Hello and welcome to the Rugged Rockhound. Today I'm here with Tim once again, and we are at Cisco and Yellow Cat, a known area for good aggots, bearite spheres, which sometimes are still bearite, sometimes are aggotized, and petrified wood. Last time we were here, we're at another spot where we found that it had wood, but the wood wasn't very good. So we're out here at another spot that we found in a geologic study that was done by a geologist decades ago talking about petrified wood in this area being nicely agotized. So that's what we're gonna look at. We're gonna look for some nice agotized wood. We came up a little way from the vehicle. I found this piece of aggot sitting there. There is some little pieces of aggot, a lot of them are white, and we're going into some pieces of petrified wood now. So there's one Tim grabbed. You can really see the wood pattern right there. I just picked that up. It looks almost like a carnelian. Yeah. All right, we were walking across and here's a claim marker. I'm not sure if it's up to date. I'd have to find the one that actually has the paperwork in it. Hopefully we'll come across that. But because I'm seeing this claim right here, and it may be active, we will move further that way and see if we can get out of the claim. All right, I just want to make a quick update. First off, I ran into one of my subscribers. He was driving along this road, Brent. He just kind of showed up and we talked to him. He kind of give us some of the knowledge he has about this area. That was pretty cool. But unfortunately, as I was walking around, I found out that this entire section is claimed, which is very unfortunate because before I came out, I kind of looked on the BLM website and this section on the website showed no claim. But BLM is known for being pretty lax. So unfortunately, this entire place is claimed. I found where they have the documentation. It's all legitimate, legal. So legally, we cannot collect anything here without permission. So we had to dump everything we had found. We spent almost two hours here and this is what we're going home with currently. Wah-wah. But that's all right. I talked to Brent and he told me that going back to where it talks about, you know, looking for stuff in the book, he knows a section where you can find okay wood, at least better than what I've seen in the past.  We'll go take a look over there, see if we can find some of that wood. Here we are in a different area. This is looking a lot more promising. So over there where we came in, over way up the edge, there was a claim over there. So we just came past it and there's this whole valley right here. We've looked through it. I don't see any claims anywhere. This looks like a great place to look around and the agate looks much better. It's a red banded agate. Yeah, it's looking pretty good. Park down there. Tim took the other side of the hill. And just to kind of show you what we're seeing up here. So you got this black rock that has a little bit of agate in it. Sometimes red, sometimes a brown color. He did see a few pieces of wood that weren't very good quality. That might be some. All right, I'm up on this hill now. The agates are getting awesome. Look at that. Wow, beautiful red banding all through it. That is gorgeous. That's a keeper. Just looking off toward the, I believe it's the Mantila Sol Mountains. And then you can see down into Arches National Park a little bit. Beautiful view. It looks like we're finding, it looks to be some wood. It's not agatized though, look at that.  Maybe it's just selenite. There's a bunch of selenite over there.  So you can see the spheres that the barite kind of made and then it got replaced by agate. The more I look at these, the more I think they might not be wood. Cause look at the cross nature of the stuff. It looks more like crystal growth. Yes. I think these are actually crystals growing. That isn't the wood. It looks like wood. But I don't think it is. There are some nice agate around here. Look at that. It's very thin. There's definitely some good looking agate here. Look at these black rocks. They're so shiny. This one's really neat. You can see the crystal terminations of what used to be the barite. Look at that. Oh yeah. So that is one of those barite spheres that's only a piece of it, but a big one. Maybe we can find something better around here. Look at that. It looks like a nice agate just sitting there like a tumbled one. Can you get it out? I don't think I'm gonna get the handle.  We are gonna stop at a river bed on the way in that I thought looked interesting that might take a look around in quickly. So I might show you that if we go do that. But so far we looked out here. A lot of good agate. I'm pleased with the agate, especially the red, there's some nice stuff. You did have to search a little bit for it. But I was unfortunately not able to find really much wood and what I did find I don't know if it's wood because there's the barite that can have that weird look. Selenite that can look kind of like it. So a lot of fakes out here. I don't know, we'll see. But so far it's like kind of what I was wanting to look for. Looks like we might have been a little too high. It might be that what we're looking for is maybe below the surface at another layer, which is why I'm hoping on the way back as we go lower again, that's why I wanna look there and just see. Cause maybe we were just too high. I filmed it but realized I was in the wrong setting. Look at that big piece I found back there. And Tim also found one kind of like this one, a little smaller, but it looks almost identical. A little bit more wood, different color. Not a very good one. Probably won't keep coming. Oh, not the prettiest color, but it's a lot of agate. I know. Oh, look at that. That looks like a pretty nice piece right there. So we need to do that. There we go. Nice. On another piece. Nice. Oh, it's pretty big too. It's nice. I love the red. I know we were just gonna stop in that one wash on the way back and then get out of here, but we came to the main wash kind of down below the main area where most people look. And Tim was like, let's just stop here really quick and check this wash again. We spent a little bit more time than we were anticipating. Take a look. So these are the ones I came up with. So it's got some nice color there, but really it's these beautiful pieces of wood. And just kind of quickly show them off to you. Found a lot of little pieces too. That was my good one. But unfortunately, Tim just blew me away. Look at that, look at the knot in that one. Nice piece. And then this one, beautiful piece. Again, and this one, this was the killer one. This was the best piece of wood we found today, by far.  Just found so many good pieces of wood. And then he found a nice big piece of agate that may or may not have been wood. I'm gonna say no, probably not wood, but nice agate. All right, I'm back from my adventure with Tim when we went down there to Cisco and Yellowcat to find the beautiful agate, agatized barite and petrified wood. I got it all cleaned off right here. Let's go ahead and take a close-up look at some of the nicer pieces. Starting off with a nice piece of banded agate that Tim found. Nice. So this, at first glance, looks like some petrified wood. But this is actually agatized barite. As you can see, it kind of makes more of a radiating pattern rather than that of typical wood. Here's another piece of that agatized barite. It's got a little bit of a blue color in it. This was the more colorful piece of agatized barite that Tim found. Yeah, it's got some nice purple in it. Up there, you can find these little nodules or concretions that filled in with agate in the middle. And sometimes the agate in the center is very nice. This is one that Tim found. Here is another piece of that nice agatized barite. Notice that radiating pattern. This is some more of that agatized barite, but instead of being red or that bluish color, blue-white, it's more of a yellow. This is some of the really nice red agate that you can find in the area. Doesn't occur usually very big. This was a nodule that had this little square that filled in with layers of agate, white and kind of colorless alternating agate and a little bit of red. Very nice. Here is a nice large piece of that barite replaced by agate. There's another one that we broke open. Nice red right in the middle. More of that agatized barite. This was my nice piece of agatized barite. Got that nice purple and other colors going through it. Very nice. This is one of the better pieces of petrified wood that I found in the wash. Another piece of petrified wood that I found in the wash. This is a piece of petrified wood that Tim found in the wash. This is one of the nice pieces of petrified wood that once again Tim found. This is a cool piece of petrified wood that Tim found. You've got a branch going through it right there. And I think it's just a couple of branches all laying together. And here is the big piece of petrified wood that Tim found. Largest piece that we found while we were out there. Very nice. And this was the favorite thing that I found. It's the barite, but look, you can see the terminations of the crystals that got replaced by the red agate. Overall, some pretty nice pieces. I like the replaced barite. Many different colors and the petrified wood was pretty good too. It was a great time. Unfortunately, there are a lot of claims down there. So we had to look around quite a bit to find areas where there weren't claim posts and claim markers. So we tried our best to avoid any claims. And if you guys do go down there, remember that there's quite a few claims out there and try to respect the claim owners as much as possible. All right, and don't collect on their claims. I hope you enjoyed this one. Remember, there's treasure everywhere.


Alright, we may have genuinely stumbled upon Bon Klai's paradise. Episode 439 and 440 of our one-piece watch through, what is going on anymore and impel down? Floor 5.5, a secret paradise where Ivan, who Bon Klai has been looking for, is apparently the leader of? I don't know. I'm hoping we're going to get some answers soon. Probably this episode. If you're new to the channel, drop us up and hit that big boy bell and let's hop into it. The build of Ivan is so confusing to me. He's enormous, but like, I don't know how to explain it. I honestly thought he was just like kind of short at first. These are some terrible moves, I'm gonna be honest. No, they're fine though! My goodness! Bro's got the mermaid man like bug shell bra. Dude, this place is actually like just Bon Klai style. Like all these people are just like so wacky. He haw. He's so stunned. He hasn't said a word since he realized it was Ivan. Oh, got a giant throne. Somebody catch Ivan's Sama! Oh, gosh. Those people are like, what is going on? Why were they moving like that? Oh, wait. Oh, son. Ivan's Sama. You get it right, Bon Klai. Ivan's Sama! Ivan's Sama! Ivan's Sama! Look at that chin! Holy guacamole! Candies! Janaiwaiya. Duki? What? Oh, my bad. My bad, Ivan. Sama. So what is a miracle worker? Like, what are the stories you've heard? Oh, is this part of the show? There's spotlights on him. Yeah, who are you? You're killing the vibe, man. 15 years ago. Oh, did he never come back? And I'm here to take revenge! Oh, his country and family. Oh, that's sad. Bro, how did you get here? Ivan's like trembling. Is he not much of a fighter? Ivan is such a troll, man. The like, like pose. Oh, what? Death wink? Oh, wait. Maybe Ivan's like cracked out of his mind. Powerful. He reminds me of Chopper. I miss that kid. Oh, my God. No form. Yo, Ivan, he's terrifying. Oh, it's just like madness. They're just clapping.  Then a word. The border called gender.  I don't want to know how this operates. Like how they get food and drinks and stuff in this random paradise down below in Pell Down. Okay, so it's definitely a double fruit. There's no way you can do that to someone without like some power. I mean, he used like nails. Again, where did that guy even come from? Bon-Boy. Aw, it's like his idol, bro. Ask the hell, Blue Fee. Can Ivan do that? How much of a miracle worker is he? Strawboy. Are we gonna see where he is? Are they keeping him? One click. It's some alone time with Ivan. They were watching them on the cameras probably. They saw what Bon-Kle was doing for Luffy. Oh. Dang, dude, Luffy was still conscious. I thought, like I said, they said they had been watching them on the cameras or some monitors. Luffy begged for them to help Bon-Kle first. Yeah. It's not luck. It's sheer willpower and determination. He sees how much he's suffering and that he asked for him to help him first. Damn. Yep. You'd ask for help. They're just strengthened. Demon and human skin. Oh. Oh, nice. What is the treatment? Dude, thank God for Ivan. What is the treatment?  That wouldn't be pretty, man. Like I said, we're just lucky to have found Ivan in the first place. Oh, my God. I knew we were going to check on these bozos eventually. Those are kind of like the stilts from Long Ring, Long Land. How'd they even make the stilter that they waxed? Are they even trying to escape at this point? Oh, my God, they broke. Has this been happening for 10 hours? And why did they lock it so securely? He has. Two more days? They don't have days. Ace's execution is scheduled for what? What did they say? Like, we're not like 26 hours or something? I honestly don't remember. What? No shot.  Is his spirit fighting, man? The what? Oh, my God. I mean, at least we found Ivan, but still. What is the range of these abilities? The female hormone, healing hormone. I'm guessing male hormone. And it's going to continue for another 48. Oh, maybe he's going to explain. It's like the inner workings of your chemistry. Change his body. Oh, my gosh. Brother, what a trial to go through. It's so crazy how none of the straw hats know what's going on right now, like the chaos involved. I think they would just make him feel worse. Why are you so worried about him? Grabs Ivan's leg. And again, we don't have two days, man. Oh, no. Is Ivan getting mad? Yeah, for real. What else? What other option is there? That's why. Oh, yeah. Why did that where the nickname comes from? He's inspiring Von Clay. I mean, that's kind of what Von Clay has been wrestling with, though, ever since he did leave Luffy. Ace. That's all he's thinking about. That's what's keeping his willpower alive, his spirit alive. How many hours? 16! Brother! We do not! All right. Well, it is just up to Buggie and Mr. Three in this case to save Ace. Will it work out? Probably not. But I can believe. Episode 440, let's get it. Yup. It's all up to Luffy. Ivan's done what can be done. And he's like, Von Clay, come on. Let's just hang out, man. Horm-horm fruit. It's a hormone fruit. Ah. Sex, body temperature, skin color, growth, animal mode. Oh my gosh. Brother! Yeah. I thought they need to answer how this was even, like, founded. Oh, no, he deathlinked them! Call some candies, bro! Yeah, so how does this demoning turn in from that to that? Like, how do you get here? So there's entrances everywhere. Oh. They just straight up have secret passages. It literally is just like an underground. It has to get kind of depressing. Like, just underground like this, though. Oh. Oh my gosh. They saw everything. They were just cheering him on. So it's kind of like they vet them before.  A whole digging ability. And it all just fell in the place. Who was able to dig these holes? That's kind of crazy. A little five ants. Okay, so there is a six. So I'm guessing six is where Ace is. That would make sense. Because he's not in that icy area. True villains are kept. Yeah, that's where they all are. Crocodile. Jembae, all of them.  Another threat in the prison. The jail manager. Shiryu of the rain? Wait, what happened? Oh, he was here beforehand. Dog. Yeah, I bet. Oh my gosh. He's got like the wings too. He looks like psychotic. Oh shoot. Wow. Magellan actually had a referendum. And this wasn't even that long ago, he said. Oh my gosh. Did they throw Shiryu into like floor six? Oh man, I bet that. Did he not even fight his way out? I was going to say a fight between them would have been nuts. Wow, he's in six? Holy. Yeah, it would have been infinitely worse if they had come when he was out. San Juan Wolf. That's a shot. I don't think we knew any of those. I figure there's a ton down there in level six that are just incredible fighters. Yes, sir.  True, just keep spinning, Panchan. Yep. Yes. Yes. Just ate the whole bone. Oh wow, even I've been saying there's no chance that Luffy can get to him. 3 p.m. today. Oh my gosh. Oh my gosh.  And Luffy's not going to be recovered by then. Yeah, two more days. Oh no. Oh my gosh. If Luffy woke up and found out it had been too late and they've already taken him, he would freak. Four-boned clay. It's emotional support, guys.  Can Luffy even hear him? Now it's just like a waiting game, dude. Like cheering him on. You had to admire it, though. You guys don't know true friendship. Keep going, Bond Clay. His spirit hears you. How long has it been? I've been starting to see who Bond Clay is and his relationship with Luffy. Bond Clay is the best, man. The best friend you could ask for. Yeah. Bond boy. It's literally the most he can do right now is yell the Luffy that he's here. Yeah, he's still insanely injured, too. Create a miracle. It's the spirit. I mean, they saw how far they went for each other unlike the monitors and stuff. They followed their whole journey. I mean, at least since they've gotten here. Inspired all of them. Now he hears all of them. That's so sweet, man. Oh, he's gone. Oh, Zoom. Oh my gosh. They alert again now. I didn't think they would even check.  There's more ships. Are they coming to get ace? Trying to create a miracle. Go back to ace. Even though they're both warlords. Nah, he's just trying to make ace feel better. Yeah, that's what I'm saying. That'd be so dumb. He's just worried. He's scared. He had that too. He knows Luffy. They're all out of breath. Yo, shout out this group, man. Oh my gosh. So much blood.  Luffy died. That's dark, man. Luffy is brother up. Shoot. He just bled his entire body clean. Not even a day. Here, Magellan, he's in the bathroom. Oh my gosh. It is actually time. Oh no. He's straight. He's an animal. To say the least.  It's like... He's back, baby. Yo, shout out Kamaland. There's a panda. Panda sighting. Yes, sir. Better than ever. Oh my gosh. That's where they came from. Well, god damn. Shout out Ivan in this place, man. I mean, you couldn't ask for more of a miracle. Without him, you wouldn't have recovered or even that quickly without Bon Clay. But the time is running out, dude. They're moving ace. At this point, like, we maybe somewhat of a fighting force if Ivan and all of them join Luffy. I mean, maybe they will since they've seen his headstrong determination. But if Ivan and all of them join, then this whole place is like bust. They're exposed. I think the only chance is they try a jailbreak of that entire floor, the entirety of six. Because how strong all those pirates are down there is just immaculate. But at the same time, what are you letting out? What kind of monsters are you letting out down there? I love this concept, though, of this secret area in the prison. It's hilarious. Even more so, the connection that it has to the island that Sanji got sent to.  But guys, that has been it for episodes 439 and 440 of our One Piece Watchthrough. Drop us up if you're new to the channel, and I'll catch you guys in the next one. Peace, peace.


I know y'all saw what I just saw. I'm heading to the next Raptors game. I said, bro, if I'm gonna be there, I might as well bring the Switch. I'm playing NBA 2K while I'm up there. What? I'm hoping the Raptors win. They've been getting played with some injuries. And I've been doing something recently. I've been like, I really wanna see iconic players play before they retire. The last chance I had to see Kobe play, he was injured. I got a chance to see LeBron play last year. It was amazing. Now, Raptors got slapped in the play. Hey, it's not about that. I said, bro, I gotta show up. Przingis going to be in town, man. Is that what you're telling me? Low key, I'm unprepared. I forgot to charge the Switch. So I'm just waiting for that to charge.  You've probably seen it coming. But shout out to SeatGeek for helping make this video possible. SeatGeek is an app that helps make buying tickets a lot easier and saves you money. You hop on the website, go to sports, NBA, your favorite team. LaSace at Atlanta Hawks. Goal to the game that you want. It'll let you know the lowest possible price you can get it for a boon. And then at that point, you're just shopping for seats. Red shows an awful deal and green shows an amazing deal. That means you're saving money and saving time. I'm actually serious, yo. This actually, it's a wavy site, bro. And then I show you a picture basically giving you a view of the exact seat that you're interested in buying. If you guys are interested, I'm gonna leave a link in the description. Use code AGENT at checkout for $20 off. It's gonna be a serious trek downtown. So let's get it going. I don't know, yo. We have to check bags now. I didn't know about that. Loki, I'm excited they let the camera in. I forgot my bipod, so I'm gonna be struggling with this vlog. She thought it was crazy when I had all this stuff. I was taking out an Nintendo Switch, a big portable charger, the camera. I was like, yo, be careful with the camera. She's like, why? I was like, it's 5,000.  Then I handed her the switch. She's like, it's not gonna fit in the bucket. I was like, nah. She's like, why do you have it? I was like, yo, honestly, you don't even want to know. It's actually not that hard to believe. Some man's just trying to hop on 2K, grind the badges on the switch. If you don't know already, I'm trying to be the number one Switch player. Everybody was looking at us like we're crazy. I'm not the crazy one. He's the one wearing shorts in this freezing weather. Winter jacket with some shorts and an Oakland Raiders cap to a Raptors game. I'm swagging, bro.  I'd expect to be this close. I don't think I've ever been this close. Nick Fens. Did she miss us? How could you say that? I know the nicks have been trashed for as long as I can remember, but you're disrespecting them and this is their year! I'm out of control. I'mma chill out. Step back. What would y'all rather do? Watch these dancers do absolutely nothing on the floor or grind your badges? I feel so disrespected, bro. Security pulled up to me and it just happens in the airport. People just confuse that I have money. They're like, yo, how did you get this ticket? He asked me for my ticket. I'm like, what you mean, bro? How did I get through all the latest security without a ticket? Of course I have a ticket. He's like, let me check. Out of all the people, he only asked me because I'm not decked out in Louis Vuitton. Damn, man. I got it. It's like the only way to fit in is to just mask people's lack of confidence with an overindulgence in designer goods.  I'm just trying to be me, bro. That's all. Just let young agent be me. If I wear a towel under my hat, that's what I do, bro. If I'm rocking, contradicting gear, that's just what I do, bro. I just want somebody to accept me for who I am. That's all. Hey, hey. You want to catch up with some 2K? I'm gonna pray for you. I'm on the ramp too. Yo, play some 2K. Bro, maybe I should dress posh in like Burberry and next time I'll be accepted. Why would you ever wear Burberry? That's what people wear, right? Uh-huh. Is that what you're wearing? No. What? I just thought of a brand. Gucci, Leviton, name it, bro. What if I like save my money for microphones and monitors? You know what I'm talking about? You save money? I say money, what you talking about? When did I ever spend money aside from food? You know what I'm talking about? Unless it's delicious Popeye's chicken, I'm not spending money. Which game am I looking to score at? I'm losing this one. This game's not important, bro, as long as I get my badges. It's like five frames per second advertisements on the switch. That's crazy. Bro, would I rather watch ankle bracelet put up shots or call out? Oh, no, no, no, no, no, no, no, no, no, no. It's gonna happen. I sense it.  It's so weird seeing the players at a level feel. I'm used to being like all the way up top because I was broke. Uh, two minutes. This is nice right here. You know, Loki would be a good job to be the hype man. You know, like between like intermissions? I'd love that job. Yo, check it out. I just made my new six foot slatcher on the switch. I'm dedicated to becoming the best switch player on the planet, you know? I just, I lost data so I just backed out. I'm just gonna come back in and grind some back. It's vicious because I'm not decked out in expensive gear. I just, I rock when I rock, man. I don't think that's ever gonna change. I'm still in the middle of cut scenes, y'all. Unbelievable. The entire halftime has took up with these advertisements. I can't even adjoin myself. Like I get we're gonna cut this part out, but, this is making it. We've been in this cut scene for a long time. Why do you think I'm gonna cut this out? I have a YouTube chat. Do you wanna see how it ended in the last one? Yo, take it how right when halftime ended. I got a chance to play it again. This is my Gwang's fault. Do you see how explosive he was? I've never in my life seen something like that. He looked like he was ready to take ankle. No, it's so weird to see like on this level, that man could really explode. The OGAZEZERO username. That's crazy. Oh, my two zeros, the OG username, baby. Why are you actually getting excited? It's my first part game, bro. What you mean? I'm trying to keep my record 100%. Good move, good take, money.  Five minutes left, three minutes left, four minutes left. I wanna watch it. So you're actually gonna pay attention? I've been paying attention, bro. Where you been after so long? Nick's, Bersingas. Y'all just let me down, man. I was like, well, Loki Wofferman's been hyping that team up, so I was expecting a little bit better.  Thanks for coming through. I'm gonna catch you guys in the next one. I'm out. Thanks.


Well, today I would like to... Can you hear me? No. Oh, it's not on. Can you hear me now? Yes. I would like to talk about another organ now. The nuclear power complex which mediates traffic in and out. As I already said to you yesterday, I'd like to quote the conducting channel. And I apologize for those who already have heard of Tom Rapport a few weeks ago showing you negatively stained images of this creature, of the protein conducting channel. I just took you through this exercise because it was those experiments which really finally convinced the public that there is a protein conducting channel. And since very few people have had these experiments, and since very few people knew, because most of the time they often see electrophysiology. I thought it was very important to give an understanding how the concept of the protein conducting channel really came about. And what I would like to say is that at this moment, two-dimensional reconstruction negatively stained the protein conducting channel and there's still not proof that it is a channel. For that you need three-dimensional reconstruction because obviously if you do that, it may look like it is a channel but may not actually be a channel. So for proving that you have a channel, you really have to do three-dimensional reconstruction. But anyway, this is just an afternoon. Now in this case of the nuclear power complex, the situation is, as the name said, very complex. And let me take through it step by step. And I apologize to those that are all just particular people like Els Gerdlund and Jim Balbert and Hans Ries who have been major contributors to the field by going through some of the rather basic slides which I'm showing here in the beginning and perhaps not discussing the latest STS trials and curves that we have but primarily focusing on concepts. We have this morning a session with Els Gerdlund and Jim and they asked me all the questions and I still am looking forward for another session and I was going to ask them all the questions but we have to schedule something. But in the first slide, maybe we should turn it up in a plunge in the target. So this is what I would really like to discuss with you. First, the nuclear power complex, what we know about destruction and what we know about the function. Then transport factors which interact with the nuclear power complex and with transport substrate to get things in and out of the nucleus. Then I would like to discuss something which is very exciting maybe that there are several distinct transport passwords. So there is not just one but there are at least four and there may be as many as ten different transport passwords of how you can get things into the nucleus. And there is also a futuristic project which we will have to do with inter-nuclear transport and I have just seen the wonderful pictures which Hans Ries has produced on inter-nuclear structures particularly tubular structures. So there is a great deal of excitement and this is a project which will receive great attention after the next couple of years and then there is just one or two slides on export and as you will see we know very little about a nuclear export as of this moment other than that there are a number of distinct passwords but the factors which mediate nuclear export remain largely undiscovered. So let me start out with this offensive slide to many of you just to say what we are talking about here. The nuclear envelope is a double envelope. Membrane, the inner membrane continues with the outer membrane and the outer membrane of course is the same as the yard. So then you have these circular openings of 100 nanometers and they are filled with a nuclear core complex and underlying this and connecting the nuclear core complexes is the laminar structure which was first described and one of the principle people who described the structure was faucet and he called it a very correctly fibrous laminar. It was already in the literature under the name of Zolina nucleon limitant if you like the names. And faucet in a very keen insight realized that these structures are fibrous in nature and in fact it was shown that the lamins which compose this structure is actually made up of intermediate filament type proteins so it is in fact fibrous laminar. And so then of course there is, and I apologize these slides are a bit dirty but there is connected to the interchromatin, to the laminar is heterochromatin and from the nuclear core complexes extending into the interior you find often the u-chromatin and that you can see in images which are taken from faucets book itself and you can see this very nicely here this is the inner nuclear envelope membrane, outer nuclear envelope membrane here are the core complexes and you can see the u-chromatin always ends up in these nuclear core complexes and heterochromatin is in between. And so here is a very dramatic case if you can focus a bit of the red cell of chicken erythrocytes and the chicken erythrocytes or the burbary erythrocytes keep the nucleus and you can see it's mostly condensed chromatin and wherever you have u-chromatin you see at the nuclear ending of outer membrane where in a membrane there's nothing much in the cytoplasm here and you see it's beautiful where the u-chromatin ends up at the nuclear periphery there's always a nuclear core complex and so this is an example of a cell which has very few nuclear core complexes and here's a cell which has a record of nuclear core complexes the amphibian oocyte which has something like 50 million nuclear core complexes where you see one after the other on the nuclear envelope and in fact it has an excess of nuclear core complexes in the cytoplasm and they're called annular lamellae and as the cell undergoes mitosis these nuclear envelope of course falls apart into vesicles and the nuclear core complexes are disassembled and so are the annular lamellae annular lamellae undergoes the same disassembly reassembly cycle in mitosis as does the nuclear envelope and these are simply stores of nuclear core complexes in the cytoplasm and it is from these images, freeze fracturing images that you can count the number of nuclear core complexes and you have Mao in particularly in the 70s they've done a great deal of counting and so that we have, you know from the size of the nucleus you can reconstruct how many nuclear core complexes there are so it is known that East for instance has two other nuclear core complexes amphibian oocyte with 50 million and the average vertebrate cell in our body has about two to three thousand such nuclear core complexes and they are not always equally distributed that is again a picture from Fawcett's book in this case you see in the spermata side you see the nuclear core complexes all at one end of the nucleus and they are not randomly distributed but again I won't go into this but from amphibian oocyte one could isolate these nuclear envelopes by simply by using squeezes one could mechanically isolate them and they give these very beautiful images in negative staining and for many other images then what has been reconstructed is the very low resolution model of the nuclear core complex and what the most important features which are shown here in these nuclear core complex models will have to be revised because it's a very low model and as I just discussed with Hans Wies there are many problems with this model I mean one of the models problem is this nuclear envelope lattice which probably does not exist and it's just a collapsed structure interpreted as a collapsed structure which consists of fibers which extend from these what Hans Wies has called fish traps which he discovered a couple of years ago extending from the nuclear core complex into the nuclear plasma Hans also discovered the fibers extending into the cytoplasm so both of these fiber structures in isolated nuclear envelopes collapsed upon the structure and therefore they're very difficult to visualize and they have now been visualized through the methods which Hans Wies has been used and he can give you a talk in detail what these structures look like they're very important structures as you will see because they play an important role in nuclear transport because it turns out that these fibers contain the docking sites for transport factors to get the protein into the nucleus the model of Christopher Ecke suggests that there is in the center of the nuclear core complex a cube and that cube has a diameter of about 26 nanometers internal diameter and it is postulated that traffic goes through this central cube of 25 nanometer internal diameter so if the protein conducting channel in the R would be 2 nanometers in diameter this is 25 nanometers in diameter which is about the size of the ribosomal subunit so even a ribosomal subunit while passing through this central tube may not have to be unfolded going across but in any case, as I said to you this is a low resolution model 90 extrem and there are lots of features which will have to be changed and I mentioned some of them for instance the nuclear envelope levels is probably this fiction and it is actually collapsed fibers which extend from this fish trap into deep into the nucleus and then here so what you essentially have in the center is this tube which is suspended by these spokes and rings in the center of this 100 nanometer wide hole so here you have the nuclear envelope membrane outer membrane, inner membrane, nervous circular 100 nanometer wide hole it is suspended in the center and anchored to this hole by rings and spokes is the central feature and emanating from these all these fibers into the cytoplasm so what do we know about the biochemistry of this structure the knowledge is by no means complete we are still in the inventory phase as I mentioned yesterday which we have passed in the case of the endoplasmic apiculum we are out of the inventory phase and now we are going into the phase of structural biology and of biophysics to understand how the structure works now again, not at this point this is a nuclear pore complex there are in East probably 60 or so nuclear pore complex proteins somewhat less than was estimated originally it was estimated maybe a hundred to 200 proteins in the nuclear pore complex our latest estimate says that maybe 60 distinct proteins making up the nuclear pore complex now, how did we get there in this, I'm showing you here a rat liver nucleus and Laura Davis which was a graduate student Rick Wozniak, also a graduate student in my lab made monoclonal antibodies against isolated nuclear pore complex laminar fraction from rat liver and what they then observed in a fluorescent is this wonderful a punctate pattern when you look on the surface of the nucleus and when you look, when you focus on the equator of the nucleus when you focus on the rim you see this beautiful punctate rim staining and this suggests that particularly this antibody was reacting with a nuclear pore complex and in fact by doing immunoelectron microscopy we could confirm that this is in fact a nuclear pore complex protein and this sort of punctate surface staining pattern has been sort of diagnostic for a nuclear pore complex protein and has facilitated identification of these proteins because it avoided doing the cumbersome electron microscopy and allowed to do the much quicker immunoporescence to diagnose what your antibody reacts with and some of these proteins were formed out and what we found in the beginning is that they shared certain features together and this was a group of proteins which reacted with a monoclonal antibody that Laura Davis and Ray Bosnack produced in our lab called MAP414 and this protein reacted with a bunch of nuclear pore complex proteins which we now call nuclear porous and these nuclear porous share these repetitive and these repetitive pathogenities which react with a monoclonal antibody giving rise to this polyspecificity and here is an immunoelectron microscopy of one of these nuclear pore complex proteins we call nuclear porous in this case it's the nuclear porous of 358 kilodalton also called DUP358 which was cloned in our lab and it's the also mission motors lab and then what we have done here is some immunoelectron microscopy and you see very nicely that it decorates the gold tip of these cytoplasmic fibers emanating from the nuclear pore complex into the cytoplasmic so if you do this sort of analysis for some of the other nuclear pore complex proteins which have been cloned in sequence what you find is that these fibers emanating into the cytoplasm are composed of different nuclear porous so there is these fiber structures are asymmetric the cytoplasmic fibers for instance at the tip have NUP 358 and then there is also NUP 214 which incidentally also turns out to be an oncogene called HAN for those who work in oncogenes and NUP NUP 368 is localized on both sides it's not known precisely where NUP 98 also an oncogene as it turns out is localized on the cytoplasmic side on the nucleoplasmic side and so is NUP 153 and so how deep the NUP 153 extends into the nucleoplasm is still being worked on HAN 3 is just no, if Werner Fakke has pictures showing that NUP 153 can extend deep into the nucleus Uli Ebi has pictures where suggesting that NUP 153 is part of the terminal ring of the fish trap so there is a this sort of sap localization is going to be a very long and laborious job to find out where all of these 60 nuclear pro-complex proteins are localized so it's for those of you know it reminds me a little bit of the story of the ribosome when I grew up and where people argued about ribosome proteins which one is in what sort of geometry are they present and how many are they and so on and so on and of course you all know that we have all the ribosome proteins now the structure and sequence of them but we still don't know exactly how they function and I'm afraid that the nuclear pro-complex will have a similar fate it will take a long time until we figure out how all of these proteins function and it will be for the next generation to really solve this problem of the nuclear pro-complex in detail but what we know is that these repeat containing nuclear porous on both sides and the nucleoplasmic side and the cytoplasmic side provide binding sites or docking sites for transport which I will talk about a bit later ok so now here's a bit on the electron microscopy of transport and this goes back to Felther's early studies where he has injected gold particles coupled to nucleoplasmic into the cytoplasm and from these sort of images you can see how the gold particles accumulate on both the nucleoplasmic side notice here this is nucleoplasmic accumulation here and as well here and as well as here as well as on the cytoplasmic side of the nuclear pro-complex so you have a high concentration of these nucleoplasmic gold particles in the nuclear pro-complex on both the nucleoplasmic and the cytoplasmic side and so it's from these images that people were convinced that traffic goes through the center of the nuclear pro-complex and actually what Felther has also done he labeled the RNA and he attached RNA to gold particle injected into the nucleus to small gold particles and protein to large gold particles injected them in the cytoplasm and he could show images where they went near the same pro-complex so the idea is that all pro-complexes are created equal that they can all be import and export and that there is no difference between these pro-complexes and this is very much the debate which also ravaged about the ribosomes for many years are all ribosomes equal or are they the same and we sort of at the field shares the opinion also I mean there may be some dissent but the pro-complexes are in a way similar and they all can do both traffic in and traffic out into the nucleus now it is not just protein traffic which they have built but also RMP traffic SNRPs for instance are assembled in the cytoplasm and go back into the nucleus and of course messenger RMP comes out then also DNA of course adenovirus particles have been shown to dock to the nuclear pro-complex and DNPs can go into the nucleus organa but the recognition probably is all protein mediated the docking and transport process is probably a protein mediated process also this has not been included either so here I am showing you the same picture with Richardson and Lasky published in 1988 in Cell and they did exactly the same experiment as they felt her they injected these nuclear plasma gold particles in the cytoplasm the preservation is not very good you see the nuclear envelope here and you see poor complexes here and you can see how extensive actually these fibers extend into the cytoplasm much longer than actually most images suggest so these fibers may be a bit longer than the images suggest but of course it could also be an artifact that they have been somehow doing the preparation of this specimen have been altered and changed but you can very nicely see the docking in multiple docking sites please remember this, multiple docking sites on the nuclear program there is a model which has recently been proposed where there is only one docking site at the end of the tyrus and the fibers bend over and it delivers the substrate to the center of the nuclear program these old images suggest that they are in fact multiple docking sites and so we favor that there are multiple docking sites and so that these fibers are like like tentacles of a jellyfish which extend into the cytoplasm and allow concentration of input substrate on these fibers and allow exclusion of proteins which do not have these so-called nuclear localization signals and this exclusion of cytoplasmic proteins is very effective for large proteins but it's not as effective for small proteins so small proteins proteins lower than 40,000 can still have access to this zone of concentration and can therefore diffuse across a nuclear core complex but we think that these fibers are really concentration devices which concentrate substrate for import subsequent diffusion perhaps down a concentration rate or some other mechanism into the difference now here is another picture of Houston Swift from 1966 I'm just showing this because these are the precursors of the more modern pictures which Bertha Danaholt is publishing recently looking at a very large messenger ampille in this case of Chironomus salivary glands insect salivary glands and they make these silk-like molecules very large molecules and you can see the messenger ampille and what Houston Swift is saying in these pictures that these large particles have to unfold while they're going through the nuclear core complex they are 50 nanometers in diameter and as I told you the central diameter of the tube is only 25 nanometers so you would have to unfold this particle to get across and here you can see these downwell-like structures and here you can see half of it is in the cytoplasm interesting enough you see some ribosomes here in the neighborhood that the 5-plamin of messenger comes out first into the cytoplasm and then engages ribosomes immediately for translation and even attachment to the endoplasmic reticulum in the case of these silk messenger RNA molecules which of course are signal patterns so there is sort of a co-transportational translation if you wish while the messenger ampille is transported through the nuclear core complex initiation of translation can start as a particle is being transported and here you can see that the process is almost completed and here the process is the particle is completely there and another particle is still approaching this on the cytoplasm of course now we know that maybe these particles don't exist as particles anymore but they immediately engage messenger RNA for translation but of course it could also be that some particles are unable to mediate, to immediately engage messenger RNA and are visible as particles so anyway, this is what Euston Swift's pictures suggest that some of these messenger RNAs do not immediately engage ribosomes so how about the biochemistry of transport and I don't want to go into the entire history but it's sort of very interesting that the channel for transport was in this field discovered first in 1959 the nuclear core complex was discovered Hans correct me if I'm wrong by somebody called Watson not James Jim Watson but another Watson who worked at Rockefeller but I would like to have a correction if I'm wrong if I'm discrediting somebody but anyway, the core complex was discovered as early as 1959 but the signal sequence which directs you to transport was discovered very very late in the year 1984 and the opposite is a case for transport across the ER the signal sequence was discovered first and the channel was discovered last and the reason for that of course is because the nuclear core complex is still big the signal sequence is not necessarily small but it was usually thought that somehow things can diffuse into the nucleus and you don't need the signal sequence at all and it was from the studies on the SP40 T antigen and mutants of an SP40 where this lysine was mutated to a threonine and where people could show Paldoron and others could show that actually this particular protein containing this mutant threonine could not be imported into the nucleus and they were subsequently able to show that this particular peptide is the region where the information for nuclear transport lies now another important progress was made when David Goldfauld in Roger Connrich's lab was able to synthesize this peptide and coupled it to a reporter protein by chemical coupling to its cysteine and it was able to for a recently label this reporter protein in this case it's human, serum, abhuman and then injected into oresides and was able to show that this reporter protein with the signal sequence, the NLS so-called nuclear localization signal co-vatedly attached to the cysteine could drive the reporter protein into the nucleus whereas if you attach a mutant signal peptide with a threonine on it you did not get import into the nuclei of these injected oresides and this was very important because unlike in secretory proteins where you cannot get translocation of a completed protein across the channel, heat shock proteins are needed to keep it in unfolded configuration and so on because the channel is only 2 nanometers estimated or 20 angstrom in diameter whereas of course the central tube is 25 nanometers so it's laughing at abhumans serum abhumans and easily get across and it doesn't need to be unfolded so this was an important breakthrough and another very important breakthrough came in Steve Adam as a post-doctor fellow in Laidu races when they developed a self-resistant that was really in my view the beginning of the biochemistry of nuclear transport they developed a self-resystem which was very ingenious, they just took tissue culture cells permeabilized the plasma membrane with digitonin which is rich in cholesterol and the digitonin of course does not touch the nuclear envelope which doesn't have much cholesterol and leaves the nuclear envelope in the endoplasmic reticulum intact but leeches out all the cytosolic component and what Steve Adam and Laidu race were able to do is to add cytosol back and they could show that the fluorescently labeled NLS containing substrate was now imported into nuclei in the presence of cytosol but was not imported in the absence of cytosol and of course they controlled the mutant peptide was not imported either so this reduced the complexity of nuclear import to the simple biochemical system I shouldn't say simple because it took quite a while to take apart what is in the cytosol fraction here Mary Moore has made major contributions, she was a post-doctor from my lab, she took the cytosol and really fractured it and what she found is that there is two fractions, fraction A and B and fraction A is involved in NLS recognition and docking of the NLS substrate to the nuclear war complex and fraction B is involved in the translocation into the nucleus and historically she purified first the components in fraction B and this was independently also done in marriage races now and so the fraction B activity was the first one which was purified and I will talk about this later because there was quite a surprise in that but I will because in our first half recognition docking I will first talk about the fraction A purification and again this was done in several laboratories simultaneously leading to several different names but the first one who purified it unequivocally the first one is Steve Adams at Northwestern and he called them NLS receptor and P97 and subsequently it was independently purified also in other labs, in Wawalski's lab in our lab, in the Japanese lab and later's lab and we all gave them independent names and we called the activity in the fraction A karyoferrin meaning bringing to or taking from the nucleus karyo of course you recognize and ferrin has a root ferrin in it the state and island ferrin bring to or take from the nucleus and we deliberately choose this very big leaf term other people like last year earlier have chosen the term but we think that important may also turn out to be exported so it's it's always more advantageous if you choose basic green names which do not make too many claims on the term and karyoferrin seem to be to us such a name and so they were often in better subunits and so we were able to make the common proteins of course other labs as well and so this is some work of Aurelian rado and they made the common in subunits any color of alpha and beta and neither of them alone gives you docking of the fluorescent substrate but when you add both of them together, alpha and beta you get beautiful docking to the nuclear periphery and this is the same sort of docking that you get with fraction A so it was clear that the fraction A activity contained these two purified proteins, karyoferrin, alpha and beta that docked the substrate to the nucleus and we now know that alpha recognizes the NLS and alpha then docks the NLS alpha binds to the dimeric complex binds to beta in fact alpha in beta form a heterodimer in the cytoplasm that picks up an NLS NLS substrate in the cytoplasm okay so what we have here then is you see here the import substrate in green and it binds to karyoferrin alpha and the karyoferrin alpha and turns to karyoferrin beta and the karyoferrin beta is able to bind to bind to these repeat containing nucleoporbents so it can bind to noob 358 it can bind to noob 214 it can bind to p62 which is probably somewhere in here it can bind to noob 153 and noob 98 which are on the nucleoplasmic side so there are nice binding sides for karyoferrin beta in all of these repeat containing nucleoporbents and they are in fact in the region of these nucleoporbents which contain these multiple peptide repeats but it is not known whether they are actually the concept that whether the docking side is by the repeat motif itself whether they are regions between the repeats that is not known at all so then let me talk about the purification of the B activity and this was in fact the first nuclear transport factor that was really purified by Mary Moore and that was a very great surprise it turned out to be Rhan that had a clone in sequence and it was named because it is a brass related nuclear protein therefore it is called Rhan and it turns out to be DPA it is a very abundant protein of the healer cell proteins is Rhan so it is very abundant and when we tried to publish the paper because we were asked to go back and back and back because the reviewers just wouldn't believe that such an abundant protein as anything to do with nuclear transport and they were suggesting to us that we should look for some other paint bands even so we have shown that the recombinant Rhan was active in the system and it was active as a purified Rhan but sometimes you have these problems with this paper and I'm sure that you have all experienced and I've made the experience that the more important the paper is the more trouble you have in publishing it the more mediocre the paper is the more relevant it is so but in any case Rhan I think was really a breakthrough because what it really said to us is that that a small GDPA is very important role in nuclear transport and as it turns out not just in nuclear import but also in nuclear export now subsequent very purified and so the literature raised a protein which interacts with Rhan which we call P10 which was also called NTF2 and I think that's all for names at the meantime but this interacts with Rhan and we know a little bit about how these proteins interact so what happens then somehow after docking Rhan and P10 get the proteins across and it looks like that the alpha subunit gets into the nucleus the substrate gets into the nucleus but the beta subunit remains behind it's indicated here to remain behind the nucleoplasmic fibers but we don't know this in fact but it's very clear that you have dissociation of alpha and beta of each other and then also of alpha from the substrate and both of them enter the nucleus and the beta remains behind at the nucleus core complex and of course this may simply be a result of kinetic partitioning it may be that the beta actually enters the nucleus and it's very quickly exported again so we cannot distinguish between these two things now a very important finding was made by Mike Rexha when he a postdoc in the lab when he found out that actually Rhan GTP but not Rhan GDP works in the dissociation of alpha from beta so if you add to alpha beta heterodyne Rhan GTP the Rhan GTP forms a stoichiometric complex a heterodyne with the carypherin beta and the alpha is dissociative so this is this gave us some indication of how Rhan may work namely after docking Rhan would dissociate the alpha and the substrate from the dock better and would then allow transport by whatever mechanism diffusion or whatever you favor into the nucleus so this time for tube of the nucleus core complex so this was a very key discovery and more recently the monocleurin and Mike Rexha have shown how this both carypherin beta and Rhan GTP is recycled and they have shown that alpha based on earlier work of Yunona Maroyano showing that alpha and Rhan bind to overlapping sites in beta that alpha can actually destabilize the interaction of Rhan GTP with beta and then this destabilization in the presence of Rhan GAP which is a Rhan GTP it's activating protein can lead to the conversion of Rhan GTP to Rhan GDP and this is how we think the beta the carypherin beta and the Rhan is recycled I don't want to go into a more mechanistic details the fact that Rhan GAP is in the cytoplasm and therefore in the cytoplasm presumably there is primarily Rhan GTP and this would make sense because you do not want in the cytoplasm a high concentration of Rhan GTP because it would lead to a dissociation of this heterodimer and it would lead to this somewhat futile dimeric complex between beta and Rhan GTP and so in the cytoplasm you have Rhan GAP localize the GTP it's activating protein which converts most of the cytoplasm into Rhan GTP also I warn you that there are no precise measurements how big the pools of Rhan GTP and GTP are in the cytoplasm but this is all extrapolation from the fact that the Rhan GAP is localized in the cytoplasm so this was an important finding another important finding in my view was the finding of Ulf Neobas and this was published in Science about a year ago that P10 plays a very important role first of all P10 can bind to repeat containing nuclear weapons very much like higher current and better parts P10 can also bind to better and most importantly P10 can also bind to Rhan GTP not to Rhan GTP and of course we know that this very low affinity Rhan GTP can also bind to better very low affinity so what Ulf has shown that if you give to this complex which was assembled from recombinant proteins if you add GTP you get an exchange of GTP for GTP and the Rhan GTP now dissociates the alpha and end the substrate of course and you would then have so what this suggests is that the explosive which is namely the Rhan GTP is not generated in the cytoplasm because you have high concentrations of Rhan GAP but the explosive is generated in C2 near the docked chirophae and henrodynam you use P10 to bring the explosive Rhan GTP in its inactive form to the chirophae and henrodynam then you do an exchange for GTP that access it explosive and dissociates the alpha from the battery what happens afterwards of course we don't know exactly but these I think are very key reactions now we thought that this is all wonderful and all very nice and this is the end of the story until Mike Rout and John Atkinson when the yeast data bank came out looked at the yeast data banks and found actually that there are homologs of the better subunit and so we were forced to turn the original better subunit better one and what we have now characterized are three more better subunits better two, better three and better four and they each go with distinct nuclear localization systems which are also known under different names and in order for conformity we will simply suggest that the NLS which is cognate for a specific time frame better simply called NLS 2 goes with better 2 NLS 3 goes with better 3 NLS 4 goes with better 4 so we don't precisely know what these NLS are the only case we know is in the case of hidden dry foods which has termed as the M9 seed plants because it was identified in a messenger on a binding protein and the corresponding protein in mammalian cells was also discovered contemporaneously in hidden dry foods is now together in also this work which we did in our lab in mammalian better to recall the protein transporting. Now I will take you through some of the yeast data regarding better 2 we will also show you some data and what Mike Rout and John A. Acheson did they replaced the endogenous copy of the yeast gene in this case it's just an open reading frame and tagged it with a protein A-tact version of the gene some selectable markets so it was forcibly possible then to take the better 2 gene copy and replace it by this protein A-tact copy of the gene and that then allowed to do even a fluorescence that allowed to show where the protein is and to isolate the interactive particles and I will quickly give you an example of this so what Mike and John A. Acheson showed is that the protein is primarily in the cytoplasm there's a little bit of a rim-staining they also could show that Mike Rout had been able to isolate nuclear pore complexes as part of the nuclear pore complexes so in a way of looking for interacting proteins what is this new eukaryoferin which has a molecular weight of 104 kD as opposed to the original beta-sarmine which has a molecular weight of 9.5 kD what does it bind to so what they did they took the protein A-tact version from the cytosol they put the cytosol in a massive blue-standing profile and here you see a light and a heavy fraction of membranes and here you see a flow-through from the cytosol through the column which contains immobilized IgG and so then you go in with a magnesium chloride gradient which is somewhere from 500 millimolar to 4 molar and you can see that in 500 millimolar there is not much coming off magnesium but then as you go to higher concentrations you get off a certain distinct proteins here and a very high magnesium concentration 4 molar you get off the carbonated 4 protein A-tact protein so this is a data 2 which is protein A-tact and we have shown this here by western and so and so then the question was what are these two proteins which are binding obviously very tightly to this better two and they turned out to be well-known messenger RNA binding proteins which in these were called NAP2 and NAP4 so these are very well known messenger binding proteins and if you look this documentary of these proteins is approximately right with regard to this documentary which was then to the carbonated 2 therefore K-104 now if you do the same thing with the K-95 protein of course you get the alpha down and you get also this has already been observed by Laura Davis you also get a certain amount of loop 2 which is a nuclear point down which may be a solid solid nuclear point which is not completely integrated into the nuclear core complex at the moment but you get alpha down when you take the K-95 and of course you get all the other substrates down but you don't get any major substrates so they don't stand out as a sharp band and it's only because these two proteins these two messenger RNA binding proteins are major substrates that they stand out and you can easily identify them so we did an actual sequence of these two proteins and we were sure that these are NAP4 and NAP2 so what these data been suggested is that this chiropherin the beta 2 or the K-104 is a transport factor or a docking factor which specifically recognizes sequences in these two yeast proteins NAP4 and NAP2 now we look whether these two proteins NAP4 and NAP2 have an M9 sequence that have been identified by drivers in the mammalian counterparts of the A1 protein and it does not there is no resemblance between the M9 sequence and the corresponding sequences in NAP4 and NAP2 so at this moment we do not know in what region of the signal sequence the NLS the NLS2 if you wish often NAP4 and NAP2 are located now so this is a way of putting out your substrates if you can lower the light a little bit because this is in the fluorescence in yeast and yeast is much more difficult to show in the fluorescence because the nucleus is much smaller if you can turn down the light a little bit and you see what John Asians and Mike Barth have done they have produced a temperature sensitive mutant in NAP4 and so they have shown that at the non permissive temperature at 37 degree after one hour you can see the protein is the great after three hours there is very little this is the rest of the protein that remains and so when they looked with a with a tagged NAP2 protein where the NAP2 protein is at zero which is this message on the binary protein you see that at zero time point it is primarily in the nucleus right you see the nucleus staining but if you look at one hour at the non permissive temperature you can see very much that the nuclear are spared the nucleus are very small so here is the nucleus right here here this would be this nucleus it is spared and also here it is spared at the non permissive temperature the the beta 2 is unable to import this message on a binary protein but it has no effect on a green fluorescent protein which is coupled to a classical NLS or to an NLS1 which would be imported by the alpha beta 1 pathway and you can see even at three hours at the non permissive temperature this particular substrate is still imported into the nucleus so it is very clear that this is another pathway which brings a subset of nuclear proteins in this case a messenger RNA binary proteins into the nucleus I want to stress however that not all messenger RNA binary proteins use this pathway because from the tremendous work that the hidden dry foods has done we know that there are also among the 20 or so messenger RNA binary proteins that have to characterize there are also some which have classical NLS1 type nuclear localization signals and so they probably can go in by the alpha than a one pathway but this has to be determined in detail for each of these proteins in the future now so here that what we have in summary is unlike the alpha beta 1 pathway where we have the beta docking directly the beta 1 docking to the nuclear pore complex and the alpha serving as a mediator for binding to the NLS1 containing substrate in this case we have the beta 2 binds to the nuclear pore complex directly into the substrate as well directly there is no mediation by an alpha sub unit involved in this case and this is also the case in the other two carrier fans beta 3 and beta 4 which I will briefly describe they also bind directly to the substrate and bind directly to the nuclear pore complex and there is no alpha sub unit involved by people that speculate in evolution what the meaning of this is but I don't want to take up too much time on this we can perhaps discuss in the discussion section now this is a couple of mammalian beta 2 experiments that we have done nearest Boniface has independently of hidden drive was also planned in mammalian proteins just going to be published very briefly and you can see the chirophera and beta which is bound to fluorescently labeled GST protein containing the chirophera protein A1 can dock to the periphery of the nuclear pore complex when you do the reaction at zero degrees if you do the reaction at room temperature the beta 2 can bring the protein into the nucleus but the run if you add run GDP you can see that you get much more increase important in the nucleus and what is particularly striking that the nucleoli are not occupied by this by this particular substrate and run GMPP and P inhibits this process and the run which doesn't have a large GDP also inhibits this process now we found that you need run only if you use higher concentrations of beta 2 such as 2 micrograms if you use lower concentrations of beta 2 like 0.5 microgram there is no difference between plus, minus run and plus run and it could be that in this case there is enough endogenous runs to do the important that when we do the higher concentration of beta we need exogenously added run but all of these things need to be figured out because it is clear that beta 2 does not bind run GDP directly as does beta 1 so we have to find out who brings the run GDP into the vicinity of beta 2 and what actually happens precisely these are all things which remain to be figured out in the future now this is just again I will skip this it binds to various nuclear plants and this is a competition experiment where we use the NLS1 substrate going in this beta 1 of run and P10 and we show that in the presence in the absence of beta 2 you have a size import but in the presence of beta 2 you have competition for import and the same is true with the other pathway if you have the GST A1 labeled and you take beta 2 and run you get in the absence of beta 1 no inhibition of import but in the presence of beta 1 you get inhibition of import and this is explained by competing that all of these better segments may compete on the nuclear points because they all bind to this repeat containing nuclear points but of course you know we are far from understanding what precisely is going on we obviously have to do mechanics here we have done something but of course again it will take too much time to go into detail so I will move on and we will quickly discuss now some other career fans in the beta 3 and beta 4 and here again the same strategy beta 4 is carb-123p again work of Mike Raul and John H. attack with protein A and the same strategy and what you see with the magnesium chloride gradient eventually because you allude the beta 4 the carb-123 and you allude also in this 500-day model region of magnesium a bunch of specific proteins and if you overload the gradient and blow this up a little bit you can see that there are actually quite a few proteins in there and we have done sequencing and mass spectrometry and they all turned out to be ribosomal proteins so what beta 4 is doing it is transported for ribosomal proteins it brings in the ribosomal proteins into the nucleus and again this was demonstrated by the usual experiments which one can do in yeast in vivo experiments that it is specific for ribosomal proteins it does not affect the import of other proteins messenger RNA binding proteins for instance or the NMS1 type containing proteins so it is really specific for these ribosomal proteins now the very big shock was when we knocked out the gene and found that this is not lethal so I mean how can that a ribosomal transporter is not required for survival that of course makes no sense so what they might have found is that cup 121 or previously also isolated as PSE1 as promoter of enhancer of secretory of protein secretion somehow this protein was thought to be involved in protein secretion enhancement but now of course we know that it is a chirophera which can bring in ribosomal proteins to the nucleus when you knock out beta 4 so beta 3 can substitute for beta 4 and when you knock out beta 3 it is lethal because beta 3 must be doing some very critical proteins of course if you knock closer obviously if you knock closer it will not tell you the truth so this brings up already sort of one of the dilemmas which we are facing namely that there may be overlap in the recognition of various substrates in these various transport pathways and that these cup 123 or the beta 4 may have developed in yeast to deal with a very large volume of ribosomal protein imported as required what Mike Routt and John Asians calculated is that knowing the number of ribosomes in yeast and knowing the generation time they calculated that there are 15 ribosomal proteins imported per second per nuclear core complex so this is only talking about ribosomal protein import not about messenger and RNA transcription factors and all sorts of things but this is a very highly active transport organa 15 per second is very respected for this sort of thing now of course they have also shown in the control that E. colar ribosomal proteins for instance do not bind at all in the overlay as well as the yeast ribosomal proteins too and in fact it turned out that the ribosomal proteins of yeast are all larger because they all have to of all eucalyptid ribosomal proteins have to carry a nuclear localization signal which E. colar ribosomal proteins don't have to carry and so the ribosomal proteins are larger in all eucalyptic cells because they have to carry an NLS to get the protein into the nucleus and the ribosomal gene is larger in eucalyptid ribosomal proteins one perspective some people may disagree this is a simple analysis of things but in any case here a sky of N better too, again like better too it binds directly to this repeat containing a few components and to the substance now the story is still not finished because further inspection of this other band we have at least 10 proteins which we think are carypherin and we are visiting identifying those substrates we have identified some substrates for them and so they indicate that there are other pathways for tRNA for instance for SNERF proteins and so on which are using separate and specific pathways and there are other carypherin and of course the fascinating question based on these results is that these pathways can be independently controlled and so the cell has a way to control the import of these various nuclear proteins in the cell at work and this is going to be a very exciting topic for the future but now I want to quickly go into what I think is even a more exciting topic perhaps for the future and that is intra-nuclear transport and people have always thought that intra-nuclear transport is just out of this mass of to the viscous mass of chromatin there were some people who have calculated that certain large particles in the viscous movement like the nucleus wouldn't diffuse very far the rate would be very very slow and so I have always been attracted by the idea that there may be a stationary transport substrates in the nucleus in the form of fibers or tubes and these stationary transport materials these fibers or tubes or whatever would be gaining the interior to the nuclear pore complex and it would be along these fibers the transport would take place and so here is a protein which Tom Mayer in our lab discovered a couple of years ago the NOP-140 is a nuclear protein I'm afraid if we don't turn on the light then we turn it off completely pitch black we don't see anything what Tom looked at is a label some immunogold electron microscopy on frozen sin sections and what you see here is very badly preserved because it's a frozen sin section you see the nuclear envelope here and you see the cytoplasm here and you see the nucleoplasm and this structure here is a nucleus and for those of you who sit in the front row I doubt that for those of you in the back row they would see it I don't know which light you turn on I'm afraid you once are you have to tip the right button maybe it's better to be visited now you see these gold particles here's one, here's one extending for a distance of microns from the nucleolus this is a nucleolus all the way to the periphery to the nuclear envelope we have actually some images where you can see these gold particles so the question is raised of whether in fact there are these curvilinear tracks and the question is raised about the transport intra-nuclear transport it's not just by diffusion in some sort of unstructured nucleoplasm but there are some stationary transport phases, fibres or tubes and what I was most fascinating by today is when Hans Wies was able to show me his pictures which he has taken and I have to ask him to give a seminar very urgently these are among the most beautiful pictures I have seen of the interior of the nucleus radio in fact C85 is emanating from the nuclear transport complex from microns into the interior of the nucleus forming actually what one could call a subway terminal system in which he has shown in some collaboration with Hans Wiedlund may actually and Jim Barber may actually be involved in transporting things to the nuclear transport complex so these structures will be very important and the question now what are these structures there is a very fascinating protein which has been discovered some time ago called TPR which stands for I forget what it stands for and this protein has been initially localized to the cytoplasm but one that was correctly localized by Volker and Cordes in Van der Franke to the nucleoplasm forming fibres which extend from the nuclear transport complex into the interior of the nucleus so the challenge now is to show whether the fibres that Hans Wies sees these 8 fibres which emanate from the fish trap as he has called from the terminal ring of the fish trap into the nucleus whether these in fact are TPR or whether they are TPR plus something else I suspected they are TPR and something else now what is very interesting about this protein is that it has a very large column of 1,500 amino acids so it could potentially form a cold cold of something like 150 nanometers but I think the periodicity of the challenge Wies has seen is only 50 nanometers so that there may be not the entire protein but as you see I'm speculating and waiting in my hands but this is very exciting and it will probably give us a tremendous number of clues for the inter-nuclear transport now my ground in Karol Wiener's zombie of the Pastilla have found yeast homologs of these proteins trying to pull out the active proteins and so on and so on doing the same strategy that we have used for the better samples so here is then what I'm saying is that these fibers probably in this cartoon which is sort of pressuring the continuum into the interior of the nucleus so that the space inter-nuclear space is some sort of tubular system not necessarily continuous indicated here but perhaps open-ended in the cytoplasm where then which serve as stocking sites for a two-dimensional diffusion if you wish along these tracks of fibers in export or import into the nucleus so the inter-nuclear phase of transport is going to be a very fascinating one now I won't throw very much into this thing about export because time is running out but what we think is that the chiropherin alpha which goes into the nucleus may also serve in taking out NLS-containing substrates from the nucleus by a different mechanism this mechanism does not involve chiropherin battle but it may and again it wouldn't take too long to go into the detail so that you actually could have a merry-go-round of certain NLS-containing substrates and the only way you can you would prevent this merry-go-round is if you anchor the substrates either in the nucleus or in the cytoplasm and NF-copper B points is a classical example of how you can anchor this protein in the cytoplasm as you know the NLS-1 of NF-copper B which is a transcription factor is covered by I-copper B and you have to first phosphorylate I-copper B then the ubiquitinated then degraded and then the NLS of this transcription factor is available for nuclear import and then somehow in the nucleus the NLS is probably inactivated because it's part of a DNA binding site and so therefore only when you take it off it probably would be subject to export so you can immobilize the protein by anchoring it in let's say chromatin or some other nuclear substrates and you can immobilize it in the cytoplasm in this case by this very complex mechanism and last slide I just would like to come back to the pictures which I showed to you early on from Houston Swift in that you have these very large messenger-on-p particles which need to be unfolded while they're going across the nuclear cortex and what we suggest is that of course these very large messenger-on-p's will have many proteins bound to them all of which may have a nuclear localization signal because we don't know of what type NLS 1, 2, 3, 4 it means to be figured out what type of proteins are the critical ones and it is with these analysis that and the corresponding chirophane and bettas that you can then be docked to these nuclear core complex repeats and this docking would be, this unfolding would be initiated at the 5 prime N perhaps via the carbide and proteins and therefore you could explain why the 5 prime N would come out first, it would be initiated at that point and then it would proceed further downstream from there by multiple docking to these multiple docking sites of these repeat container nuclear points you would convert across sounds or to speak in a millipede and you would therefore allow its diffusion across the center of the tube but this is for its wild speculation and this is just a model and at this moment it became other models but this this is I think all the slides which I have to present so just in summary, we have a long way to go to understanding the structure of the nuclear core complex this will be a challenge which will go on for a long time Mike Raug and Chris Ackier doing vitreous eye-selecting microscopy we have now a resolution which the thing is down to 25 angstrom and we are eagerly awaiting their new models on these isolated core complexes but again, the complication there as Hans Ries has pointed out when you isolate nuclear core complex or when you take nuclear interlocks these fibers collapse upon the structure in addition you have carrier fan balance plus transport substrate bound to the fibers so you get a harm of a mess which is very difficult to sort out so ideally what you would like to look at, you would like to look at a core complex which perhaps doesn't have any fibers in which perhaps doesn't have any transport substrate bound to them and then you would get an idea of what the core of the core complex looks like and you would like to study the fibers independently and independently of the transport substrate so you can see that in spite of the efforts of very powerful electron structural biologists we are at a very low resolution of the nuclear core complex and that is of course the key if we want to understand our transport works and if we want to develop hypotheses of how the transport works now in transport factors we thought it would be a simple story and we ended up with this multitude of transport factors and transport pathways distinct pathways and this would also take a long time to all figure out and it would be a challenging it would be a great challenge to figure out how these pathways are all regular and then I mentioned briefly inter nuclear transport will play a very big role and then I have not discussed much in export I just wanted to say that there is a signal which has been called nuclear export signal NES which is presumably involved but other than identifying such an NES there is not much more progress of how this NES works it is also possible that the NES is actually not a signal sequence by itself but is a piggy bagging sequence which attaches these proteins to bona fide 1, 2, 3, 4, 5 whatever containing proteins that the export of these NES containing proteins is actually not via direct interactions export factors but with some other factors so in summary you see that this is a field which is very much a turmoil and a very lot of hand waving but for these reasons it is very exciting because as of this moment there are several hypotheses and everybody has their own favorite ones how it works and this is good in a way sort of like coming back to Mao Zedongi in some other fashion instead of let a thousand flowers bloom and let a thousand hypothesis bloom eventually one of them will be supported by enough data several want to be supported by enough data so that we can acquire credibility and so this is giving you this lecture was trying to give you a short overview over where we stand in understanding this whole thing and I apologize for those of you who are special as they learned and they were hearing a lecture which they all were worried about and there was not much new very much like the lecture I gave yesterday which wasn't that much new either in other words I refrain from showing our latest sort of data slides on that but it was more important for me to get across the concepts and for those people who do not work in the field and for those people who work in the field I'm happy to go and discuss details but thank you very much for your attention I'm sorry


Here's the three things you have to do in this world. I'll show you the specific tactics I would use. The first thing you have to do, adapt, adopt, and persist. People don't make money because, A, they're not adaptable. They're like, my mom and dad told me this is how you make money and I'm gonna stick to it. So they're not adaptable. They're not adaption evolutionary machine. Number two, adopt. Meaning basically copy what's working for other people. Pablo Picasso said, good artist copy, great artist steal. Okay? So you find somebody who's making money in real estate. You find somebody who's making money with social media and you know what you do? You literally adopt, clone their exact system. And then the last thing is persist. Like people give up too easy. Now like TikTok and Instagram reels it's like three second attention span. Something doesn't catch people's attention three seconds they're moving on. People aren't persistent like they used to be. So adapt, adopt, and persist.


The UEFA Europa Conference League quarter-final dream continues for Victoria Pleasant and Florentina. See this first leg match on Thursday, April 11, at 11.45am for the Eastern Caribbean, 10.45pm in Jamaica. It's on SportsMax Plus, on the app. Your game, your way, get the SportsMax app today.