions of the software will be made available at http://lccb.hms.harvard.edu/software.html.
Generation of dynamin‐1 KO H1299 cell lines by CRISPR‐Cas9n and reconstitution with Dyn1‐EGFP constructs
Genome editing of H1299 cells was performed using a double‐nicking strategy to knockout (KO) endogenous dynamin‐1. Two pairs of 20‐bp‐long dynamin‐1 sgRNAs (single guide) were designed using the CRISPR design tool available at www.genome-engineering.org/crispr (Ran et al, 2013). The guide RNA pairs (+ and − strands) were cloned into a bicistronic expression vector (pX335) containing a human codon‐optimized Cas9n and necessary RNA components (Addgene). Top two double nickase "hits" targeting exon 1 of human dynamin‐1 were chosen for cloning into pX335 vector. The sgRNA pair (A) for Clone A9 and Clone A19: 5′‐TTCCATGCCGCGGTTGCCCATGG‐3′ and 5′‐ATCTCATCCCGCTGGTCAACCGG‐3′ and the sgRNA pair (B) for Clone B22: 5′‐CCGGCTGCCGCTAGCGCTCCCGG‐3′ and 5′‐AGCCATGGGCAACCGCGGCATGG‐3′ were used.
The single‐guide RNAs (sgRNAs) in the pX335 vector (1 μg each for a sgRNA pair) were mixed with pmaxGFP plasmid (0.2 μg; Lonza) and co‐transfected into H1299 cells using lipofectamine 2000 transfection reagent (Life Technologies). Forty‐eight hours post‐transfection, the cells were trypsinized, washed with PBS and resuspended in PBS containing 2% serum. GFP‐positive cells were single‐cell sorted by FACS (The Moody Foundation Flow Cytometry Facility, UT Southwestern Medical Center, Dallas, TX) into a 96‐well plate format into RPMI containing 5% FBS. Single clones were expanded and screened for dynamin‐1 expression by Western blotting using the anti‐dynamin‐1 rabbit monoclonal antibody (EP772Y, Abcam).
Stable reconstitution with WT Dyn1‐EGFP and S774A Dyn1‐EGFP fusion proteins in H1299 Dyn1 KO cells (Clone B22) was performed as previously described (Liu et al, 2008). Briefly, WT Dyn1 containing an N‐terminal HA tag and C‐terminal EGFP fusion protein were cloned into pMIEG3 retroviral vector. Virus was produced in 293T packaging cells and the retroviral supernatant was used to infect H1299 Dyn1 KO cells. Cells were collected 48 h post‐infection and FACS sorted for low expression levels based on GFP intensity. The S774A mutation was introduced into the above retroviral vector via site directed mutagenesis and similarly infected and sorted for comparable expression levels.
Author contributions
CRR and PHC designed the project, performed the experiments and analyzed the results. CRR and SLS wrote the manuscript. SS generated and validated the H1299 Dyn1 KO and reconstituted cell lines. MM assisted with experiments and provided reagents. FA performed the computational analysis of APPL1 and EEA1 by TIRFM. SLS conceived the project, supervised and directed the research. All authors discussed the results and commented on the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Figure S1 [embj201591518-sup-0001-FigS1.pdf]
Supplementary Figure S2 [embj201591518-sup-0002-FigS2.pdf]
Supplementary Figure S3 [embj201591518-sup-0003-FigS3.pdf]
Supplementary Figure S4 [embj201591518-sup-0004-FigS4.pdf]
Supplementary Figure S5 [embj201591518-sup-0005-FigS5.pdf]
Supplementary Figure S6 [embj201591518-sup-0006-FigS6.pdf]
Supplementary Figure Legends [embj201591518-sup-0007-Legends.docx]
We are grateful to Gaudenz Danuser for critical contributions and discussions leading to the checkpoint hypothesis. We thank the members of the Schmid and Danuser laboratory for discussions and technical assistance, and Mike Henne and Peter Michaely for critically reading the manuscript. This research was supported by National Institutes of Health grants R01 GM73165 (G.D. and S.L.S.) and MH61345 and GM42455 (to S.L.S.). P‐HC was supported by a Taiwan National Science Council Grant 103‐2917‐I‐564‐029.
FundingNational Institutes of HealthR01 GM73165MH61345GM42455
- © 2015 The Authors


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