1 00:00:15 --> 00:00:19 OK. I think we'll get started. We're moving into another phase of 2 00:00:19 --> 00:00:24 the course today talking about cell biology. There are going to be 3 00:00:24 --> 00:00:29 three lectures on cell biology covering a variety of topics. 4 00:00:29 --> 00:00:41 So we've been talking about cells in 5 00:00:41 --> 00:00:46 various contexts over the course of several lectures, 6 00:00:46 --> 00:00:50 actually, but more or less we've talked about them as static objects 7 00:00:50 --> 00:00:55 that carry out replication and transcription and translation and so 8 00:00:55 --> 00:01:00 on and so forth. But, in fact, as I'm sure you are 9 00:01:00 --> 00:01:05 aware, cells are highly dynamic structures which do many things at 10 00:01:05 --> 00:01:10 all times. And this is a movie from which shows the movement of cells. 11 00:01:10 --> 00:01:14 Cells will move randomly when you place them in a dish, 12 00:01:14 --> 00:01:19 but they'll also move in a directed fashion in response to factors in 13 00:01:19 --> 00:01:24 their environment that attract them or they'll move away from factors 14 00:01:24 --> 00:01:29 that repel them. So cell movement is an example of a 15 00:01:29 --> 00:01:33 dynamic process. Cells in the immune system, 16 00:01:33 --> 00:01:37 for example, are cells that do a lot of very critical movement to get to 17 00:01:37 --> 00:01:41 the site of a wound, to get to the site of an infection, 18 00:01:41 --> 00:01:45 and then to elicit the proper response to that infection. 19 00:01:45 --> 00:01:49 It's a late arriving crowd today. Another aspect of the dynamic 20 00:01:49 --> 00:01:53 nature of cells is the fact that they divide. And I've actually 21 00:01:53 --> 00:01:57 shown you this movie previously. From early embryogenesis through the 22 00:01:57 --> 00:02:02 formation of the fully formed organism and then throughout adult 23 00:02:02 --> 00:02:06 life, cells are receiving signals to divide at the appropriate times and 24 00:02:06 --> 00:02:11 to stop dividing when it's not longer necessary. 25 00:02:11 --> 00:02:16 This is a process of signal transduction, receipt of a signal 26 00:02:16 --> 00:02:21 which tells the cell it's time to do something, and then to process that 27 00:02:21 --> 00:02:26 signal in a way to leads to the proper cellular response. 28 00:02:26 --> 00:02:30 Maybe one of the most well understood or familiar examples of 29 00:02:30 --> 00:02:34 this process of signal transduction is the neuron depicted here in its 30 00:02:34 --> 00:02:38 actual state filled with a dye so you can see its various processes 31 00:02:38 --> 00:02:42 and diagramed here. Neurons, of course, 32 00:02:42 --> 00:02:46 are part of the information transfer in the nervous system where signals 33 00:02:46 --> 00:02:50 are received from these structures here called dendrites. 34 00:02:50 --> 00:02:54 That signal is then propagated through the body of the neuron 35 00:02:54 --> 00:02:58 through these long structures called axons and then released. 36 00:02:58 --> 00:03:01 The signal finally acts upon the structures at the very end of these 37 00:03:01 --> 00:03:05 nerve terminals leading to the release, for example, 38 00:03:05 --> 00:03:08 of neurotransmitters. These are impressive cells for a variety of 39 00:03:08 --> 00:03:12 reasons. And Professor Sive will actually give you some lectures 40 00:03:12 --> 00:03:16 which deal more specifically about signaling in the nervous system. 41 00:03:16 --> 00:03:19 One of the ways that they're impressive is their sheer length. 42 00:03:19 --> 00:03:23 You have neurons in your body that are more than a meter in length, 43 00:03:23 --> 00:03:27 and whales have neurons in their body that are tens of 44 00:03:27 --> 00:03:31 meters in length. So these are remarkable cells which 45 00:03:31 --> 00:03:36 carry out very specific functions. So basically what we want to talk 46 00:03:36 --> 00:03:42 about over the course of today's lecture, we'll get there eventually, 47 00:03:42 --> 00:03:47 we have to do some other stuff first, is the process by which cells 48 00:03:47 --> 00:03:56 respond to a stimulus. 49 00:03:56 --> 00:04:01 And then, much as a computer would do, to process that signal in very 50 00:04:01 --> 00:04:07 sophisticated ways, which I'll introduce to you this 51 00:04:07 --> 00:04:13 time and talk about in greater detail next time as well, 52 00:04:13 --> 00:04:19 to lead to a particular cellular response. And I've given you three 53 00:04:19 --> 00:04:25 examples of cellular responses in the movies and images this morning. 54 00:04:25 --> 00:04:29 For example, the cells might respond by moving in a particular directed 55 00:04:29 --> 00:04:33 fashion. They might divide because it's time for them to divide. 56 00:04:33 --> 00:04:37 Or they might, in response to this signal, release a neurotransmitter 57 00:04:37 --> 00:04:47 -- 58 00:04:47 --> 00:04:51 In the peripheral nervous system or in the brain. So what we want to 59 00:04:51 --> 00:04:56 understand is what does this really mean? What are the mechanisms that 60 00:04:56 --> 00:05:00 cells use to process signals? And you'll actually see analogies 61 00:05:00 --> 00:05:05 to integrated circuits as we go through some examples. 62 00:05:05 --> 00:05:10 Before we get there, we need to understand a little bit 63 00:05:10 --> 00:05:15 about how cells are built. Largely, but not entirely, 64 00:05:15 --> 00:05:20 the structure of a cell is related to its function. 65 00:05:20 --> 00:05:25 So neurons are an example. Their particular structures allow 66 00:05:25 --> 00:05:30 them to do the things that they do. 67 00:05:30 --> 00:05:34 Erythrocytes are another example. Small red blood cells, which are 68 00:05:34 --> 00:05:38 oxygen-carrying cells, are built the way they're built to 69 00:05:38 --> 00:05:42 do what they need to do. And largely, but not entirely, 70 00:05:42 --> 00:05:46 a cell's structure is determined by what proteins are expressed. 71 00:05:46 --> 00:05:54 And also where those proteins are 72 00:05:54 --> 00:05:57 found inside the cell. Where those proteins are localized 73 00:05:57 --> 00:06:00 inside the cell. So I want to spend a little bit of 74 00:06:00 --> 00:06:03 time talking about protein localization. 75 00:06:03 --> 00:06:06 So I want to spend a little bit of time talking about protein 76 00:06:06 --> 00:06:09 localization, how is it that proteins within your cells get to 77 00:06:09 --> 00:06:13 where they're going, in a sense know where they're 78 00:06:13 --> 00:06:22 supposed to go. 79 00:06:22 --> 00:06:26 And a critical tool for understanding this process of 80 00:06:26 --> 00:06:35 protein localization -- 81 00:06:35 --> 00:06:45 -- is fluorescence imaging. 82 00:06:45 --> 00:06:48 And so I think you're all aware of standard light microscopy where you 83 00:06:48 --> 00:06:52 pass white light through a sample, let's say a cell or a collection of 84 00:06:52 --> 00:06:56 cells, and based on the absorbance of the light through the different 85 00:06:56 --> 00:07:00 structures within those cells you're actually able to visualize what the 86 00:07:00 --> 00:07:04 cells look like. With fluorescence microscopy, 87 00:07:04 --> 00:07:08 what you do is shine UV light onto the cell, which brings energy to the 88 00:07:08 --> 00:07:13 cell and allows molecules that will fluoresce in response to that 89 00:07:13 --> 00:07:18 wavelength of UV to be visualized. So if you have a fluorescent signal 90 00:07:18 --> 00:07:22 within the cell, you can see where that signal is 91 00:07:22 --> 00:07:27 coming from using fluorescence microscopy or fluorescence 92 00:07:27 --> 00:07:32 imaging. So how is it that we get 93 00:07:32 --> 00:07:37 fluorescence from the particular structures, proteins or other things 94 00:07:37 --> 00:07:42 that we want to see inside the cell? Well, there are two general ways of 95 00:07:42 --> 00:07:47 doing that. The first is fluorescent antibodies. 96 00:07:47 --> 00:07:57 So let's imagine we have a protein 97 00:07:57 --> 00:08:03 of interest. We want to know where it is inside the cell. 98 00:08:03 --> 00:08:08 And we're able to make that protein, say, in bacteria. 99 00:08:08 --> 00:08:13 We can inject that protein into a bunny, that's a bunny, 100 00:08:13 --> 00:08:19 or into a mouse, that's a mouse. OK. These are the common organisms 101 00:08:19 --> 00:08:24 used for production of antibodies. We'll actually talk about how 102 00:08:24 --> 00:08:30 antibodies are made and how they recognize the things they recognize 103 00:08:30 --> 00:08:34 in subsequent lectures. But I think you all know that 104 00:08:34 --> 00:08:38 antibodies are made by the body to recognize foreign stuff. 105 00:08:38 --> 00:08:42 In this case, this protein that we've produced is foreign to the 106 00:08:42 --> 00:08:46 bunny or to the mouse. And so in response to that it makes 107 00:08:46 --> 00:08:50 an antibody, or a series of antibodies. These are proteins 108 00:08:50 --> 00:08:54 which have a particular structure that we often diagram like this. 109 00:08:54 --> 00:08:58 They have four chains, two big ones and two little ones. So 110 00:08:58 --> 00:09:02 these are antibodies. And importantly the antibodies are 111 00:09:02 --> 00:09:06 specific. That is they will bind to this protein but they won't bind to 112 00:09:06 --> 00:09:10 other proteins. So they specifically recognize this 113 00:09:10 --> 00:09:14 protein which is called the antigen in the context of antibodies. 114 00:09:14 --> 00:09:18 We can purify these antibodies and then chemically modify them to add a 115 00:09:18 --> 00:09:36 fluorochrome. 116 00:09:36 --> 00:09:38 A chemical moiety that will fluoresce in response to a 117 00:09:38 --> 00:09:41 particular UV wavelength of light. We can then apply those 118 00:09:41 --> 00:09:44 fluorescently labeled antibodies to cells that we've put onto a glass 119 00:09:44 --> 00:09:47 slide and actually poked holes in to allow the antibody to access the 120 00:09:47 --> 00:09:50 inside of the cell. And then we can visualize where the 121 00:09:50 --> 00:09:53 fluorescence comes from within the cell using fluorescence 122 00:09:53 --> 00:09:57 imaging microscopy. So, for example, 123 00:09:57 --> 00:10:01 if the protein of interest is in the nucleus, and we have labeled our 124 00:10:01 --> 00:10:06 antibody with a fluorochrome that fluoresces in the green wavelength, 125 00:10:06 --> 00:10:11 now when we visualize this cell by fluorescence microscopy the nucleus, 126 00:10:11 --> 00:10:15 if the protein is nuclear, will look green. OK? And this has been used 127 00:10:15 --> 00:10:20 in cell biology for a very long time, and it's still used today. 128 00:10:20 --> 00:10:25 It's a very powerful method for determining protein localization. 129 00:10:25 --> 00:10:30 This method is called immunofluorescence. 130 00:10:30 --> 00:10:40 Immuno for the antibody and 131 00:10:40 --> 00:10:45 fluorescence. And you can actually use multiple antibodies labeled with 132 00:10:45 --> 00:10:50 different colored fluorochromes and find multiple proteins in relation 133 00:10:50 --> 00:10:55 to one another. So that's one method. 134 00:10:55 --> 00:11:00 To an extent, this method has been replaced by a new method which 135 00:11:00 --> 00:11:05 relies on the fact that there are naturally fluorescent molecules. 136 00:11:05 --> 00:11:11 Naturally fluorescent proteins, 137 00:11:11 --> 00:11:16 I should say. You've actually all seen these. If you go to the beach, 138 00:11:16 --> 00:11:21 you might have encountered jelly fish which fluoresce. 139 00:11:21 --> 00:11:26 And they fluoresce because they make a protein which itself 140 00:11:26 --> 00:11:32 fluoresces when activated by the proper UV wavelength. 141 00:11:32 --> 00:11:36 These proteins have been isolated from these species, 142 00:11:36 --> 00:11:40 cloned into plasmids, and then used in recombinant DNA 143 00:11:40 --> 00:11:44 techniques, like I told you about before, to visualize where other 144 00:11:44 --> 00:11:48 proteins go inside the cell. And I'll give you some examples of 145 00:11:48 --> 00:11:52 that in a minute. But here's the jelly fish naturally. 146 00:11:52 --> 00:11:56 Here are these structures. The jelly fish actually makes a 147 00:11:56 --> 00:12:00 light which then causes these protein bundles to fluoresce green. 148 00:12:00 --> 00:12:05 The gene was cloned, as I said. It was given the name 149 00:12:05 --> 00:12:21 GFP for green fluorescence protein. 150 00:12:21 --> 00:12:25 And a cDNA copy of the mRNA is used in molecular biology all over the 151 00:12:25 --> 00:12:30 place to do an experiment like this. And I'll take you through an 152 00:12:30 --> 00:12:35 example of another one like this in a moment where you can actually fuse 153 00:12:35 --> 00:12:40 your gene of interest, gene X, cDNA corresponding to that 154 00:12:40 --> 00:12:45 gene with the GFP cDNA to make a fusion protein that has your gene of 155 00:12:45 --> 00:12:50 interest, plus GFP as one long polypeptide. 156 00:12:50 --> 00:12:53 And now this protein will fluoresce. And you'll be able to visualize it. 157 00:12:53 --> 00:12:56 And, again, I'll give you an example of how that's done in a 158 00:12:56 --> 00:12:59 moment. We've also used this, we, the field has used this to make 159 00:12:59 --> 00:13:03 organisms, other organisms which fluoresce. 160 00:13:03 --> 00:13:07 In my lab, for example, we've mad green mice which express 161 00:13:07 --> 00:13:12 the GFP protein in the cells of the skin of the mouse. 162 00:13:12 --> 00:13:17 So if you shine UV light on the mouse it turns green. 163 00:13:17 --> 00:13:22 It's kind of cool. And others have made pets that fluoresce. 164 00:13:22 --> 00:13:27 Surprising, but true, you can actually buy a fluorescent rabbit. 165 00:13:27 --> 00:13:32 I'm not sure who sells them, but they are available commercially. 166 00:13:32 --> 00:13:36 And I decided to show you a picture of one, of a rabbit called Alba the 167 00:13:36 --> 00:13:40 fluorescent bunny, which should be showing here. 168 00:13:40 --> 00:13:44 But Alba is actually a little bit shy so she doesn't always come. 169 00:13:44 --> 00:13:48 Alba the fluorescent bunny. Oh, here she comes. So this is actually 170 00:13:48 --> 00:13:52 a fluorescent rabbit which was made by introducing this GFP gene into 171 00:13:52 --> 00:13:56 the cells of the skin, such that it expressed in the cells 172 00:13:56 --> 00:14:02 of the skin of the rabbit. So it's a very powerful technique, 173 00:14:02 --> 00:14:08 but let me give you a few more examples of how it might be useful. 174 00:14:08 --> 00:14:15 I know. Everybody now wants one. So if you have a cDNA for GFP, 175 00:14:15 --> 00:14:21 and actually there are clever molecular engineers who have 176 00:14:21 --> 00:14:27 modified this gene GFP to produce fluorescent proteins that fluoresce 177 00:14:27 --> 00:14:33 in the red wavelength. So they're RFP, 178 00:14:33 --> 00:14:37 red fluorescent proteins. There are yellow fluorescent 179 00:14:37 --> 00:14:41 proteins. There are blue called scion CFP fluorescent proteins. 180 00:14:41 --> 00:14:46 So there's a whole kit, a whole coloring box, if you will, 181 00:14:46 --> 00:14:50 of different color fluorescent proteins that you can play with. 182 00:14:50 --> 00:14:54 You can then add that, in techniques that you are familiar 183 00:14:54 --> 00:14:59 with, to a cDNA of your gene of interest. 184 00:14:59 --> 00:15:07 So this is a cDNA of your gene of 185 00:15:07 --> 00:15:13 interest. If you now combine those using recombinant DNA techniques 186 00:15:13 --> 00:15:20 that we told you about in previous lectures, you could make a plasmid 187 00:15:20 --> 00:15:26 which has GFP fused directly in frame, to make a long open reading 188 00:15:26 --> 00:15:33 frame with your gene of interest. OK? And in that plasmid you would 189 00:15:33 --> 00:15:39 also include a promoter to allow the expression of this fusion. 190 00:15:39 --> 00:15:45 And it's also necessary to have, at the other end, a polyadenylation 191 00:15:45 --> 00:15:51 sequence, again, to insure proper expression of this 192 00:15:51 --> 00:15:57 fusion protein in the cells. And this would then be housed 193 00:15:57 --> 00:16:02 inside of a plasmid vector. OK? We could then transfect, 194 00:16:02 --> 00:16:07 introduce, using methods similar to transformation, 195 00:16:07 --> 00:16:12 but now not into bacteria. Instead into mammalian cells. 196 00:16:12 --> 00:16:18 We could introduce this plasmid into cells in culture so that in the 197 00:16:18 --> 00:16:23 nucleus of these cells that I've modified, I now have this new gene 198 00:16:23 --> 00:16:28 present which can express a green fusion protein with my 199 00:16:28 --> 00:16:33 gene of interest. Now, importantly, 200 00:16:33 --> 00:16:37 the goal of this experiment is to understand where the protein that's 201 00:16:37 --> 00:16:41 encoded by my gene of interest is localized. And the way that I'm 202 00:16:41 --> 00:16:45 going to do that is to follow where the green color goes. 203 00:16:45 --> 00:16:49 If it's a cytoplasmic protein the green will be in the cytoplasm. 204 00:16:49 --> 00:16:53 If it's a nuclear protein the green will be in the nucleus and so on. 205 00:16:53 --> 00:16:57 So if I now look at this cell by fluorescence microscopy, 206 00:16:57 --> 00:17:02 I will see the green signal in the nucleus. 207 00:17:02 --> 00:17:06 OK? It's a very, very powerful technique that allows 208 00:17:06 --> 00:17:10 us to follow the products of genes of interest wherever they might go 209 00:17:10 --> 00:17:14 inside of cells. And I just made a collection of 210 00:17:14 --> 00:17:19 images from the literature. Here you can see, actually, 211 00:17:19 --> 00:17:23 two different proteins being visualized simultaneously. 212 00:17:23 --> 00:17:27 The blue is actually a DNA stain. That shows you were the nuclei are. 213 00:17:27 --> 00:17:31 The green is one particular protein. And you can see that it's largely 214 00:17:31 --> 00:17:35 cytoplasmic. And the red is another protein rimming the plasma membrane 215 00:17:35 --> 00:17:39 of these cells. There are a whole bunch of cells 216 00:17:39 --> 00:17:43 here. You can see their shape by where the red color is. 217 00:17:43 --> 00:17:46 And the green is then marking the other protein which is largely 218 00:17:46 --> 00:17:50 cytoplasmic. In this example, similar to what I drew on the board, 219 00:17:50 --> 00:17:54 the fusion protein is in the nucleus. So the fusion protein is a nuclear 220 00:17:54 --> 00:17:58 protein. It drags the GFP with it into the nucleus. 221 00:17:58 --> 00:18:01 This is a structure called the endoplasmic reticulum, 222 00:18:01 --> 00:18:04 the ER. We'll come back to it later in the lecture. 223 00:18:04 --> 00:18:07 It's involved in the sorting of proteins to different parts of the 224 00:18:07 --> 00:18:11 cell. And here's an ER protein that's been fussed to GFP. 225 00:18:11 --> 00:18:14 So now the signal is coming from this structure inside the cell, 226 00:18:14 --> 00:18:17 the endoplasmic reticulum. And here is a protein which resides in the 227 00:18:17 --> 00:18:21 mitochondria, that energy-producing organelle inside the cell. 228 00:18:21 --> 00:18:24 You can drag GFP into the mitochondria to highlight 229 00:18:24 --> 00:18:28 those structures. Here's a red example, 230 00:18:28 --> 00:18:32 RFP fused to a cytoskeletal protein, so you now see these long filaments 231 00:18:32 --> 00:18:36 inside the cell now fluorescing red. And here's another kind of neat 232 00:18:36 --> 00:18:40 example. This is a protein that moves. It's normally present in the 233 00:18:40 --> 00:18:44 cytoplasm. As you can see here it's filling the cytoplasm of the cell 234 00:18:44 --> 00:18:48 and it's not in the nucleus which is labeled green. 235 00:18:48 --> 00:18:52 However, in response to a signal the protein moves. 236 00:18:52 --> 00:18:56 It goes from the cytoplasm into the nucleus. It might be, 237 00:18:56 --> 00:19:00 for example, a transcription factor, which is kept in the cytoplasm so 238 00:19:00 --> 00:19:04 that it won't turn on the expression of its target genes. 239 00:19:04 --> 00:19:08 But in response to the signal it now moves from the cytoplasm into the 240 00:19:08 --> 00:19:12 nucleus such that it can access the DNA and begin to turn on the 241 00:19:12 --> 00:19:17 expression of those genes. OK? So proteins can be followed 242 00:19:17 --> 00:19:21 dynamically. And you can actually do movies of these things 243 00:19:21 --> 00:19:30 over time. OK. 244 00:19:30 --> 00:19:36 So proteins can be moved from place to place. How do they get there? 245 00:19:36 --> 00:19:42 What is the mechanism by which proteins move from one compartment 246 00:19:42 --> 00:19:48 of the cell to another? How do they know where to go? 247 00:19:48 --> 00:19:54 There are two answers to this question. One is signals 248 00:19:54 --> 00:20:02 in the polypeptide. 249 00:20:02 --> 00:20:06 Signals within the polypeptide tell the protein where to go. 250 00:20:06 --> 00:20:11 These signals can be short, a few amino acids, four, five amino 251 00:20:11 --> 00:20:15 acids, they can be a little longer, 15 or 20 amino acids, but they 252 00:20:15 --> 00:20:20 mostly function as a molecular zip code. They can be read by the 253 00:20:20 --> 00:20:25 cell's protein sorting machinery. Like a post office reads a zip code, 254 00:20:25 --> 00:20:30 it knows to send certain letters to certain places. 255 00:20:30 --> 00:20:34 This machinery reads these signals and sends proteins to different 256 00:20:34 --> 00:20:38 places within the cell. There's also a separate and 257 00:20:38 --> 00:20:45 sometimes related mechanism -- 258 00:20:45 --> 00:20:48 -- known as post-translational modification. 259 00:20:48 --> 00:20:53 Here it's not a sequence within the 260 00:20:53 --> 00:20:56 protein amino acid sequence. It's not a signal within the amino 261 00:20:56 --> 00:20:59 acid sequence, but rather it's something that's 262 00:20:59 --> 00:21:02 added onto the protein after it's been made. That's why it's called 263 00:21:02 --> 00:21:06 post-translational. And, again, this can influence where 264 00:21:06 --> 00:21:11 the protein ends up inside the cell. So let me give you some examples of 265 00:21:11 --> 00:21:16 what I'm talking about here. With respect to signals that are 266 00:21:16 --> 00:21:22 contained within the polypeptide, proteins that are synthesized in the 267 00:21:22 --> 00:21:27 cytoplasm on ribosomes, this is a ribosome, this is a mRNA, 268 00:21:27 --> 00:21:32 5 prime end, 3 prime end, will give rise, through the process of 269 00:21:32 --> 00:21:36 translation, to proteins. And when this process is completed, 270 00:21:36 --> 00:21:40 those proteins will be released into the cytoplasm of the cell. 271 00:21:40 --> 00:21:44 OK? Most translation occurs freely inside the cytoplasm of the cell. 272 00:21:44 --> 00:21:48 There are exceptions to that, as I'll show you in a moment. 273 00:21:48 --> 00:21:52 Most of it takes place freely in the cytoplasm of the cell, 274 00:21:52 --> 00:21:56 so when the process is done protein winds up in the cytoplasm. 275 00:21:56 --> 00:22:00 These proteins have two fates generally speaking. 276 00:22:00 --> 00:22:04 One is that they remain cytoplasmic. And I gave you some examples of 277 00:22:04 --> 00:22:08 cytoplasmic proteins earlier. Lots of the enzymes that are 278 00:22:08 --> 00:22:12 present inside your cells just hang out inside the cytoplasm and do 279 00:22:12 --> 00:22:17 their job. Other proteins are synthesized first in the cytoplasm 280 00:22:17 --> 00:22:21 will end up going to the nucleus, and these proteins are 281 00:22:21 --> 00:22:25 distinguishable by virtue of the fact that the ones that end up in 282 00:22:25 --> 00:22:30 the nucleus have a little tag on them. 283 00:22:30 --> 00:22:37 And this little tag has a name called the nuclear localization 284 00:22:37 --> 00:22:47 sequence. 285 00:22:47 --> 00:22:52 This is one of the short tags. It can be just four or five amino 286 00:22:52 --> 00:22:58 acids, usually positively charged. And inside the cytoplasm of this 287 00:22:58 --> 00:23:04 cell there are proteins which will recognize that little tag. 288 00:23:04 --> 00:23:10 Attached to it these proteins belong to a class known as importins. 289 00:23:10 --> 00:23:16 And what the importins do is drag the protein that has an NLS to the 290 00:23:16 --> 00:23:22 nucleus where they interact with a structure within the membrane of the 291 00:23:22 --> 00:23:28 nucleus known as the nuclear pore complex. 292 00:23:28 --> 00:23:37 And importin then drags the protein 293 00:23:37 --> 00:23:41 into the nucleus. It imports it into the nucleus. 294 00:23:41 --> 00:23:45 So nuclear proteins get into the nucleus by virtue of the fact that 295 00:23:45 --> 00:23:49 they have a nuclear localization sequence on them. 296 00:23:49 --> 00:23:53 OK? And you might imagine, and probably you will think hard 297 00:23:53 --> 00:23:57 about how you could visualize this process of nuclear importation, 298 00:23:57 --> 00:24:01 for example, in a problem set. So that's how cytoplasmic resident 299 00:24:01 --> 00:24:05 proteins are distinguished from nuclear proteins. 300 00:24:05 --> 00:24:08 What about the proteins that fully leave the cell? 301 00:24:08 --> 00:24:12 Many proteins that you make actually get secreted. 302 00:24:12 --> 00:24:15 They leave the cell entirely. Still other proteins wind up on the 303 00:24:15 --> 00:24:19 membrane itself. We're going to talk later in the 304 00:24:19 --> 00:24:22 lecture and next time about receptors that sit on the outside of 305 00:24:22 --> 00:24:26 your cell waiting for signals to be sent. Those proteins have to get 306 00:24:26 --> 00:24:30 there, and there's a special mechanism that insures that. 307 00:24:30 --> 00:24:43 So this applies to both secreted, 308 00:24:43 --> 00:24:49 as well as what we refer to as transmembrane proteins. 309 00:24:49 --> 00:24:57 And here the situation is a little 310 00:24:57 --> 00:25:01 bit different. In this case, when the ribosomes 311 00:25:01 --> 00:25:05 synthesize the very first few amino acids, the very first polypeptide 312 00:25:05 --> 00:25:09 chain, at the end, the end terminal end of these 313 00:25:09 --> 00:25:13 polypeptides, as they're emerging from the ribosome there is a longer, 314 00:25:13 --> 00:25:17 more like 15 or 20 amino acid sequence known as the 315 00:25:17 --> 00:25:25 signal sequence. 316 00:25:25 --> 00:25:28 If a protein has a signal sequence on its end you know it's going to 317 00:25:28 --> 00:25:31 end up as a secreted protein or a transmembrane protein. 318 00:25:31 --> 00:25:34 And when it emerges from the bottom of the ribosome, 319 00:25:34 --> 00:25:37 as depicted here, there's a specialized protein that 320 00:25:37 --> 00:25:40 will bind to it, it's actually a complex of proteins 321 00:25:40 --> 00:25:44 called the signal recognition particle. 322 00:25:44 --> 00:25:55 SRP has a very important function, 323 00:25:55 --> 00:25:59 actually two important functions. Firstly, when it binds to the 324 00:25:59 --> 00:26:03 ribosome, when it binds to the signal sequence like this it leads 325 00:26:03 --> 00:26:11 to translational arrest. 326 00:26:11 --> 00:26:17 It stops translation. It blocks the process of 327 00:26:17 --> 00:26:24 translation right at this point. OK? The next thing that happens is 328 00:26:24 --> 00:26:30 that it drags this complex of the ribosome with its little nascent 329 00:26:30 --> 00:26:37 polypeptide to a structure known as the endoplasmic reticulum. 330 00:26:37 --> 00:26:51 In the membrane of the endoplasmic 331 00:26:51 --> 00:26:57 reticulum. That was really loud. Is it that phone? I got to do this 332 00:26:57 --> 00:27:02 the other day in a class. Hello. Oh, they hung up. 333 00:27:02 --> 00:27:06 Last time I was teaching a class and the computer guy left his cell 334 00:27:06 --> 00:27:11 phone, and it rang. And the name of the person calling 335 00:27:11 --> 00:27:16 actually showed up on the screen, so I picked it up and I said Herb. 336 00:27:16 --> 00:27:20 And we actually talked for about five minutes and I told him I had to 337 00:27:20 --> 00:27:25 go and keep teaching a class. Anyway, inside the endoplasmic 338 00:27:25 --> 00:27:30 reticulum there's another receptor, not unlike the nuclear pore complex. 339 00:27:30 --> 00:27:35 This is called the SRP receptor. It binds to this thing SRP, signal 340 00:27:35 --> 00:27:41 recognition particle. And this then allows SRP to go away 341 00:27:41 --> 00:27:47 and allows translation to continue allowing the protein to be 342 00:27:47 --> 00:27:53 synthesized through this membrane and end up inside the endoplasmic 343 00:27:53 --> 00:27:59 reticulum. So the end result of this process is that the protein 344 00:27:59 --> 00:28:05 will now reside within this structure, this membrane-bound 345 00:28:05 --> 00:28:12 structure. Subsequently, the protein leaves 346 00:28:12 --> 00:28:24 this structure in a vesicle. 347 00:28:24 --> 00:28:29 It buds off the membrane, gets carried like cargo in a little 348 00:28:29 --> 00:28:34 pod coming off the mother ship here. It goes to another sorting 349 00:28:34 --> 00:28:39 structure called the Golgi apparatus. And it eventually goes to the 350 00:28:39 --> 00:28:45 plasma membrane where it fuses to the plasma membrane allowing this 351 00:28:45 --> 00:28:50 secreted protein to leave the cell. I realize that this is a little bit 352 00:28:50 --> 00:28:56 messy, but there are comparable pictures in your book. 353 00:28:56 --> 00:28:59 The point being that these proteins have a signal which drags them to 354 00:28:59 --> 00:29:03 the ER. There's then a sorting process that takes them out of the 355 00:29:03 --> 00:29:07 cell, takes them to the plasma membrane and then out of the cell. 356 00:29:07 --> 00:29:11 So that's how you deal with secreted proteins. 357 00:29:11 --> 00:29:14 How do you deal with proteins that end up in the membrane? 358 00:29:14 --> 00:29:18 If they don't get secreted they wind up resident in the plasma 359 00:29:18 --> 00:29:22 membrane. This happens by virtue of another signal. 360 00:29:22 --> 00:29:25 And I'm going to pick up this process, the beginning of the 361 00:29:25 --> 00:29:29 process is exactly the same. I'm going to pick up this process 362 00:29:29 --> 00:29:33 at a slightly later point where we have the SRP receptor 363 00:29:33 --> 00:29:37 bound by a ribosome. It's actually partway through the 364 00:29:37 --> 00:29:42 process of producing a polypeptide that has wound up inside the ER. 365 00:29:42 --> 00:29:47 For proteins that wind up ultimately in the membrane there's 366 00:29:47 --> 00:29:53 another sequence called a stop transfer sequence. 367 00:29:53 --> 00:30:02 And when a stop transfer sequence is 368 00:30:02 --> 00:30:06 hit and recognized by this complex it causes the ribosome to 369 00:30:06 --> 00:30:11 disassociate from the mRNA, or rather allows the synthesis to 370 00:30:11 --> 00:30:16 occur without continuous transfer through this complex. 371 00:30:16 --> 00:30:20 So the protein just gets built on this side of the membrane. 372 00:30:20 --> 00:30:25 And the result of that then is a protein that ends up halfway in and 373 00:30:25 --> 00:30:30 halfway out of the endoplasmic reticulum. 374 00:30:30 --> 00:30:35 If you then take this through the sort of sorting steps that I just 375 00:30:35 --> 00:30:40 outlined for secreted proteins, this protein will end up with part 376 00:30:40 --> 00:30:46 of it on the outside and part of it on the inside of the cell. 377 00:30:46 --> 00:30:53 OK? So that's how we deal with 378 00:30:53 --> 00:30:57 proteins that are transmembrane. And you'll actually see proteins 379 00:30:57 --> 00:31:01 that go through the membrane multiple times. 380 00:31:01 --> 00:31:05 That's because they have signal sequences and stop transfer sequence, 381 00:31:05 --> 00:31:10 signal sequences and stop transfer sequences that allow them to 382 00:31:10 --> 00:31:15 transition through the membrane more than once. Many of the proteins 383 00:31:15 --> 00:31:20 just once but other proteins multiple times. 384 00:31:20 --> 00:31:25 OK. So that's the way it's done in terms of signal recognition. 385 00:31:25 --> 00:31:32 I also mentioned that there is this 386 00:31:32 --> 00:31:38 class of posttranslational modification. Proteins get modified 387 00:31:38 --> 00:31:45 after they're made by translation. And the examples include 388 00:31:45 --> 00:31:51 proteolysis. The protein might actually get cleaved, 389 00:31:51 --> 00:31:58 cut. Half of it goes one place. The other half goes somewhere else. 390 00:31:58 --> 00:32:07 Phosphorylation. 391 00:32:07 --> 00:32:13 Addition of a phosphate can determine the localization of a 392 00:32:13 --> 00:32:19 protein. Glycosylation. Addition of sugar groups, 393 00:32:19 --> 00:32:25 carbohydrates onto the protein can determine where the protein goes. 394 00:32:25 --> 00:32:33 And finally lipid addition. 395 00:32:33 --> 00:32:37 Addition of a lipid molecule can determine where the protein goes. 396 00:32:37 --> 00:32:41 And I'll give you one example of that last one. 397 00:32:41 --> 00:32:46 A protein that we work on in my lab, the protein called RAS, 398 00:32:46 --> 00:32:50 which will actually come up in the next lecture. It's a signaling 399 00:32:50 --> 00:32:55 protein, which actually turns out to be very important in cancer. 400 00:32:55 --> 00:32:59 In its unmodified state the protein is cytoplasmic. 401 00:32:59 --> 00:33:04 However, it gets modified by an enzyme called farnesyltransferase. 402 00:33:04 --> 00:33:12 Which modifies the very end of the 403 00:33:12 --> 00:33:18 protein, the C terminal end of the protein, and it sticks onto it a 404 00:33:18 --> 00:33:24 lipid group, about a 20 carbon lipid group, which is very much like the 405 00:33:24 --> 00:33:30 fatty acid side chains in the plasma membrane. 406 00:33:30 --> 00:33:33 And, in fact, this is a very hydrophobic structure. 407 00:33:33 --> 00:33:37 And it ends up dragging the protein to the plasma membrane. 408 00:33:37 --> 00:33:41 Here's the plasma membrane. I'm drawing it now as a lipid 409 00:33:41 --> 00:33:45 bilayer, whereas I drew it as a single line previously. 410 00:33:45 --> 00:33:48 And that lipid tail, which has been stuck on the RAS protein, 411 00:33:48 --> 00:33:52 inserts into the lipid bilayer and localizes RAS then to the plasma 412 00:33:52 --> 00:33:56 membrane. OK? So here are examples of 413 00:33:56 --> 00:34:00 post-translational modifications that effect where the protein 414 00:34:00 --> 00:34:04 ends up going. So we can determine where the 415 00:34:04 --> 00:34:08 protein goes, that's very important. I also want to make the point 416 00:34:08 --> 00:34:12 before we move onto the next topic that protein-protein interactions 417 00:34:12 --> 00:34:16 are very important in determining function. These proteins that we've 418 00:34:16 --> 00:34:20 been talking about almost never, probably never do their functions by 419 00:34:20 --> 00:34:24 themselves. They interact with other proteins. 420 00:34:24 --> 00:34:28 And these are determined, again, by sequences that bind to one 421 00:34:28 --> 00:34:31 another between the two proteins. So this is actually a protein 422 00:34:31 --> 00:34:35 structure from a colleague here at MIT, Thomas Schwartz, 423 00:34:35 --> 00:34:39 solved by him. What you're going to look at hopefully, 424 00:34:39 --> 00:34:42 this is a ribbon diagram of two proteins, the one in blue and the 425 00:34:42 --> 00:34:46 one in pink. And you can see that these two proteins bind to each 426 00:34:46 --> 00:34:49 other based on amino acid residues at this interface between the two 427 00:34:49 --> 00:34:53 proteins. This ribbon diagram will be replaced by a space-filling model. 428 00:34:53 --> 00:34:57 This is what the protein really would like inside the cell. 429 00:34:57 --> 00:35:00 And here's that interface more clearly visualized with residues in 430 00:35:00 --> 00:35:04 light blue on one side, pink on the other that allows the 431 00:35:04 --> 00:35:07 two proteins to interact. So protein-protein interaction is 432 00:35:07 --> 00:35:11 extremely important, allowing proteins to interact either 433 00:35:11 --> 00:35:15 for a long time because they carry out some essential function together 434 00:35:15 --> 00:35:19 or very transiently. Protein-protein interactions can be 435 00:35:19 --> 00:35:23 long-lasting or very transient. And to just give you a visual 436 00:35:23 --> 00:35:27 reminder of that, the transient interaction, 437 00:35:27 --> 00:35:31 this I took a couple of years ago, Johnny Damon getting run into by 438 00:35:31 --> 00:35:35 Damian Jackson. A very transient but actually quite 439 00:35:35 --> 00:35:39 important interaction between these two baseball players. 440 00:35:39 --> 00:35:43 And then there can be longer lasting interactions. 441 00:35:43 --> 00:35:48 Here's Brad Pitt, well, it's not that long-lasting, 442 00:35:48 --> 00:35:52 I guess, but the point is that proteins do interact either very 443 00:35:52 --> 00:35:56 transiently or in a long-lasting way. Jennifer actually did the cutting 444 00:35:56 --> 00:36:01 out herself of this picture that she sent me. 445 00:36:01 --> 00:36:08 OK. Finally, proteins can change 446 00:36:08 --> 00:36:12 their shape. Proteins can change their shape in response to binding 447 00:36:12 --> 00:36:16 of other proteins or binding of cofactors that they interact with. 448 00:36:16 --> 00:36:21 And here's another movie from Thomas Schwartz. 449 00:36:21 --> 00:36:25 This is a signaling protein, much like the RAS protein actually. 450 00:36:25 --> 00:36:29 And you'll see that upon binding to a nucleotide here, 451 00:36:29 --> 00:36:34 the protein dramatically changes its shape. 452 00:36:34 --> 00:36:38 It actually unwinds a little bit and exposes a new structure for binding. 453 00:36:38 --> 00:36:42 And this is one of the critical ways that proteins participate in 454 00:36:42 --> 00:36:46 this signal transduction, signal processing function. 455 00:36:46 --> 00:36:50 They receive signals, change their shape, and this allows them to bind 456 00:36:50 --> 00:36:54 to other proteins or carry out other functions. OK. 457 00:36:54 --> 00:36:58 So I want to move now to the signaling introduction for the last 458 00:36:58 --> 00:37:02 12 minutes or so. I started the day with this slide of 459 00:37:02 --> 00:37:06 cells moving around. They're receiving signals from one 460 00:37:06 --> 00:37:10 another or from the medium and they're responding to it. 461 00:37:10 --> 00:37:14 I also gave you examples of cells dividing or not dividing, 462 00:37:14 --> 00:37:18 cells differentiating, turning into one type of end-stage cell or 463 00:37:18 --> 00:37:22 another, changing their function. We'll talk later about cells that 464 00:37:22 --> 00:37:26 receive signals and kill other cells, cells that receive signals and 465 00:37:26 --> 00:37:30 commit suicide in a couple of more lectures. 466 00:37:30 --> 00:37:36 So cells will do very complicated things in receipt of various signals. 467 00:37:36 --> 00:37:42 And this general process is known as signal transduction. 468 00:37:42 --> 00:37:49 And basically it's the ability of cells to respond to things in their 469 00:37:49 --> 00:37:55 extracellular environment, stimuli in their extracellular 470 00:37:55 --> 00:38:01 environment. These could be physical stimuli, 471 00:38:01 --> 00:38:06 light, odorants, or they could be biological stimuli, 472 00:38:06 --> 00:38:12 peptides, proteins, hormones, which then get sent first from the 473 00:38:12 --> 00:38:17 outside of the cell and then to the inside of the cell leading to either 474 00:38:17 --> 00:38:29 short-term responses -- 475 00:38:29 --> 00:38:33 I'll give you an example of glucose secretion in response to a hormone 476 00:38:33 --> 00:38:37 epinephrine or adrenalin. This happens exclusively inside the 477 00:38:37 --> 00:38:42 cytoplasm of the cell. The signal is received, 478 00:38:42 --> 00:38:46 processed, and the result is the secretion of glucose. 479 00:38:46 --> 00:38:51 Other signals will make their way into the nucleus of the cell, 480 00:38:51 --> 00:38:55 and this might lead to changes in gene expression. 481 00:38:55 --> 00:39:00 New genes are turned. Others might be turned off. 482 00:39:00 --> 00:39:04 Changes in gene expression. And this might lead to a longer 483 00:39:04 --> 00:39:09 term response. And we'll give you an example of 484 00:39:09 --> 00:39:14 this in the next lecture. So how does this work? Well, 485 00:39:14 --> 00:39:19 it's different in every way. Each example is slightly different. 486 00:39:19 --> 00:39:24 I show you this slide to give you sort of a visual picture of what 487 00:39:24 --> 00:39:28 it's like, we think. Like one of these, 488 00:39:28 --> 00:39:32 what do they call them, Kineto-something or rather 489 00:39:32 --> 00:39:35 sculptures that you see at Logan Airport, for example, 490 00:39:35 --> 00:39:39 where a small perturbation at one end is propagated through a series 491 00:39:39 --> 00:39:42 of devices and leads to sounds and whistles and movements taking place 492 00:39:42 --> 00:39:46 within the structure. It's not unlike dominos. 493 00:39:46 --> 00:39:49 When you flip a domino at one end it can spread, 494 00:39:49 --> 00:39:53 increasing its effect because of the involvement of additional dominoes 495 00:39:53 --> 00:39:57 as it goes along a complex structure. 496 00:39:57 --> 00:40:01 We actually found, as we were looking at the Web for 497 00:40:01 --> 00:40:06 pictures of this movie of Mr. Domino, does anybody know what Mr. 498 00:40:06 --> 00:40:10 Domino is? I had no idea. It's a computer game, 499 00:40:10 --> 00:40:15 and they have a cool little animation which I thought I would 500 00:40:15 --> 00:40:19 show you. Here's Mr. Domino. And it's actually kind of 501 00:40:19 --> 00:40:24 relevant because he'll go and his signal is being transmitted along a 502 00:40:24 --> 00:40:29 linear path involving other types of molecules spreading out. 503 00:40:29 --> 00:40:34 Again, other types of molecules being involved, 504 00:40:34 --> 00:40:39 and so on and so forth. It's not a great example but it is 505 00:40:39 --> 00:40:44 kind of fun. OK. So before I give you a specific 506 00:40:44 --> 00:40:49 example, I want to give you some principles about signal transduction. 507 00:40:49 --> 00:40:55 And, again, the principles that are relevant here in biology are not 508 00:40:55 --> 00:41:00 dissimilar to signal propagation in electrical engineering, 509 00:41:00 --> 00:41:05 for example. Signal transduction in its simplest 510 00:41:05 --> 00:41:11 form means that something which takes some form, 511 00:41:11 --> 00:41:16 like a ligand binding to a receptor on the surface of the cell, 512 00:41:16 --> 00:41:22 changes its form inside the cell. It causes a change at the membrane 513 00:41:22 --> 00:41:28 to produce a different type of response inside the cell. 514 00:41:28 --> 00:41:32 Importantly, for almost all of these signal transduction pathways, 515 00:41:32 --> 00:41:37 what happens at the membrane is usually inadequate to change 516 00:41:37 --> 00:41:42 whatever process is to be changed inside the cell such that it is 517 00:41:42 --> 00:41:47 necessary for signal amplification to take place. 518 00:41:47 --> 00:41:52 And we'll give you examples of how signal amplification occurs in 519 00:41:52 --> 00:41:57 biology. But rather than making one square, in fact, you might 520 00:41:57 --> 00:42:02 make multiple squares. There's also the opportunity, 521 00:42:02 --> 00:42:07 and it's often the case for signal diversification, 522 00:42:07 --> 00:42:12 so that in response to the same signal, in fact, 523 00:42:12 --> 00:42:16 processed slightly different ways temporally or spatially with inside 524 00:42:16 --> 00:42:21 the cell you might make, sorry, let's make this a circle. 525 00:42:21 --> 00:42:26 You might make two different responses that coordinate the 526 00:42:26 --> 00:43:00 overall cellular response. 527 00:43:00 --> 00:43:03 There's the opportunity for what we call signal integration. 528 00:43:03 --> 00:43:11 So with respect to signal 529 00:43:11 --> 00:43:16 integration, you might imagine one signal which produces a particular 530 00:43:16 --> 00:43:21 response. A separate signal which produces a distinct response. 531 00:43:21 --> 00:43:26 But if you add the two together, that is if both signals are received 532 00:43:26 --> 00:43:32 simultaneously, you get some third response. 533 00:43:32 --> 00:43:35 So signal integration, again, not unlike electrical 534 00:43:35 --> 00:43:39 engineering. And finally signal modulation. 535 00:43:39 --> 00:43:47 So let's imagine again that we have, 536 00:43:47 --> 00:43:52 in some circumstances, a particular signal giving a particular response. 537 00:43:52 --> 00:43:57 If, in a different context with a different signal being received by 538 00:43:57 --> 00:44:02 the cell at the same time, instead of producing this response, 539 00:44:02 --> 00:44:08 there's an inhibitory effect so that nothing is produced. 540 00:44:08 --> 00:44:12 This signal inhibits the processing of this signal, 541 00:44:12 --> 00:44:17 so this is inhibitory. And you could imagine, 542 00:44:17 --> 00:44:21 and we know exists, the opposite such that in the presence of this 543 00:44:21 --> 00:44:26 signal plus now a distinct signal there's an augmentation of the 544 00:44:26 --> 00:44:31 response so that now you make much more of whatever you were making. 545 00:44:31 --> 00:44:36 So this is augmentation. So all of these things happen in 546 00:44:36 --> 00:44:42 the process of cellular signal transduction, cellular signal 547 00:44:42 --> 00:44:47 processing. At the beginning of this process, for at least the 548 00:44:47 --> 00:44:53 examples we're going to talk about, things happen at the plasma membrane. 549 00:44:53 --> 00:45:05 So here is the plasma membrane again. 550 00:45:05 --> 00:45:09 You can think of the plasma membrane as sort of the outside of 551 00:45:09 --> 00:45:13 your house with the antennas sticking up into the air waiting for 552 00:45:13 --> 00:45:18 signals to be received. Well, in biological circumstances, 553 00:45:18 --> 00:45:22 those antennas are polypeptide receptors. These are in this 554 00:45:22 --> 00:45:26 category. Or proteins that have transmembrane domains, 555 00:45:26 --> 00:45:31 get stuck in the membrane, and end up on the plasma membrane. 556 00:45:31 --> 00:45:35 These polypeptide receptors are sitting out there. 557 00:45:35 --> 00:45:40 There are often thousands of a given polypeptide receptor type on 558 00:45:40 --> 00:45:45 the outside of a cell. And these bind to ligands that fit 559 00:45:45 --> 00:45:49 inside them. There's complementarity between the shape of 560 00:45:49 --> 00:45:54 the ligand and the receptor such that you get specific binding of the 561 00:45:54 --> 00:45:59 ligand to the receptor. The consequences of binding of 562 00:45:59 --> 00:46:03 ligand to receptor we generally think of now as changing the shape 563 00:46:03 --> 00:46:08 of the receptor. Something that happens on the 564 00:46:08 --> 00:46:13 outside through binding of the ligand to the receptor is propagated 565 00:46:13 --> 00:46:17 through this polypeptide chain to a change in the structure at the 566 00:46:17 --> 00:46:22 inside. OK? It's as though I took this guy's head and I twisted it. 567 00:46:22 --> 00:46:27 Eventually his feet are going to move. The same thing here. 568 00:46:27 --> 00:46:31 If I make a change out here, I can propagate that change through 569 00:46:31 --> 00:46:36 the polypeptide chain and change the inside structure. 570 00:46:36 --> 00:46:41 So if you imagine a ligand which has a particular structure, 571 00:46:41 --> 00:46:45 I'm sorry a receptor that has a particular structure, 572 00:46:45 --> 00:46:50 when you add the ligand you now change the structure. 573 00:46:50 --> 00:46:55 And take a protein that might be inactive, this might have some 574 00:46:55 --> 00:47:00 enzymatic activity, from inactive to one that's active. 575 00:47:00 --> 00:47:05 Or another example, if I take this receptor and I bind 576 00:47:05 --> 00:47:11 the ligand, change its structure, I might now allow, based on this 577 00:47:11 --> 00:47:16 change in its structure, an interaction between this protein 578 00:47:16 --> 00:47:22 and some other signaling molecule, some other signaling protein. And, 579 00:47:22 --> 00:47:28 again, we know examples of all of those things. 580 00:47:28 --> 00:47:44 Usually, maybe always, 581 00:47:44 --> 00:47:48 the change that takes place at the plasma membrane, 582 00:47:48 --> 00:47:52 like the first domino that you flip, doesn't have enough energy 583 00:47:52 --> 00:47:56 associated with it to change the biological response. 584 00:47:56 --> 00:48:00 It's necessary to propagate that signal and to amplify that signal 585 00:48:00 --> 00:48:05 through various stages. This process of signal amplification 586 00:48:05 --> 00:48:11 is very important. And there are two general classes 587 00:48:11 --> 00:48:17 of signal amplification. One is the production of secondary 588 00:48:17 --> 00:48:23 signaling molecules called second messengers. And the example that 589 00:48:23 --> 00:48:30 we'll give you next time is the second messenger cyclic AMP -- 590 00:48:30 --> 00:48:34 -- which can be produced in large amounts following the binding of a 591 00:48:34 --> 00:48:39 ligand to a receptor, thereby amplifying the signal. 592 00:48:39 --> 00:48:43 And the other, which we'll give you as two examples next time, 593 00:48:43 --> 00:48:48 is enzymatic, very often kinase cascades. Enzymatic cascades where 594 00:48:48 --> 00:48:52 you turn on the activity of one enzyme, which turns on the activity 595 00:48:52 --> 00:48:57 of more other enzymes, which turn on the activity of still 596 00:48:57 --> 00:49:01 more other enzymes and so on. So, we'll talk about specifics 597 00:49:01 --> 00:49:04 related to these next time.