1 00:00:17,040 --> 00:00:21,160 BARBARA IMPERIALI: I always like to just remind you that 2 00:00:21,160 --> 00:00:22,620 the sixth -- 3 00:00:22,620 --> 00:00:24,810 it's kind of an assignment, but the numbers-- 4 00:00:24,810 --> 00:00:28,260 we're going to do this news brief project, where 5 00:00:28,260 --> 00:00:30,930 it's a teamwork project if you choose. 6 00:00:30,930 --> 00:00:35,030 If you take a look at the piece that you have in your hands 7 00:00:35,030 --> 00:00:38,190 now, it asks you for a little bit of information 8 00:00:38,190 --> 00:00:40,710 on that, who you're going to be working with, 9 00:00:40,710 --> 00:00:42,720 if you choose to work with someone. 10 00:00:42,720 --> 00:00:44,110 Or you can work on your own. 11 00:00:44,110 --> 00:00:45,137 That's fine. 12 00:00:45,137 --> 00:00:46,970 And we're looking to get a news brief that's 13 00:00:46,970 --> 00:00:51,510 of significance to research going on in the life sciences. 14 00:00:51,510 --> 00:00:53,580 And I've given you-- there are a couple of links 15 00:00:53,580 --> 00:00:56,880 in the sidebar of the website, so good places where you 16 00:00:56,880 --> 00:00:59,040 can find interesting material. 17 00:00:59,040 --> 00:01:02,150 What I'm super interested in for you, 18 00:01:02,150 --> 00:01:05,930 as a group where many of you are in the engineering fields, 19 00:01:05,930 --> 00:01:08,760 is to find something really cool at the interface 20 00:01:08,760 --> 00:01:11,240 between the life sciences and engineering, 21 00:01:11,240 --> 00:01:15,240 where engineering has a huge impact on the life sciences. 22 00:01:15,240 --> 00:01:16,810 You have alternatives. 23 00:01:16,810 --> 00:01:19,500 You can download the coordinates of a protein 24 00:01:19,500 --> 00:01:22,470 and print it on a 3D printer and give us 25 00:01:22,470 --> 00:01:25,360 a summary of what the protein is, what it does, 26 00:01:25,360 --> 00:01:27,420 and submit your 3D print. 27 00:01:27,420 --> 00:01:28,920 I'll give it back to you afterwards, 28 00:01:28,920 --> 00:01:30,240 once we've had a look at it. 29 00:01:30,240 --> 00:01:33,180 But actually submit the 3D print. 30 00:01:33,180 --> 00:01:35,160 And then the other opportunity is-- 31 00:01:35,160 --> 00:01:37,290 I think you'll remember back to when 32 00:01:37,290 --> 00:01:40,600 we were talking about molecular biology of the cell. 33 00:01:40,600 --> 00:01:43,080 I did kind of a clunky demo at the front 34 00:01:43,080 --> 00:01:45,540 of the class, nothing like Professor Martin's demos 35 00:01:45,540 --> 00:01:46,440 at all. 36 00:01:46,440 --> 00:01:49,230 This was me with the ethernet cables showing you 37 00:01:49,230 --> 00:01:51,270 what topoisomerase did. 38 00:01:51,270 --> 00:01:53,250 But in my demo, I didn't show you 39 00:01:53,250 --> 00:01:56,850 how topo also cuts a strand of DNA, 40 00:01:56,850 --> 00:01:59,940 holds it while the supercoiling unwinds, 41 00:01:59,940 --> 00:02:01,490 and then stitches it together. 42 00:02:01,490 --> 00:02:03,007 So I thought some of the engineers 43 00:02:03,007 --> 00:02:04,590 might be able to come up was something 44 00:02:04,590 --> 00:02:07,210 that was really better than that for me to use, 45 00:02:07,210 --> 00:02:09,310 for us to use, next year in class. 46 00:02:09,310 --> 00:02:13,680 So I'm really laying down the challenge there. 47 00:02:13,680 --> 00:02:16,470 So I always like things in the news. 48 00:02:16,470 --> 00:02:18,120 I thought this was kind of interesting 49 00:02:18,120 --> 00:02:21,630 that the first vertebrates evolved in shallow waters. 50 00:02:21,630 --> 00:02:25,470 I thought those were really cool first vertebrates. 51 00:02:25,470 --> 00:02:27,900 I'd love to get one of them in a fish tank and keep it. 52 00:02:27,900 --> 00:02:32,270 But anyway, that's that. 53 00:02:32,270 --> 00:02:35,730 It's truly amazing what you can see in the science 54 00:02:35,730 --> 00:02:37,710 reports, news briefs. 55 00:02:37,710 --> 00:02:39,660 I look at them whenever they come in. 56 00:02:39,660 --> 00:02:41,940 I get the posts every two or three days. 57 00:02:41,940 --> 00:02:43,920 And I'm kind of pleased to see that there's 58 00:02:43,920 --> 00:02:46,890 a lot of things that are in those news briefs 59 00:02:46,890 --> 00:02:49,260 that I feel that we're enabling you to read 60 00:02:49,260 --> 00:02:51,570 with some appreciation because of what 61 00:02:51,570 --> 00:02:53,440 we're covering in the class. 62 00:02:53,440 --> 00:02:56,310 So what we're doing now is we're really taking a leap forward 63 00:02:56,310 --> 00:03:00,000 here into cells and organisms, with respect 64 00:03:00,000 --> 00:03:10,140 to understanding how structure and function 65 00:03:10,140 --> 00:03:14,130 of individual macromolecules, proteins, nucleic acids, 66 00:03:14,130 --> 00:03:18,420 sugars, determine life, determine the dynamics of life 67 00:03:18,420 --> 00:03:22,530 that are necessary for an organism to really go 68 00:03:22,530 --> 00:03:25,920 through a life cycle, divide, have cells divide, 69 00:03:25,920 --> 00:03:27,660 go forward, have cells move. 70 00:03:27,660 --> 00:03:29,070 So what we're going to be talking 71 00:03:29,070 --> 00:03:32,400 about in the next lectures, which is section 6, 72 00:03:32,400 --> 00:03:34,680 is cellular trafficking and signaling. 73 00:03:34,680 --> 00:03:38,370 And so for the first lecture, which is 19 that we're on now-- 74 00:03:38,370 --> 00:03:40,680 so we're past the midway mark-- 75 00:03:40,680 --> 00:03:42,600 I'm going to be talking about trafficking. 76 00:03:42,600 --> 00:03:44,730 And that is how, within a cell, things 77 00:03:44,730 --> 00:03:49,260 get to where they need to be, or they get exported from a cell. 78 00:03:49,260 --> 00:03:51,690 Because all of the actions of a cell-- 79 00:03:51,690 --> 00:03:54,180 I really like thinking about the cell 80 00:03:54,180 --> 00:03:56,910 as a circuit board, where there's a receiver that 81 00:03:56,910 --> 00:03:58,530 gets information. 82 00:03:58,530 --> 00:04:03,000 And then the complex circuitry determines what outcome 83 00:04:03,000 --> 00:04:04,740 you get at the end of the day. 84 00:04:04,740 --> 00:04:07,680 So many of the proteins that we've talked about 85 00:04:07,680 --> 00:04:12,300 need to be in specific places for the cell to function. 86 00:04:12,300 --> 00:04:15,820 We have to have DNA polymerase in the nucleus. 87 00:04:15,820 --> 00:04:18,730 It's not going to be useful in the cytoplasm. 88 00:04:18,730 --> 00:04:24,270 We have to have a transcription factor that helps transcription 89 00:04:24,270 --> 00:04:27,540 go to the nucleus at the right time for transcription 90 00:04:27,540 --> 00:04:28,530 to occur. 91 00:04:28,530 --> 00:04:30,330 But we don't want it there all the time, 92 00:04:30,330 --> 00:04:32,205 because otherwise you'd have the light switch 93 00:04:32,205 --> 00:04:33,600 on the entire time. 94 00:04:33,600 --> 00:04:34,620 That wouldn't be useful. 95 00:04:34,620 --> 00:04:39,060 So we need to regulate where certain macromolecules are. 96 00:04:39,060 --> 00:04:42,570 We need to have the receivers on the surface of the cell 97 00:04:42,570 --> 00:04:45,010 to receive signals from outside. 98 00:04:45,010 --> 00:04:48,760 This is not just pertinent for multicellular organisms. 99 00:04:48,760 --> 00:04:51,420 It's pertinent for unicellular organisms, 100 00:04:51,420 --> 00:04:53,700 for them to sense their environment, 101 00:04:53,700 --> 00:04:55,640 know what's going on around them. 102 00:04:55,640 --> 00:04:57,690 Is the salt concentration changing? 103 00:04:57,690 --> 00:04:58,830 Is it getting very hot? 104 00:04:58,830 --> 00:05:00,240 Is it getting cold? 105 00:05:00,240 --> 00:05:02,010 Is there enough oxygen? 106 00:05:02,010 --> 00:05:05,910 Even unicellular organisms need to receive signals and respond 107 00:05:05,910 --> 00:05:06,780 to them. 108 00:05:06,780 --> 00:05:10,950 Multicellular organisms are way more complicated. 109 00:05:10,950 --> 00:05:14,010 Because you need to establish organs 110 00:05:14,010 --> 00:05:16,770 and different parts of a multicellular organism 111 00:05:16,770 --> 00:05:18,630 that have specialized function. 112 00:05:18,630 --> 00:05:21,540 So trafficking really is about what 113 00:05:21,540 --> 00:05:26,220 happens after you've made a replicated DNA in the nucleus, 114 00:05:26,220 --> 00:05:30,660 transcribed it, made a mature messenger that goes out 115 00:05:30,660 --> 00:05:32,520 to the cytoplasm in most cases. 116 00:05:32,520 --> 00:05:35,070 We'll talk about the exceptions to that case. 117 00:05:35,070 --> 00:05:38,250 And then in the cytoplasm, when proteins are expressed, 118 00:05:38,250 --> 00:05:40,800 all the different things that happen that 119 00:05:40,800 --> 00:05:44,070 guarantee that the protein gets to a proper destination 120 00:05:44,070 --> 00:05:45,270 for function. 121 00:05:45,270 --> 00:05:47,400 And some of those are quite complicated. 122 00:05:47,400 --> 00:05:49,620 Because remember, if I'm going to park 123 00:05:49,620 --> 00:05:52,470 a receiver in the cellular membrane 124 00:05:52,470 --> 00:05:55,320 with the signals being captured from outside, 125 00:05:55,320 --> 00:05:56,910 I've got to get from the cytoplasm 126 00:05:56,910 --> 00:06:00,780 out there in a reliable way. 127 00:06:00,780 --> 00:06:04,470 In lectures 20 and 21, I'll talk to you 128 00:06:04,470 --> 00:06:08,610 about cellular signaling with a focus on mammalian cells 129 00:06:08,610 --> 00:06:10,890 and the sorts of signaling processes 130 00:06:10,890 --> 00:06:15,600 that may go awry in cells, for example, proliferating cells. 131 00:06:15,600 --> 00:06:17,820 And then Professor Martin will really 132 00:06:17,820 --> 00:06:22,620 focus in on neuronal cells, optogenetics in lecture 22. 133 00:06:22,620 --> 00:06:26,280 So this bundle really allows you to call in the things 134 00:06:26,280 --> 00:06:28,740 that you've learned until now and apply them 135 00:06:28,740 --> 00:06:32,280 into much more intriguing and complex situations. 136 00:06:32,280 --> 00:06:34,380 So here's a wonderful, sort of silly drawing 137 00:06:34,380 --> 00:06:36,390 of a triangular cell. 138 00:06:36,390 --> 00:06:41,490 There's always a joke in cell biologists, 139 00:06:41,490 --> 00:06:45,300 when they're trying to talk to mathematicians 140 00:06:45,300 --> 00:06:48,000 and mathematicians want to simplify everything. 141 00:06:48,000 --> 00:06:50,040 And so everything gets-- imagine a cell, 142 00:06:50,040 --> 00:06:52,470 and there's this box shows up on a screen. 143 00:06:52,470 --> 00:06:54,540 Well, we all know that cells aren't 144 00:06:54,540 --> 00:06:56,310 triangular or box-shaped. 145 00:06:56,310 --> 00:06:59,310 But nevertheless, I thought this one was particularly cool. 146 00:06:59,310 --> 00:07:02,730 And so trafficking, the process of trafficking, 147 00:07:02,730 --> 00:07:07,770 is really all about, where is the information encoded 148 00:07:07,770 --> 00:07:11,670 into the protein that ensures that the protein is where 149 00:07:11,670 --> 00:07:16,920 it needs to be for the dynamics that we observe 150 00:07:16,920 --> 00:07:19,930 in living system? 151 00:07:19,930 --> 00:07:22,020 We've talked a lot about static things. 152 00:07:22,020 --> 00:07:23,268 We make the protein. 153 00:07:23,268 --> 00:07:24,060 Here's the protein. 154 00:07:24,060 --> 00:07:25,410 The protein folds. 155 00:07:25,410 --> 00:07:28,470 We've talked a lot about things that are kind of fixed 156 00:07:28,470 --> 00:07:29,590 in time and space. 157 00:07:29,590 --> 00:07:31,380 But what we want to do is understand 158 00:07:31,380 --> 00:07:35,760 what makes a cell programmed to undergo a new function. 159 00:07:35,760 --> 00:07:38,580 For example, something as simple as cell division, 160 00:07:38,580 --> 00:07:43,800 we have to orchestrate a huge variety of activities in order 161 00:07:43,800 --> 00:07:46,770 for the cell division process to start to occur. 162 00:07:46,770 --> 00:07:50,190 Something as really simple a cell mobility, think about, 163 00:07:50,190 --> 00:07:52,230 how do cells move? 164 00:07:52,230 --> 00:07:54,540 They're not moving all the time, but sometimes they 165 00:07:54,540 --> 00:07:56,190 will move towards a signal. 166 00:07:56,190 --> 00:07:58,690 What triggers that kind of interactions? 167 00:07:58,690 --> 00:08:02,580 So in looking at the cell, these are some of the older images, 168 00:08:02,580 --> 00:08:06,120 where certain organelles, for example, 169 00:08:06,120 --> 00:08:08,610 are stained so that you can see them. 170 00:08:08,610 --> 00:08:12,030 So peroxisomes are where degradation happens. 171 00:08:12,030 --> 00:08:14,340 The golgi and the ER are a part of what's 172 00:08:14,340 --> 00:08:16,890 known as the endomembrane system. 173 00:08:16,890 --> 00:08:19,980 You'll see a lot about this towards the later part 174 00:08:19,980 --> 00:08:22,470 of the class, where we talk about how 175 00:08:22,470 --> 00:08:26,310 things get outside the cell through the endomembrane 176 00:08:26,310 --> 00:08:27,210 system. 177 00:08:27,210 --> 00:08:29,850 There's the surface plasma membrane. 178 00:08:29,850 --> 00:08:34,470 The cytoplasm is this sort of not really aqueous-- 179 00:08:34,470 --> 00:08:35,850 it's an open space. 180 00:08:35,850 --> 00:08:37,200 But it really isn't open. 181 00:08:37,200 --> 00:08:39,870 It's highly congested with all kinds 182 00:08:39,870 --> 00:08:43,780 of molecules, all kinds of structural proteins and so on. 183 00:08:43,780 --> 00:08:46,950 So don't think of the cytoplasm as a solution, 184 00:08:46,950 --> 00:08:50,520 but think of it as a much more gel-like structure with a lot 185 00:08:50,520 --> 00:08:53,400 of things happening in it. 186 00:08:53,400 --> 00:08:57,330 The nucleus itself is also surrounded by a membrane, 187 00:08:57,330 --> 00:08:59,380 as is the endomembrane system. 188 00:08:59,380 --> 00:09:01,530 So this would be the nuclear envelope. 189 00:09:01,530 --> 00:09:03,480 Within the nucleus, you have a structure 190 00:09:03,480 --> 00:09:06,360 called the nucleolus, where aspects 191 00:09:06,360 --> 00:09:11,310 of the nucleic acids necessary for protein biosynthesis 192 00:09:11,310 --> 00:09:12,060 are made. 193 00:09:12,060 --> 00:09:13,620 Then there are structural proteins 194 00:09:13,620 --> 00:09:16,480 like microtubules and actin. 195 00:09:16,480 --> 00:09:18,360 But now, in this day and age, we don't 196 00:09:18,360 --> 00:09:20,940 have to deal with these vanilla images. 197 00:09:20,940 --> 00:09:23,070 We can actually use the methods that you've 198 00:09:23,070 --> 00:09:27,000 learned about in the last section, recombinant biology, 199 00:09:27,000 --> 00:09:30,070 to create new versions of proteins that 200 00:09:30,070 --> 00:09:34,550 have along with their sequence a marker that gives them 201 00:09:34,550 --> 00:09:36,600 a fluorescence-colored marker. 202 00:09:36,600 --> 00:09:38,700 So we are, later on in the semester, 203 00:09:38,700 --> 00:09:42,180 going to spend three lectures on fluorescence and cellular 204 00:09:42,180 --> 00:09:46,890 imaging, where you'll learn more about these fabulous proteins 205 00:09:46,890 --> 00:09:50,190 beyond just saying we've got a green one and a red one. 206 00:09:50,190 --> 00:09:52,770 We're going to give you all the background on the protein 207 00:09:52,770 --> 00:09:56,695 engineering that enabled those to become tools for biology. 208 00:09:56,695 --> 00:09:58,320 But for now, I'm just going to show you 209 00:09:58,320 --> 00:10:01,920 how much more interesting the images of the subcellular 210 00:10:01,920 --> 00:10:04,290 structures are when you've labeled, 211 00:10:04,290 --> 00:10:08,550 for example, a particular protein that goes exclusively 212 00:10:08,550 --> 00:10:12,120 to the nucleolus with a blue fluorescent protein, 213 00:10:12,120 --> 00:10:13,680 or to the mitochondria. 214 00:10:13,680 --> 00:10:15,700 Remember, Professor Martin told you 215 00:10:15,700 --> 00:10:17,950 we always think of these as-- and I'm not 216 00:10:17,950 --> 00:10:19,180 going to do the push-up. 217 00:10:19,180 --> 00:10:22,210 I'm just going to say it, powerhouse of the cell. 218 00:10:22,210 --> 00:10:26,920 I'm not doing-- [LAUGHS] I'm not great with push-ups, 219 00:10:26,920 --> 00:10:28,480 to be honest. 220 00:10:28,480 --> 00:10:32,650 But you see these sort of more tangled, extended structures. 221 00:10:32,650 --> 00:10:35,410 Vimentin is more of a structural protein. 222 00:10:35,410 --> 00:10:38,560 Here are the golgi, the endoplasmic reticulum, 223 00:10:38,560 --> 00:10:39,800 and the nucleus. 224 00:10:39,800 --> 00:10:43,150 So the colored fluorophore proteins, 225 00:10:43,150 --> 00:10:45,520 or the fluorescent fluorophore proteins, 226 00:10:45,520 --> 00:10:50,290 actually allow us, in real time, to observe dynamics. 227 00:10:50,290 --> 00:10:53,650 Once a protein is made, where does it go? 228 00:10:53,650 --> 00:10:58,300 If we add a trigger to the cell to cause an interaction, 229 00:10:58,300 --> 00:11:01,000 can we observe that protein, for example, 230 00:11:01,000 --> 00:11:03,280 migrating to the plasma membrane. 231 00:11:03,280 --> 00:11:06,670 Can we watch proteins being made through the ER? 232 00:11:06,670 --> 00:11:10,360 A variety of different things that allow us in modern biology 233 00:11:10,360 --> 00:11:15,110 to really look at dynamics, not just static information. 234 00:11:15,110 --> 00:11:18,010 And so what I'm going to talk to you about 235 00:11:18,010 --> 00:11:20,890 is the ways in which proteins are 236 00:11:20,890 --> 00:11:25,870 coded very early on in their genesis, in their biogenesis, 237 00:11:25,870 --> 00:11:29,860 in order to go to certain locales within the cell. 238 00:11:29,860 --> 00:11:34,540 So let me just give you a bit of a road map here with a protein. 239 00:11:38,590 --> 00:11:40,710 And where things may start-- 240 00:11:40,710 --> 00:11:42,410 so we have some options. 241 00:11:42,410 --> 00:11:46,860 Do we want to send the protein outside the cell 242 00:11:46,860 --> 00:11:51,660 or keep it inside the cell? 243 00:11:51,660 --> 00:11:54,690 Obviously, two big default differences, if you're 244 00:11:54,690 --> 00:11:58,770 going to go to a particular venue inside the cell. 245 00:11:58,770 --> 00:12:01,840 Are we going to just stay in the cytosol? 246 00:12:05,030 --> 00:12:07,470 That's a sort of simple-- 247 00:12:07,470 --> 00:12:09,510 actually, that is the default position. 248 00:12:09,510 --> 00:12:12,960 Because you want to remember that most proteins 249 00:12:12,960 --> 00:12:16,530 are made on ribosomes in the cytosol of the cell. 250 00:12:16,530 --> 00:12:20,100 But the statistics are that about 50% of proteins 251 00:12:20,100 --> 00:12:23,070 end up somewhere else than the cytoplasm. 252 00:12:23,070 --> 00:12:25,410 They may end up in an organelle, back 253 00:12:25,410 --> 00:12:27,900 in the nucleus on the surface, or secreted. 254 00:12:27,900 --> 00:12:28,860 So there's a lot-- 255 00:12:28,860 --> 00:12:32,280 so it's a good, solid 50% that don't end up 256 00:12:32,280 --> 00:12:36,540 staying in the cytosol, where they were originally made. 257 00:12:36,540 --> 00:12:39,300 Their alternative is to go to organelles. 258 00:12:42,720 --> 00:12:45,600 And if you're going to an organelle, remember, 259 00:12:45,600 --> 00:12:48,900 the ribosome is not membrane. 260 00:12:48,900 --> 00:12:50,850 It doesn't have a membrane perimeter. 261 00:12:50,850 --> 00:12:54,360 But many of the organelles do have membrane perimeters. 262 00:12:54,360 --> 00:12:57,090 So we're talking here about the mitochondria. 263 00:13:06,830 --> 00:13:08,950 That is far too long of a word. 264 00:13:08,950 --> 00:13:12,280 The nucleus-- so I'm going to abbreviate things 265 00:13:12,280 --> 00:13:18,070 like peroxisomes, or various membrane-bordered organelles, 266 00:13:18,070 --> 00:13:20,470 where we're going to have to figure out, if something 267 00:13:20,470 --> 00:13:22,840 is made in the cytoplasm, how does it 268 00:13:22,840 --> 00:13:25,130 get into those organelles? 269 00:13:25,130 --> 00:13:27,310 Now we've spoken a little bit about the fact 270 00:13:27,310 --> 00:13:29,790 that some proteins are made in the mitochondria. 271 00:13:29,790 --> 00:13:31,790 I'm going to get back to that in a moment. 272 00:13:31,790 --> 00:13:34,030 But all the proteins in the mitochondria 273 00:13:34,030 --> 00:13:36,370 are not made in the mitochondria. 274 00:13:36,370 --> 00:13:38,230 Some of them are shipped in. 275 00:13:38,230 --> 00:13:42,130 Remember the thing the endosymbiont theory, 276 00:13:42,130 --> 00:13:44,410 where we said that mitochondria may 277 00:13:44,410 --> 00:13:49,330 have originated from bacteria and been engulfed into cells. 278 00:13:49,330 --> 00:13:53,260 Those bacteria obviously were originally self-sufficient. 279 00:13:53,260 --> 00:13:55,540 But a lot of the proteins that were expressed 280 00:13:55,540 --> 00:13:58,720 in the mitochondria were dispensed with, 281 00:13:58,720 --> 00:14:01,630 and mitochondria now use proteins 282 00:14:01,630 --> 00:14:04,330 that are encoded by the nuclear DNA rather 283 00:14:04,330 --> 00:14:05,680 than the mitochondrial. 284 00:14:05,680 --> 00:14:09,040 But to this day, some proteins remain encoded 285 00:14:09,040 --> 00:14:10,730 within the mitochondria. 286 00:14:10,730 --> 00:14:14,920 So these are opportunities for where that may be. 287 00:14:14,920 --> 00:14:19,150 And I'm going to talk very specifically about signals 288 00:14:19,150 --> 00:14:22,540 that can get proteins into the mitochondria 289 00:14:22,540 --> 00:14:24,130 and into the nucleus. 290 00:14:24,130 --> 00:14:26,710 And it turns out that the barriers 291 00:14:26,710 --> 00:14:29,200 around those organelles are pretty different. 292 00:14:29,200 --> 00:14:30,880 I'll come back to that in a second 293 00:14:30,880 --> 00:14:33,310 when we get on the next slide. 294 00:14:33,310 --> 00:14:35,980 With respect to going outside the cell, 295 00:14:35,980 --> 00:14:37,550 there are two options. 296 00:14:37,550 --> 00:14:41,890 One option is for the protein to remain in the plasma membrane 297 00:14:41,890 --> 00:14:45,860 but with part of its structure outside the cell. 298 00:14:45,860 --> 00:14:56,900 So the other option is for the protein actually 299 00:14:56,900 --> 00:15:00,680 to be spit out of the cell as a soluble entity that 300 00:15:00,680 --> 00:15:04,340 can travel around an organism, for example, in the bloodstream 301 00:15:04,340 --> 00:15:05,930 and go to a remote site. 302 00:15:05,930 --> 00:15:08,750 And that becomes very important in signaling. 303 00:15:08,750 --> 00:15:13,610 So we would call those proteins secreted and soluble. 304 00:15:13,610 --> 00:15:16,310 So these would be membrane-bound. 305 00:15:16,310 --> 00:15:18,920 These would end up being soluble proteins. 306 00:15:18,920 --> 00:15:22,010 Let's take a look at the structure of the cell 307 00:15:22,010 --> 00:15:25,500 and look at where these various components are. 308 00:15:25,500 --> 00:15:28,880 So if you see these dots, those are free ribosomes 309 00:15:28,880 --> 00:15:30,350 in the cytoplasm. 310 00:15:30,350 --> 00:15:33,930 They would start to express different proteins. 311 00:15:33,930 --> 00:15:36,500 A lot of proteins are expressed in the ribosome. 312 00:15:36,500 --> 00:15:39,170 But in some cases, proteins become 313 00:15:39,170 --> 00:15:42,590 expressed on ribosomes that are associated 314 00:15:42,590 --> 00:15:44,930 with the endoplasmic reticulum. 315 00:15:44,930 --> 00:15:47,210 And therefore, you start a process 316 00:15:47,210 --> 00:15:50,540 whereby proteins end up being shipped 317 00:15:50,540 --> 00:15:52,410 to the outside of the cell. 318 00:15:52,410 --> 00:15:56,740 So where you see the speckles here, 319 00:15:56,740 --> 00:15:58,930 the free ribosome, and then the ribosomes 320 00:15:58,930 --> 00:16:02,200 bound to the rough endoplasmic reticulum, here, 321 00:16:02,200 --> 00:16:05,110 your destinies are on the right-hand side 322 00:16:05,110 --> 00:16:06,220 of that picture. 323 00:16:06,220 --> 00:16:08,920 And here, the destiny of these proteins 324 00:16:08,920 --> 00:16:12,190 ends up on the left-hand side of this sort of family tree 325 00:16:12,190 --> 00:16:13,580 that I'm showing you. 326 00:16:13,580 --> 00:16:16,150 There's obviously one more place where proteins are made, 327 00:16:16,150 --> 00:16:18,040 and that's in the mitochondria. 328 00:16:18,040 --> 00:16:21,130 And if you remember the first question on your exam, 329 00:16:21,130 --> 00:16:24,520 it described the DNA that's in the mitochondria. 330 00:16:24,520 --> 00:16:26,560 Going back to the endosymbiont theory, 331 00:16:26,560 --> 00:16:29,410 that's a circular piece of DNA. 332 00:16:29,410 --> 00:16:30,970 And it sets it apart. 333 00:16:30,970 --> 00:16:33,000 And the ribosomes in the mitochondria 334 00:16:33,000 --> 00:16:35,800 look more like bacterial ribosomes than you 335 00:16:35,800 --> 00:16:36,970 eukaryotic ribosomes. 336 00:16:36,970 --> 00:16:39,010 So remember, all along, we're going 337 00:16:39,010 --> 00:16:41,800 to try in the second half of the course 338 00:16:41,800 --> 00:16:44,380 to bring back knowledge we've taught you, 339 00:16:44,380 --> 00:16:46,390 but sort of, in a sense, endlessly 340 00:16:46,390 --> 00:16:49,270 remind you to keep the big picture in mind. 341 00:16:49,270 --> 00:16:51,670 Because we've already spoken to you about it. 342 00:16:57,580 --> 00:17:01,030 So this now is a nice pictorial vision 343 00:17:01,030 --> 00:17:02,920 of what I've just described to you. 344 00:17:02,920 --> 00:17:05,560 And I'm going to first of all talk about proteins 345 00:17:05,560 --> 00:17:07,680 that are made in the cytoplasm and may 346 00:17:07,680 --> 00:17:10,980 be shipped to various organelles, 347 00:17:10,980 --> 00:17:12,940 and how that's accomplished. 348 00:17:12,940 --> 00:17:14,980 And then in the second part of the class, 349 00:17:14,980 --> 00:17:18,250 I'll talk about how proteins are shipped to cell surface, 350 00:17:18,250 --> 00:17:20,290 or through expulsion from the cell. 351 00:17:22,810 --> 00:17:26,349 So the key mechanisms whereby proteins 352 00:17:26,349 --> 00:17:28,600 are trafficked to new locations are first 353 00:17:28,600 --> 00:17:41,710 of all using targeting sequences that are 354 00:17:41,710 --> 00:17:45,405 part of the protein sequence. 355 00:17:50,020 --> 00:17:54,340 And this is a very common way in which proteins are trafficked. 356 00:17:54,340 --> 00:17:55,940 They are part of the sequence. 357 00:17:55,940 --> 00:18:00,325 They may be at the amino or the carboxy terminus. 358 00:18:03,950 --> 00:18:06,810 But they are woven into the structure of your protein. 359 00:18:06,810 --> 00:18:10,730 So your protein comes along with a barcode saying where it's 360 00:18:10,730 --> 00:18:12,760 going to necessarily end up. 361 00:18:12,760 --> 00:18:28,430 And for the nucleus mitochondria and peroxisomes, 362 00:18:28,430 --> 00:18:32,690 for example, people have done extensive work 363 00:18:32,690 --> 00:18:37,550 with bioinformatics to basically look up protein sequences 364 00:18:37,550 --> 00:18:41,570 and find common themes of particular sequences 365 00:18:41,570 --> 00:18:44,900 that may be common to where a set of proteins may end up. 366 00:18:44,900 --> 00:18:46,670 Sometimes those sequences may not 367 00:18:46,670 --> 00:18:49,190 be easy to see just at first glance. 368 00:18:49,190 --> 00:18:52,640 But now there are websites that you can very, very readily 369 00:18:52,640 --> 00:18:55,970 put your protein sequence into the web site, and it will say, 370 00:18:55,970 --> 00:18:58,640 it's got a nuclear localization sequence, 371 00:18:58,640 --> 00:19:01,460 or a mitochondrial-targeting sequence. 372 00:19:01,460 --> 00:19:04,190 So we can either do this by eye or we 373 00:19:04,190 --> 00:19:06,380 can use informatics analysis. 374 00:19:06,380 --> 00:19:08,840 Informatics analysis is very valuable 375 00:19:08,840 --> 00:19:12,290 because sometimes information may be a bit more encrypted. 376 00:19:12,290 --> 00:19:14,210 And it may be a real struggle to slog 377 00:19:14,210 --> 00:19:16,080 through a lot of sequences. 378 00:19:16,080 --> 00:19:21,800 So you can really find out about the targeting sequences 379 00:19:21,800 --> 00:19:23,240 through bioinformatics. 380 00:19:25,850 --> 00:19:31,010 Because nowadays, the genomes of dozens and thousands 381 00:19:31,010 --> 00:19:34,010 of organisms are available readily online. 382 00:19:34,010 --> 00:19:36,890 And you can literally parse out information 383 00:19:36,890 --> 00:19:39,230 from the genomic information that gives you 384 00:19:39,230 --> 00:19:41,870 the proteomic information. 385 00:19:41,870 --> 00:19:45,560 So that's one way, so with sequences that are targeted. 386 00:19:45,560 --> 00:19:48,590 In some cases, those targeting sequences 387 00:19:48,590 --> 00:19:50,780 remain part of the protein. 388 00:19:50,780 --> 00:19:53,240 But in other cases, in order to ensure 389 00:19:53,240 --> 00:19:56,900 that the protein stays put, the targeting sequences 390 00:19:56,900 --> 00:19:58,050 are removed. 391 00:19:58,050 --> 00:20:00,230 So that's another important point. 392 00:20:08,580 --> 00:20:10,800 You may keep the targeting sequence, 393 00:20:10,800 --> 00:20:14,430 or you may lose it through the action of another enzyme that 394 00:20:14,430 --> 00:20:16,890 cuts off the targeting sequence when 395 00:20:16,890 --> 00:20:19,080 destination has been reached. 396 00:20:19,080 --> 00:20:24,270 Now, there's a second way that we can 397 00:20:24,270 --> 00:20:27,300 program where a protein may go. 398 00:20:27,300 --> 00:20:30,540 And these are rather useful transformations that 399 00:20:30,540 --> 00:20:32,560 make things even more dynamic. 400 00:20:32,560 --> 00:20:35,760 So let me walk you through a concept. 401 00:20:35,760 --> 00:20:38,580 If you think of a protein that's made on the ribosome, 402 00:20:38,580 --> 00:20:40,680 it's got a targeting sequence. 403 00:20:40,680 --> 00:20:43,410 In order to get that protein to destination, 404 00:20:43,410 --> 00:20:45,960 you've got to make a new batch of protein that's 405 00:20:45,960 --> 00:20:47,840 going to go to its destination. 406 00:20:47,840 --> 00:20:50,330 It's going to end up in the mitochondria. 407 00:20:50,330 --> 00:20:53,250 You've got to make the protein de novo. 408 00:20:53,250 --> 00:20:56,790 Sometimes when we need to have the action of a cell 409 00:20:56,790 --> 00:20:58,170 we can't wait that long. 410 00:20:58,170 --> 00:21:01,920 We can do things quickly and expect the cell 411 00:21:01,920 --> 00:21:04,950 to suddenly change what it's doing. 412 00:21:04,950 --> 00:21:07,320 Because we're sitting around waiting for the ribosome 413 00:21:07,320 --> 00:21:09,730 to make new copies of the protein. 414 00:21:09,730 --> 00:21:12,240 So the second way in which proteins 415 00:21:12,240 --> 00:21:14,970 are targeted to new destinations is 416 00:21:14,970 --> 00:21:17,760 through what's known as post-translational 417 00:21:17,760 --> 00:21:18,840 modifications. 418 00:21:24,370 --> 00:21:26,500 This is so unfair, Adam. 419 00:21:29,110 --> 00:21:30,670 I saw you using the middle boards, 420 00:21:30,670 --> 00:21:33,610 but it looked so much easier. 421 00:21:33,610 --> 00:21:37,960 So the second way to target a protein to a destination 422 00:21:37,960 --> 00:21:45,840 is using post-translational modification. 423 00:21:48,550 --> 00:21:49,730 What does this mean? 424 00:21:49,730 --> 00:21:52,390 What it means is that the protein is made. 425 00:21:52,390 --> 00:21:53,470 It's ready. 426 00:21:53,470 --> 00:21:54,670 It's waiting. 427 00:21:54,670 --> 00:21:58,480 But we haven't engaged its final destiny. 428 00:21:58,480 --> 00:22:01,400 We haven't triggered it to go where it needs to be. 429 00:22:01,400 --> 00:22:04,650 But we're waiting for an enzyme to just carry out 430 00:22:04,650 --> 00:22:07,540 a seemingly minor modification of that protein. 431 00:22:07,540 --> 00:22:10,390 And then the protein will go to its destiny. 432 00:22:10,390 --> 00:22:12,940 And I've shown you here examples of three 433 00:22:12,940 --> 00:22:14,860 types of modifications. 434 00:22:14,860 --> 00:22:18,760 One we will talk about today, because it's very simple 435 00:22:18,760 --> 00:22:21,910 to understand, lipidation. 436 00:22:21,910 --> 00:22:23,740 And then the other two, we'll talk 437 00:22:23,740 --> 00:22:32,050 about next time, phosphorylation and ubiquitination. 438 00:22:38,360 --> 00:22:41,790 And these are all what are known as PTMs, 439 00:22:41,790 --> 00:22:45,070 Post-Translational Modifications. 440 00:22:45,070 --> 00:22:48,960 And they are changes that occur to an amino acid side 441 00:22:48,960 --> 00:22:53,700 chain within an already made protein to alter its destiny. 442 00:22:53,700 --> 00:22:55,740 And I'd like to talk about lipidation 443 00:22:55,740 --> 00:23:01,080 first, because I get to remind you about cellular membranes. 444 00:23:01,080 --> 00:23:06,330 So remember, we've talked about these semipermeable barriers 445 00:23:06,330 --> 00:23:11,180 that are around organelles and around cells. 446 00:23:11,180 --> 00:23:13,200 And let's say that this is a membrane-- 447 00:23:13,200 --> 00:23:15,510 I've got to put my-- 448 00:23:15,510 --> 00:23:20,490 that exists between the cytoplasm 449 00:23:20,490 --> 00:23:23,370 and the outside of a cell. 450 00:23:23,370 --> 00:23:27,900 And let's say I have a protein lurking around 451 00:23:27,900 --> 00:23:30,920 in the cytoplasm, but I need it at the membrane. 452 00:23:30,920 --> 00:23:33,990 I need it to get involved in a signaling process. 453 00:23:33,990 --> 00:23:36,400 And I need it now to be there. 454 00:23:36,400 --> 00:23:38,880 If I have a soluble protein, it's not 455 00:23:38,880 --> 00:23:41,100 associated with the membrane. 456 00:23:41,100 --> 00:23:44,040 But I can use another enzyme to attach 457 00:23:44,040 --> 00:23:48,280 a hydrophobic, greasy tail to that protein. 458 00:23:48,280 --> 00:23:50,160 So what it really wants to do is to get 459 00:23:50,160 --> 00:23:52,080 to the hydrophobic membrane. 460 00:23:52,080 --> 00:23:54,750 Lipidation is such a modification. 461 00:23:54,750 --> 00:24:03,150 It's just the modification with a long-chain, often C16, C18, 462 00:24:03,150 --> 00:24:08,880 fatty acid that then renders the protein lipophilic 463 00:24:08,880 --> 00:24:12,750 and makes it want to move, and insert this lipophilic tail 464 00:24:12,750 --> 00:24:18,190 into the membrane, and part the protein of the plasma membrane. 465 00:24:18,190 --> 00:24:20,880 So the information is still, though, 466 00:24:20,880 --> 00:24:23,130 encoded within the protein. 467 00:24:23,130 --> 00:24:24,900 How could that happen? 468 00:24:24,900 --> 00:24:28,170 How could I have made that information be in the protein? 469 00:24:28,170 --> 00:24:30,540 What might be the strategy there? 470 00:24:30,540 --> 00:24:34,280 It's still encoded, but it's secret. 471 00:24:34,280 --> 00:24:35,310 It's cryptic. 472 00:24:35,310 --> 00:24:35,810 Any ideas? 473 00:24:41,830 --> 00:24:47,350 So I'm not going to just glom this group onto a protein. 474 00:24:47,350 --> 00:24:49,530 I'm going to put it somewhere specific. 475 00:24:49,530 --> 00:24:53,370 And so oftentimes, lipidation reactions 476 00:24:53,370 --> 00:24:58,140 occur site-specifically at particular sites 477 00:24:58,140 --> 00:25:02,400 within a sequence, and an enzyme recognizes that site 478 00:25:02,400 --> 00:25:05,400 and transfers the lipidic molecule to it. 479 00:25:05,400 --> 00:25:09,220 So lipidation actually may occur, for example, 480 00:25:09,220 --> 00:25:11,820 of the amino terminus of a protein. 481 00:25:11,820 --> 00:25:14,880 But if there are certain features within that protein, 482 00:25:14,880 --> 00:25:17,710 you may then attach the lipidic group. 483 00:25:17,710 --> 00:25:21,120 So once again, using bioinformatics, 484 00:25:21,120 --> 00:25:24,300 you can look at the target protein of interest 485 00:25:24,300 --> 00:25:28,110 and predict that it's the target of a post-translational 486 00:25:28,110 --> 00:25:29,790 modification reaction. 487 00:25:29,790 --> 00:25:32,730 So once again, the information is 488 00:25:32,730 --> 00:25:36,270 programmed into the sequence, but it's quite cryptic. 489 00:25:36,270 --> 00:25:38,970 It could be within the middle of the sequence. 490 00:25:38,970 --> 00:25:41,520 There could only maybe be a couple of clues. 491 00:25:41,520 --> 00:25:43,200 But the clues are there nonetheless 492 00:25:43,200 --> 00:25:47,070 that can be parsed out using computer learning and screening 493 00:25:47,070 --> 00:25:50,670 of sequences to say that is a target for lipidation, 494 00:25:50,670 --> 00:25:53,970 or phosphorylation or such. 495 00:25:53,970 --> 00:25:56,340 Is that clear to people? 496 00:25:56,340 --> 00:25:57,660 Does that make sense? 497 00:25:57,660 --> 00:26:01,350 The information is encoded, but you can't see that it's there. 498 00:26:01,350 --> 00:26:03,420 But the advantage of the post-translational 499 00:26:03,420 --> 00:26:06,690 modifications is that they occur on demand, 500 00:26:06,690 --> 00:26:10,680 as opposed to making a new protein de novo, 501 00:26:10,680 --> 00:26:14,160 and then having it go to a particular cellular location. 502 00:26:14,160 --> 00:26:17,460 Later on, when we talk about phosphorylation, 503 00:26:17,460 --> 00:26:19,570 you will see that phosphorylation 504 00:26:19,570 --> 00:26:22,800 is the bread and butter of cellular signaling. 505 00:26:22,800 --> 00:26:24,690 It's the light switch in every room 506 00:26:24,690 --> 00:26:28,110 in the cell that turns on and off in order to make functions 507 00:26:28,110 --> 00:26:29,700 happen within the cell. 508 00:26:29,700 --> 00:26:33,510 And that's a really major, dynamic post-translational 509 00:26:33,510 --> 00:26:37,440 modification that has significant meaning. 510 00:26:37,440 --> 00:26:40,650 So the reason on this little image-- 511 00:26:40,650 --> 00:26:42,600 I just wanted to show you the membrane 512 00:26:42,600 --> 00:26:46,530 and just remind you that the membrane is a supramolecular 513 00:26:46,530 --> 00:26:50,070 structure that's assembled with a hydrophobic core 514 00:26:50,070 --> 00:26:52,560 and polar head groups on both faces, 515 00:26:52,560 --> 00:26:55,410 as I've sort of indicated in this cartoon. 516 00:26:55,410 --> 00:26:57,600 So let's start with sequences that 517 00:26:57,600 --> 00:26:59,205 might take us to the nucleus. 518 00:27:02,110 --> 00:27:05,100 Now, the nuclear membrane is rather a strange entity. 519 00:27:10,110 --> 00:27:13,410 Because the nuclear membrane isn't a simple membrane 520 00:27:13,410 --> 00:27:15,220 like the plasma membrane. 521 00:27:15,220 --> 00:27:17,920 It's actually a double-layered membrane. 522 00:27:17,920 --> 00:27:20,880 So if you look at a nuclear membrane-- and I'm 523 00:27:20,880 --> 00:27:27,540 just going to do a job of showing a portion 524 00:27:27,540 --> 00:27:29,310 of the nuclear membrane. 525 00:27:29,310 --> 00:27:31,170 Within the nuclear membrane, there 526 00:27:31,170 --> 00:27:35,400 are pores, quite launch openings. 527 00:27:35,400 --> 00:27:39,510 And the membrane is actually a double membrane, where 528 00:27:39,510 --> 00:27:42,840 all of these lipid bilayers. 529 00:27:42,840 --> 00:27:44,265 So it's not a single membrane. 530 00:27:48,090 --> 00:27:56,465 It's a double membrane with large openings. 531 00:28:02,690 --> 00:28:05,180 And you might say, well, that's no use. 532 00:28:05,180 --> 00:28:08,750 There's just these great big, gaping holes in the nucleus. 533 00:28:08,750 --> 00:28:11,120 Anything can come and go if it wants. 534 00:28:11,120 --> 00:28:14,780 But the nuclear pores are kind of a special structure. 535 00:28:14,780 --> 00:28:18,920 Because they have a protein that's kind of disordered, 536 00:28:18,920 --> 00:28:21,740 that creates a tangled network. 537 00:28:21,740 --> 00:28:24,500 That means that that pore isn't totally open, 538 00:28:24,500 --> 00:28:26,150 but there's some stuff that something's 539 00:28:26,150 --> 00:28:29,120 got to get through to get from one side to the other. 540 00:28:29,120 --> 00:28:32,630 And my colleague Thomas Schwartz in biology works 541 00:28:32,630 --> 00:28:35,360 on the macromolecular structure of nuclear 542 00:28:35,360 --> 00:28:37,820 pores to understand these structures. 543 00:28:37,820 --> 00:28:41,060 Because these are also made through the auspices 544 00:28:41,060 --> 00:28:45,840 of having a lot of proteins that help create this structure. 545 00:28:45,840 --> 00:28:47,720 Otherwise, that membrane wouldn't 546 00:28:47,720 --> 00:28:53,090 stay in its proper format. 547 00:28:53,090 --> 00:28:56,190 So in order for a protein to get into the nucleus, 548 00:28:56,190 --> 00:28:58,490 if it needs to, or leave the nucleus, 549 00:28:58,490 --> 00:29:00,950 it has to have some kind of mechanism 550 00:29:00,950 --> 00:29:04,070 to get through this structure that's 551 00:29:04,070 --> 00:29:05,540 plugging the nuclear pore. 552 00:29:05,540 --> 00:29:08,580 So this would be the inside of the nucleus. 553 00:29:08,580 --> 00:29:11,610 And this would be the cytoplasm. 554 00:29:11,610 --> 00:29:15,160 So as shown on this slide, the nucleus, 555 00:29:15,160 --> 00:29:17,570 there's a particular protein sequence 556 00:29:17,570 --> 00:29:19,520 that's appended to a protein. 557 00:29:19,520 --> 00:29:24,120 That's known as the Nuclear Localization Sequence, or NLS. 558 00:29:27,510 --> 00:29:30,200 And what an NLS sequence is, it's 559 00:29:30,200 --> 00:29:32,750 a short sequence of amino acids that 560 00:29:32,750 --> 00:29:36,170 enables a protein to get to its proper destination. 561 00:29:36,170 --> 00:29:39,260 And these sequences are quite well recognized. 562 00:29:39,260 --> 00:29:42,320 They may end up being highly basic sequences. 563 00:29:45,860 --> 00:29:49,601 So an example of an NLS would be Lys-- 564 00:29:49,601 --> 00:29:51,290 it's not very exciting, but it just 565 00:29:51,290 --> 00:29:59,810 goes on, Lys, Lys, Lys, arginine, lysine. 566 00:29:59,810 --> 00:30:04,640 And it may be bounded by hydrophobic residues 567 00:30:04,640 --> 00:30:05,460 or other types. 568 00:30:05,460 --> 00:30:10,520 So that would be a typical NLS sequence that's in a protein. 569 00:30:10,520 --> 00:30:19,090 And I want to remind you that lysine and arginine all have 570 00:30:19,090 --> 00:30:21,630 side chains that at physiological pH 571 00:30:21,630 --> 00:30:22,970 are positively charged. 572 00:30:22,970 --> 00:30:25,670 So the nuclear localization sequence 573 00:30:25,670 --> 00:30:28,210 is something that's easily recognized because 574 00:30:28,210 --> 00:30:31,060 of this sort of short sequence that 575 00:30:31,060 --> 00:30:32,740 may be at the N- or C-terminus. 576 00:30:32,740 --> 00:30:34,540 I think there's either possibility. 577 00:30:34,540 --> 00:30:36,250 But it's a very clear sequence. 578 00:30:36,250 --> 00:30:38,500 You could look at your protein sequence and say, 579 00:30:38,500 --> 00:30:40,630 there's an NLS on that sequence. 580 00:30:40,630 --> 00:30:43,480 And it's the NLS sequence alone that's 581 00:30:43,480 --> 00:30:46,330 responsible for getting the proteins in 582 00:30:46,330 --> 00:30:48,250 and out of the nuclear pore. 583 00:30:48,250 --> 00:30:51,385 Let's mostly focus on getting into the nucleus. 584 00:30:55,300 --> 00:30:59,710 Basically, you have a protein structure 585 00:30:59,710 --> 00:31:02,560 that has an NLS sequence at one terminus. 586 00:31:02,560 --> 00:31:06,010 And that NLS sequence binds to another protein. 587 00:31:13,410 --> 00:31:15,640 Creatively, you had a little bit of chance 588 00:31:15,640 --> 00:31:18,250 to give proteins names in the exam. 589 00:31:18,250 --> 00:31:19,750 It's called importin. 590 00:31:19,750 --> 00:31:24,040 So it's an import protein that binds to the NLS, 591 00:31:24,040 --> 00:31:27,490 and as a consequence of that, will carry cargo. 592 00:31:27,490 --> 00:31:31,090 It will escort cargo into the nucleus of the cell. 593 00:31:31,090 --> 00:31:35,920 And it sends it through this meshwork of proteins. 594 00:31:35,920 --> 00:31:39,250 That's a very loose mesh work of proteins. 595 00:31:39,250 --> 00:31:41,350 And they're not ordered proteins. 596 00:31:41,350 --> 00:31:43,690 They're highly disordered proteins. 597 00:31:43,690 --> 00:31:46,455 So they make more of a filter than a plug. 598 00:31:46,455 --> 00:31:47,830 But they are definitely something 599 00:31:47,830 --> 00:31:50,500 that doesn't allow any old protein to go 600 00:31:50,500 --> 00:31:52,090 through that nuclear pore. 601 00:31:52,090 --> 00:31:55,840 NLS tags are very easy to recognize, once again, 602 00:31:55,840 --> 00:31:58,413 through bioinformatics analysis. 603 00:31:58,413 --> 00:31:59,830 And what's really cool is that you 604 00:31:59,830 --> 00:32:02,320 can reprogram a protein to be where you 605 00:32:02,320 --> 00:32:04,930 want by manipulating the NLS. 606 00:32:04,930 --> 00:32:07,880 So this is rather a nice set of experiments. 607 00:32:07,880 --> 00:32:11,290 Let's say we have a protein that we're going to micro-inject 608 00:32:11,290 --> 00:32:13,540 into the cytoplasm of the cell. 609 00:32:13,540 --> 00:32:17,080 And we want to program it to either go into the nucleus 610 00:32:17,080 --> 00:32:19,150 or stay outside the nucleus. 611 00:32:19,150 --> 00:32:21,730 That can be done readily by attaching 612 00:32:21,730 --> 00:32:24,940 a nuclear localization sequence to a protein 613 00:32:24,940 --> 00:32:29,020 along with a fluorophore dye or fluorescent protein that 614 00:32:29,020 --> 00:32:31,900 will allow you to observe that experiment. 615 00:32:31,900 --> 00:32:35,230 If you micro-inject into the cytoplasm, 616 00:32:35,230 --> 00:32:39,940 that protein that's got an NLS will get run into the nucleus 617 00:32:39,940 --> 00:32:43,250 through association of the NLS with importin. 618 00:32:43,250 --> 00:32:47,140 But if you chop that NLS, the protein the stuck, 619 00:32:47,140 --> 00:32:49,570 remains out in the cytoplasm. 620 00:32:49,570 --> 00:32:51,610 Let's say you want to study a new protein. 621 00:32:51,610 --> 00:32:54,010 I just want to show you that these NLS 622 00:32:54,010 --> 00:32:58,480 sequence are totally independent of the cargo they carry. 623 00:32:58,480 --> 00:33:01,270 You can just stick an NLS on your favorite protein 624 00:33:01,270 --> 00:33:03,030 who you want to interrogate. 625 00:33:03,030 --> 00:33:04,850 Let's take pyruvate kinase. 626 00:33:04,850 --> 00:33:08,140 It doesn't have anything to do with specific transport 627 00:33:08,140 --> 00:33:09,220 to the nucleus. 628 00:33:09,220 --> 00:33:11,370 But nevertheless, if you put-- 629 00:33:11,370 --> 00:33:14,080 if it doesn't have an NLS, it's fluorescently labeled, 630 00:33:14,080 --> 00:33:16,870 it stays outside in the cytoplasm. 631 00:33:16,870 --> 00:33:18,700 But if you put an analysis on it, 632 00:33:18,700 --> 00:33:21,470 you concentrate into that region of the cell. 633 00:33:21,470 --> 00:33:23,830 So these experiments show you that what 634 00:33:23,830 --> 00:33:26,020 we know about these targeting sequences 635 00:33:26,020 --> 00:33:30,820 can be manipulated and used to enable you to move things 636 00:33:30,820 --> 00:33:31,930 around in the cell. 637 00:33:31,930 --> 00:33:35,140 So that's one particular type of mechanism. 638 00:33:35,140 --> 00:33:39,400 The next mechanism I want to describe to you 639 00:33:39,400 --> 00:33:45,610 is the mechanism that's used for mitochondrial transports. 640 00:33:45,610 --> 00:33:48,055 And it's a little bit different in its strategy. 641 00:33:53,570 --> 00:33:58,090 So to get into the mitochondria, there is, again, a recognition 642 00:33:58,090 --> 00:34:07,130 sequence, in this case, a mitochondrial localization 643 00:34:07,130 --> 00:34:11,400 sequence that has particular characteristics. 644 00:34:11,400 --> 00:34:17,940 In this case, the mitochondrial localization sequence, 645 00:34:17,940 --> 00:34:22,310 let's say it's at the N-terminus of your protein. 646 00:34:22,310 --> 00:34:26,510 And it would be something that might be a mix of charges. 647 00:34:26,510 --> 00:34:32,750 Some Arg, Glu, Arg, Glu. 648 00:34:32,750 --> 00:34:35,449 So that's a typical MLS sequence. 649 00:34:35,449 --> 00:34:39,980 And in this case, the charge at physiological pH 650 00:34:39,980 --> 00:34:43,429 is different from the nuclear localization sequence, 651 00:34:43,429 --> 00:34:46,670 because it's an alternating positive and negative charge. 652 00:34:46,670 --> 00:34:48,530 So this is pretty different from this. 653 00:34:48,530 --> 00:34:51,179 It doesn't say bioinformatics to figure that one out. 654 00:34:51,179 --> 00:34:54,679 So you can then pick out mitochondrial localization 655 00:34:54,679 --> 00:34:55,949 sequences. 656 00:34:55,949 --> 00:34:59,090 And so in this case, remember, mitochondria 657 00:34:59,090 --> 00:35:03,140 make some of their own proteins on their circular DNA. 658 00:35:03,140 --> 00:35:06,710 But they've abandoned expressing all the proteins that 659 00:35:06,710 --> 00:35:08,540 are needed in the mitochondria. 660 00:35:08,540 --> 00:35:11,060 And some proteins are transported 661 00:35:11,060 --> 00:35:14,780 into the mitochondria using these types of sequences. 662 00:35:14,780 --> 00:35:16,940 But the approach, the strategy, is 663 00:35:16,940 --> 00:35:19,670 different from getting into the nucleus. 664 00:35:19,670 --> 00:35:24,710 In this case, the MLS sequence associates 665 00:35:24,710 --> 00:35:28,520 with a protein channel that is in a closed state. 666 00:35:28,520 --> 00:35:30,090 So here's a membrane. 667 00:35:30,090 --> 00:35:31,790 Here's the makings of a channel. 668 00:35:31,790 --> 00:35:33,530 But it's in a closed state. 669 00:35:33,530 --> 00:35:37,370 But once the protein with the NLS sequence binds to that, 670 00:35:37,370 --> 00:35:39,830 that channel opens. 671 00:35:39,830 --> 00:35:42,320 It's triggered by the binding of that sequence 672 00:35:42,320 --> 00:35:47,420 to a portion of the protein that's outside that membrane. 673 00:35:47,420 --> 00:35:51,590 And that then allows the protein to be unfolded and transported 674 00:35:51,590 --> 00:35:55,940 into the mitochondria, where that sequence may be removed. 675 00:35:55,940 --> 00:35:59,070 And then protein refolds in the mitochondria. 676 00:35:59,070 --> 00:36:01,070 So it's a very different strategy 677 00:36:01,070 --> 00:36:03,860 for that and the nuclear localization sequence. 678 00:36:03,860 --> 00:36:07,850 So you'll find, for many different organelles 679 00:36:07,850 --> 00:36:11,900 in the cell, there might be very specific localization sequences 680 00:36:11,900 --> 00:36:14,250 that you could look up and learn about. 681 00:36:14,250 --> 00:36:16,220 But one thing I want to mention to you 682 00:36:16,220 --> 00:36:19,850 is that these localization details are very important. 683 00:36:19,850 --> 00:36:23,840 And many diseases in cells are a consequence 684 00:36:23,840 --> 00:36:27,570 of proteins not being localized to the right place. 685 00:36:27,570 --> 00:36:30,260 If you're not in the right place at the right time, 686 00:36:30,260 --> 00:36:32,510 then things will start to go wrong 687 00:36:32,510 --> 00:36:35,730 with the signaling or the processes of the cell. 688 00:36:35,730 --> 00:36:39,470 So diseases are frequently associated 689 00:36:39,470 --> 00:36:41,360 with mislocalization. 690 00:36:41,360 --> 00:36:48,450 So now what we're going to do is basically say, 691 00:36:48,450 --> 00:36:51,720 we've taken care of understanding things made 692 00:36:51,720 --> 00:36:52,680 in the cell. 693 00:36:52,680 --> 00:36:54,570 They either stay in the cytosol or they'll 694 00:36:54,570 --> 00:36:58,950 go to organelles based on particular types of strategies 695 00:36:58,950 --> 00:37:03,450 that are largely dependent on short tagging sequences, 696 00:37:03,450 --> 00:37:07,380 but in other cases, may be dependent on post translational 697 00:37:07,380 --> 00:37:09,360 modification. 698 00:37:09,360 --> 00:37:09,860 All right. 699 00:37:09,860 --> 00:37:12,990 So here is a cartoon. 700 00:37:12,990 --> 00:37:16,020 But actually, I want to do something slightly different 701 00:37:16,020 --> 00:37:19,530 if it doesn't take too long. 702 00:37:19,530 --> 00:37:22,710 Now, when we first talked about translation 703 00:37:22,710 --> 00:37:26,010 on the ribosome, what you see there in green and yellow 704 00:37:26,010 --> 00:37:27,210 is the ribosome. 705 00:37:27,210 --> 00:37:29,790 The dark band is a messenger RNA. 706 00:37:29,790 --> 00:37:32,550 The dark blue are transfer RNAs that 707 00:37:32,550 --> 00:37:37,050 are being helped with elongation factors to get to the ribosome. 708 00:37:37,050 --> 00:37:38,910 But what I want to point out here 709 00:37:38,910 --> 00:37:42,420 is the emerging sequence of polypeptide 710 00:37:42,420 --> 00:37:44,970 coming out through a tunnel on the ribosome. 711 00:37:44,970 --> 00:37:49,710 Now, if a protein is going to be destined outside the cell, 712 00:37:49,710 --> 00:37:53,050 it is expressed with what's known as a signal sequence. 713 00:37:53,050 --> 00:37:55,380 It's about a 20-amino acid residue 714 00:37:55,380 --> 00:37:59,400 sequence that is recognized by the signal recognition 715 00:37:59,400 --> 00:38:00,630 particle. 716 00:38:00,630 --> 00:38:04,290 And then translation slows down and clamps 717 00:38:04,290 --> 00:38:06,990 the ribosome on the endoplasmic reticulum 718 00:38:06,990 --> 00:38:10,350 membrane so that the new peptide starts 719 00:38:10,350 --> 00:38:14,010 being threaded into the endoplasmic reticulum 720 00:38:14,010 --> 00:38:16,680 through what's known as the translocon. 721 00:38:16,680 --> 00:38:21,120 So you're now not sending the protein out to the cytoplasm, 722 00:38:21,120 --> 00:38:23,400 but you're rather sending the protein 723 00:38:23,400 --> 00:38:26,050 into the endoplasmic reticulum. 724 00:38:26,050 --> 00:38:31,050 And you're also sending it down this branch of the protein 725 00:38:31,050 --> 00:38:32,760 biosynthesis pathway. 726 00:38:32,760 --> 00:38:35,400 You see this piece of protein emerging. 727 00:38:35,400 --> 00:38:39,510 This hatched portion is the cytoplasm. 728 00:38:39,510 --> 00:38:42,720 The gray portion is the endoplasmic reticulum. 729 00:38:42,720 --> 00:38:46,470 So there is a complex machinery at play 730 00:38:46,470 --> 00:38:50,460 that enables proteins to be made in the cytoplasm 731 00:38:50,460 --> 00:38:53,772 but now targeted to a completely new location. 732 00:38:53,772 --> 00:38:55,230 And these are the proteins that are 733 00:38:55,230 --> 00:38:58,980 going to be destined to either stay in the plasma membrane 734 00:38:58,980 --> 00:39:01,050 or be secreted from the cell. 735 00:39:01,050 --> 00:39:04,680 And this view here gives you a little bit more 736 00:39:04,680 --> 00:39:05,910 than the cartoon. 737 00:39:05,910 --> 00:39:08,640 So ribosome-- a signal peptide is 738 00:39:08,640 --> 00:39:11,490 made that is a green peptide sequence that's 739 00:39:11,490 --> 00:39:13,890 about 20 amino acids long. 740 00:39:13,890 --> 00:39:17,160 That is actually called a signal peptide. 741 00:39:17,160 --> 00:39:19,500 It's signaling for synthesis through 742 00:39:19,500 --> 00:39:21,750 the endomembrane network. 743 00:39:21,750 --> 00:39:26,160 That causes the ribosomes to dock down on the cytosol ER 744 00:39:26,160 --> 00:39:29,040 membrane and keep on being synthesized 745 00:39:29,040 --> 00:39:32,880 so that proteins are made into that endomembrane system. 746 00:39:32,880 --> 00:39:35,190 And you can think of this cavernous endomembrane 747 00:39:35,190 --> 00:39:39,510 system as your tunnels out of a cell for either display 748 00:39:39,510 --> 00:39:43,170 on the surface of the cell or for secretion entirely 749 00:39:43,170 --> 00:39:44,350 in vesicles. 750 00:39:44,350 --> 00:39:47,550 So let's take a look at how that occurs. 751 00:39:47,550 --> 00:39:49,920 When you make a protein in that way, 752 00:39:49,920 --> 00:39:53,400 see the dark dots, the rough ER? 753 00:39:53,400 --> 00:39:57,180 These are ribosomes that are attached to the membrane. 754 00:39:57,180 --> 00:39:59,550 Proteins are made into the membrane. 755 00:39:59,550 --> 00:40:03,120 And then the endomembrane system is not really 756 00:40:03,120 --> 00:40:06,540 just a tunnel or a labyrinth. 757 00:40:06,540 --> 00:40:08,610 But actually, each of those layers 758 00:40:08,610 --> 00:40:12,120 spits off vesicles that fuse with next layers 759 00:40:12,120 --> 00:40:15,840 to gradually make their way outside of the cells. 760 00:40:15,840 --> 00:40:17,850 So here you see there are vesicles. 761 00:40:17,850 --> 00:40:20,430 You're always keeping proteins associated 762 00:40:20,430 --> 00:40:24,310 with membrane as you go through the endomembrane system. 763 00:40:24,310 --> 00:40:27,120 And here is a vesicle that's got protein in it. 764 00:40:27,120 --> 00:40:30,570 It may either release it to the outside of the cell, 765 00:40:30,570 --> 00:40:34,650 or the protein may be associated with the membrane 766 00:40:34,650 --> 00:40:38,760 of the vesicle and stay parked in the plasma membrane. 767 00:40:38,760 --> 00:40:42,990 And so I just want to give you one final slide where 768 00:40:42,990 --> 00:40:46,310 I talk about the biogenesis of membrane proteins. 769 00:40:46,310 --> 00:40:48,180 Now, this is pretty complicated stuff. 770 00:40:48,180 --> 00:40:51,540 Because you have to remember what's inside and out. 771 00:40:51,540 --> 00:40:56,490 So I spent more time than I should have on this cartoon 772 00:40:56,490 --> 00:40:59,430 to show you which end of the protein 773 00:40:59,430 --> 00:41:02,100 ends up outside the cell and which inside the cell, 774 00:41:02,100 --> 00:41:05,490 and how you make multi-membrane-spanning 775 00:41:05,490 --> 00:41:06,130 proteins. 776 00:41:06,130 --> 00:41:09,030 So let's take a look at this in detail now, looking-- 777 00:41:09,030 --> 00:41:10,620 here's the ribosome. 778 00:41:10,620 --> 00:41:12,720 Here's the protein emerging. 779 00:41:12,720 --> 00:41:14,580 If there's signal sequence there, 780 00:41:14,580 --> 00:41:17,280 that ribosome docks down on the membrane 781 00:41:17,280 --> 00:41:21,300 and starts translating the protein, amino terminus 782 00:41:21,300 --> 00:41:23,730 first, into the endoplasmic reticulum. 783 00:41:23,730 --> 00:41:25,800 We'll all OK with that. 784 00:41:25,800 --> 00:41:30,576 As synthesis continues, we may reach the stop codon 785 00:41:30,576 --> 00:41:32,640 on the messenger RNA. 786 00:41:32,640 --> 00:41:35,820 And what may happen is that the protein may remain 787 00:41:35,820 --> 00:41:37,710 associated with membrane. 788 00:41:37,710 --> 00:41:40,020 The amino terminus will be in the ER. 789 00:41:40,020 --> 00:41:42,910 And the C-terminus will remain on the other side. 790 00:41:42,910 --> 00:41:46,650 There are a number of different configurations. 791 00:41:46,650 --> 00:41:50,490 But if we want to start to transport this protein 792 00:41:50,490 --> 00:41:53,280 to the surface of the cell, that will then 793 00:41:53,280 --> 00:41:55,890 stay associated with membrane but not 794 00:41:55,890 --> 00:41:59,330 in the form of the flat membrane that it was delivered into. 795 00:41:59,330 --> 00:42:04,320 But that membrane may pinch off into a spherical vesicle. 796 00:42:04,320 --> 00:42:06,720 But you still have the C-terminus outside 797 00:42:06,720 --> 00:42:09,000 and the N-terminus inside. 798 00:42:09,000 --> 00:42:11,730 That will then work its way through 799 00:42:11,730 --> 00:42:16,110 the endomembrane system, and ultimately, fuse 800 00:42:16,110 --> 00:42:17,190 with the cytosol. 801 00:42:17,190 --> 00:42:20,190 This is the really fun part. 802 00:42:20,190 --> 00:42:22,305 And then, once it's fused with the cytosol, 803 00:42:22,305 --> 00:42:24,660 it has the option to be displayed 804 00:42:24,660 --> 00:42:26,410 on the outside of the cell. 805 00:42:26,410 --> 00:42:26,910 Why? 806 00:42:26,910 --> 00:42:28,620 You have a protein. 807 00:42:28,620 --> 00:42:30,450 The N-terminus is on the outside. 808 00:42:30,450 --> 00:42:32,610 The C-terminus is on the inside. 809 00:42:32,610 --> 00:42:36,090 So that shows you the biogenesis of the cell surface 810 00:42:36,090 --> 00:42:38,130 protein that's stuck in the membrane 811 00:42:38,130 --> 00:42:41,580 through its membrane-associated domain. 812 00:42:41,580 --> 00:42:43,900 If you're not going to stay with the membrane, 813 00:42:43,900 --> 00:42:46,530 you can actually also simply release this 814 00:42:46,530 --> 00:42:50,010 into the vesicle for release of a soluble protein. 815 00:42:50,010 --> 00:42:51,330 I will not go through this. 816 00:42:51,330 --> 00:42:53,940 But there are miraculous steps that 817 00:42:53,940 --> 00:42:58,320 end up in the biogenesis of multi-transmembrane proteins. 818 00:42:58,320 --> 00:43:00,870 Because each of those transmembrane domains 819 00:43:00,870 --> 00:43:04,400 gets made in the translocon and gets shuttled sideways. 820 00:43:04,400 --> 00:43:07,620 And you start piling up transmembrane domains 821 00:43:07,620 --> 00:43:09,060 that span the membrane. 822 00:43:09,060 --> 00:43:10,800 And in the next class, we're going 823 00:43:10,800 --> 00:43:15,090 to see how useful these proteins are in cellular signaling. 824 00:43:15,090 --> 00:43:18,990 So those are very important proteins to think about. 825 00:43:18,990 --> 00:43:22,020 One last thing-- so let's think about this. 826 00:43:22,020 --> 00:43:27,090 For either configuration, either post-translational modification 827 00:43:27,090 --> 00:43:31,440 or using targeting sequences, when do we define where 828 00:43:31,440 --> 00:43:33,660 the protein's going to end up? 829 00:43:33,660 --> 00:43:35,625 Where's the information first defined? 830 00:43:39,020 --> 00:43:42,730 Anyone want to answer me and explain why? 831 00:43:42,730 --> 00:43:43,424 Yes? 832 00:43:43,424 --> 00:43:45,400 AUDIENCE: Would it be B, the mRNA sequence, 833 00:43:45,400 --> 00:43:47,353 because that would have a significant portion 834 00:43:47,353 --> 00:43:48,020 of the splicing? 835 00:43:51,910 --> 00:43:53,490 BARBARA IMPERIALI: It's a good try. 836 00:43:53,490 --> 00:43:56,760 But you want to remember, yes, splicing is important. 837 00:43:56,760 --> 00:43:59,400 But when was the sequence actually 838 00:43:59,400 --> 00:44:00,660 in the entire pre-mRNA? 839 00:44:03,773 --> 00:44:05,190 When would that have been defined? 840 00:44:07,980 --> 00:44:09,040 Yeah? 841 00:44:09,040 --> 00:44:09,540 Sorry. 842 00:44:09,540 --> 00:44:10,350 Carmen? 843 00:44:10,350 --> 00:44:13,570 AUDIENCE: Is it in the genomic DNA sequence? 844 00:44:13,570 --> 00:44:14,560 BARBARA IMPERIALI: Yes. 845 00:44:14,560 --> 00:44:18,040 Because you never have information in the RNA 846 00:44:18,040 --> 00:44:19,490 that wasn't in the DNA. 847 00:44:19,490 --> 00:44:21,790 So the DNA has got the information there. 848 00:44:21,790 --> 00:44:24,880 Yeah, it may need a bit of splicing to put things 849 00:44:24,880 --> 00:44:26,390 in the right place. 850 00:44:26,390 --> 00:44:28,780 But the information is there in the DNA. 851 00:44:28,780 --> 00:44:33,280 So you want to remember, for all of this targeting information, 852 00:44:33,280 --> 00:44:36,310 it's in the genomic information most commonly. 853 00:44:36,310 --> 00:44:37,870 It's the genomic information that 854 00:44:37,870 --> 00:44:41,050 has the patterns of sequences for post-translational 855 00:44:41,050 --> 00:44:42,150 modification. 856 00:44:42,150 --> 00:44:44,470 It's the genomic information that has 857 00:44:44,470 --> 00:44:46,990 things like NLSes and MLSes. 858 00:44:46,990 --> 00:44:48,320 They're already there. 859 00:44:48,320 --> 00:44:50,350 But they are often encrypted. 860 00:44:50,350 --> 00:44:52,510 And there was a very nice point there, though. 861 00:44:52,510 --> 00:44:58,240 If you want to send to make a single chunk of a genome that 862 00:44:58,240 --> 00:45:02,350 encodes either a protein that's going to be exported 863 00:45:02,350 --> 00:45:06,430 through the secretory pathway or stay in the cytosol, 864 00:45:06,430 --> 00:45:10,120 you might splice in or out a signal sequence. 865 00:45:10,120 --> 00:45:13,750 So that's a really good way, using the same original DNA 866 00:45:13,750 --> 00:45:16,420 sequence, to actually get to proteins 867 00:45:16,420 --> 00:45:19,870 that fulfill different final destinies within the cell. 868 00:45:19,870 --> 00:45:22,120 So next time, we're going to talk about signaling. 869 00:45:22,120 --> 00:45:24,240 It's going to be a blast.