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Remember, mitosis is the process of cell division, but it’s just a portion of the full cell cycle. Figure 1 shows approximately how long a cell spends in each stage of the cell cycle:
As you can see, cells spend most of their time in interphase.
Video Review: The Cell Cycle
This video reviews all the steps of mitosis; seeing it all together is a great review at this stage.
A YouTube element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/biom1/?p=330
An improved MS2 system for accurate reporting of the mRNA life cycle
The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.
Conflict of interest statement
Competing Financial Interest
The material in this manuscript is the subject of a provisional application to the US Patent and Trademark Office. It has not been licensed to any corporation, and the authors (E.T, M.V. and R.H.S) are the sole inventors.
Figure 1. Current MBS systems resist degradation…
Figure 1. Current MBS systems resist degradation in yeast
(a, b) Scheme of ASH1 and…
500 cells per experiment, distribution of the mRNAs was generated using the same binning. (g) MBS aggregates in the cytoplasm are detected as bright fluorescent spots by smFISH. The percentage indicates cells positive for MBS aggregates. Yellow arrows = MBS aggregates. Scale bar = 5μm. (h) MBS aggregates are detected in the cytoplasm of living cells co-expressing MCP. (White arrows = single ASH1 (left) and MDN1 (right) mRNAs. Yellow arrows = MBS aggregates. Scale bar = 5μm.
Figure 2. Design and characterization of a…
Figure 2. Design and characterization of a new MS2-MCP system
(a) RNA stem loop sequences…
Figure 3. 12xMBSV6 faithfully reports the rapid…
Figure 3. 12xMBSV6 faithfully reports the rapid degradation of GAL1 mRNA
Figure 4. MBSV6-MCP maintains single mRNA resolution…
Figure 4. MBSV6-MCP maintains single mRNA resolution in living cells
100 cells per experiment. Non-parametric Mann-Whitney test does not show significant difference in brightness between 24xMBSV6 or 24xMBSORF populations, P= 0.6753. (d) Frequency distribution of MDN1 mRNAs per cell tagged with either 24xMBSV6 or 24xMBSORF. Single mRNA molecules counted from sample in 4b. Mean and SD of three independent cultures, n=
100 cells per experiment. (e) Scheme of the mixed cultures to compare the intensity of single MDN1 mRNAs tagged with either 24xMBSV6 or 12xMBSV6. Cells expressing 24xMBSV6-MCP co-express Nup49-tdTomato marking the nuclear envelope (red circle). (f) Two color live imaging of the mixed cultures. MERGE shows the mRNA signal (gray) and Nup49tdTomato (red). White arrows = single mRNAs. (g) Plot of the intensities of single MDN1 mRNAs tagged with either 24xMBSV6 (n= 1684) or 12xMBSV6 (n=861). Mean and SD of three independent cultures, n =
100 cells per experiment. Non-parametric Mann-Whitney test shows significant difference in brightness between 24xMBSV6 or 12xMBSV6 populations, P <0.0001. (h) Frequency distribution of MDN1 mRNAs per cell tagged with either 24xMBSV6 (red) or 12MBSV6 (blue). Single mRNA molecules counted from sample in 4f. Mean and SD of three independent cultures, n=
Figure 5. MBSV6-MCP enables single mRNA imaging…
Figure 5. MBSV6-MCP enables single mRNA imaging under stress conditions
Figure 6. MBSV6-MCP quantitatively reports ASH1 mRNA…
Figure 6. MBSV6-MCP quantitatively reports ASH1 mRNA levels throughout the cell cycle
Dr. Leland Hartwell is a former president and director of the Fred Hutchinson Cancer Research Center in Seattle, Washington. He earned his bachelor’s of Science from the California Institute of Technology in 1961. Hartwell continued his graduate education at Massachusetts Institute of Technology in Boston under the mentorship of Dr. Boris Magasanik. After his graduation… Continue Reading
Dr. Sue Biggins studied biology as an undergraduate at Stanford University and initially thought she would apply to medical school after receiving her degree. However, after a summer working in a research lab, she changed her mind and decided to apply to graduate school. Biggins received her PhD in molecular biology from Princeton and was… Continue Reading
More Talks in Genetics and Gene Regulation
The Four Stages of Complete Metamorphosis
Complete metamorphosis gives insects greater advantages in terms of survival, with each stage characterized by its behavioral, anatomical and physiological changes. Even the environment in which each form exists can differ.
Various theories exist as to the triggers for the passing from one stage into the next, including starvation, critical weight, gene upregulation, temperature, hormonal stimulation and time. However, the presence, quantity and balance of 20-hydroxyecdysone (20E) and juvenile hormone (JH) are probably the most important chemical guides of the process of complete metamorphosis.
Egg Stage of Complete Metamorphosis
The egg provides the genetic information necessary for all growth and function, including the blueprints for imaginal discs. Imaginal discs are present in insect embryos and eventually become anatomical parts of adult forms. Imaginal discs have the capacity to develop into carapaces, compound eyes, mandibles and exoskeletons, for example.
Insect eggs are produced in large numbers and deposited by way of the female ovipositor on protected, hidden surfaces. Where the egg is laid depends on the diet of the larval form. Butterflies lay their eggs on the underside of specific leaf types which their young will consume. One example is the cabbage white butterfly, who’s larvae decimate cabbage leaves as they gather the energy required for the next stage of complete metamorphosis.
The shell of an insect egg – the chorion – is tough. It forms within the adult female before fertilization, meaning sperm must enter by way of a network of channels or micropyles that provide access to the center of the egg via the chorion. Similarly, oxygen is transported in and carbon dioxide out through aeropyles. Aeropyles are not always present in the chorion of eggs laid under the surface of water. Instead, gases diffuse passively through multiple pores.
A further structure found on the chorion of both terrestrial and submerged insect eggs is the plastron network or the chorionic plastron which holds a thin sheet of air close to the surface of the egg. This ensures a supply of oxygen, even when the egg is covered by water.
Larval Stage of Complete Metamorphosis
The worm or maggot shape of an insect larva is usually a far cry from its adult form. Upon hatching from the egg, its primary goal is to consume energy in preparation for the huge morphological changes of the next stage of complete metamorphosis. This means that the most developed part of any larva’s anatomy is the alimentary canal.
Larvae also present with imaginal discs or imaginal buds that later form parts of the adult anatomy. The majority of larvae will go through at least one instar or larval stage, where it is necessary for the larva to cast off its skin to give it room to grow. In larva, this process has two stages: the separating of the cuticle from the underlying cells (apolysis), and the shedding or molting of the skin (ecdysis).
The final larval stage is known as the prepupa here the constant urge to feed stops and the larva becomes inactive.
Pupal Stage of Complete Metamorphosis
In the pupal stage, the imaginal discs of the insect embryo and larva become active. A carefully timed process of cell death and cell proliferation occurs, where larval cells die off and are broken down to provide energy for the countless processes involved in the development of an adult insect. An adult must be able to reproduce, and it is at this stage that the reproductive organs develop. The image below shows the various life stages of the ant.
The above image shows the different forms of the ant, from egg to pupal form.
It is important to distinguish between the pupal stage and the pupa structure. In the stage between larva and adult the insect is called a pharate. The protective housing that surrounds the pharate is known generically as a pupa this is often derived from the hardened cuticle of the now immobile larva. Other names including chrysalis, cocoon and tumbler depend on the insect type or additional covering materials, such as silk.
Imago Stage of Complete Metamorphosis
The emergence of an adult insect from the pupa is termed eclosion. Hormones released at the end of the pupal stage soften the shell wall, allowing the adult insect to emerge. The pupal case is left behind as an empty shell, and for a time the adult insect finds itself particularly exposed to the elements and predators.
This is because all wings are crumpled and damp, and the adult insect is unable to fly. Until the venous network of the wings has first been filled with meconium, and then with hemolymph through the pumping actions of the abdomen, an adult winged insect is very much at risk.
When the wings have unfolded, structures within dissolve and only small amounts of hemolymph are required to circulate within the wing veins, keeping them very lightweight and efficient. The insect is now mobile and able to fulfill its goal – to reproduce.
Trichinellosis (trichinosis) is caused by nematodes (roundworms) of the genus Trichinella. In addition to the classical agent T. spiralis (found worldwide in many carnivorous and omnivorous animals), several other species of Trichinella are now recognized, including T. pseudospiralis (mammals and birds worldwide), T. nativa (Arctic bears), T. nelsoni (African predators and scavengers), T. britovi (carnivores of Europe and western Asia), and T. papuae (wild and domestic pigs of Papua New Guinea and Thailand). Trichinella zimbabwensis is found in crocodiles in Africa but to date there are no known associations of this species with human disease.
Depending on the classification used, there are several species of Trichinella: T. spiralis, T. pseudospiralis, T. nativa, T. murelli, T. nelsoni, T. britovi, T. papuae, and T. zimbabwensis, all but the last of which have been implicated in human disease. Adult worms and encysted larvae develop within a single vertebrate host, and an infected animal serves as a definitive host and potential intermediate host. A second host is required to perpetuate the life cycle of Trichinella. The domestic cycle most often involved pigs and anthropophilic rodents, but other domestic animals such as horses can be involved. In the sylvatic cycle, the range of infected animals is great, but animals most often associated as sources of human infection are bear, moose and wild boar.
Trichinellosis is caused by the ingestion of undercooked meat containing encysted larvae (except for T. pseudospiralis and T. papuae, which do not encyst) of Trichinella species . After exposure to gastric acid and pepsin, the larvae are released from the cysts and invade the small bowel mucosa where they develop into adult worms . Females are 2.2 mm in length males 1.2 mm. The life span in the small bowel is about four weeks. After 1 week, the females release larvae that migrate to striated muscles where they encyst . Diagnosis is usually made based on clinical symptoms, and is confirmed by serology or identification of encysted or non-encysted larvae in biopsy or autopsy specimens.
10.27: The Complete Cycle - Biology
Remember, mitosis is the process of cell division, but it’s just a portion of the full cell cycle. Figure 1 shows approximately how long a cell spends in each stage of the cell cycle:
Figure 1. The cell cycle consists of interphase and the mitotic phase. During interphase, the cell grows and the nuclear DNA is duplicated. Interphase is followed by the mitotic phase. During the mitotic phase, the duplicated chromosomes are segregated and distributed into daughter nuclei. The cytoplasm is usually divided as well, resulting in two daughter cells.
As you can see, cells spend most of their time in interphase.
Video Review: The Cell Cycle
This video reviews all the steps of mitosis seeing it all together is a great review at this stage.
Part 2: Controlling the Cell Cycle: Cdk Substrates
00:00:02.03 So, hello, my name is Dave Morgan. I'm from the University of California in San Francisco.
00:00:06.04 And in this lecture I'm going to go over some of my own work on studies of
00:00:09.23 how the cyclin-dependent kinases drive the events of the eukaryotic cell division cycle.
00:00:15.29 Now itâ€™s well established at this point that the major regulators
00:00:19.11 of the eukaryotic cell cycle are the cyclin dependent kinases or Cdks.
00:00:23.13 And the basic idea is that a series of Cdk-cyclin complexes are activated
00:00:28.11 in a specific sequence during the cell cycle
00:00:30.08 to trigger the events of the cell cycle in the appropriate order.
00:00:33.21 And so, for example, S-phase Cdk-cyclin complex is formed in late mitosis or in late G1
00:00:39.15 and are then activated in the beginning of S-phase to initiate DNA synthesis.
00:00:43.03 And then M-phase Cdk-cyclin complexes form at the end of G2
00:00:47.06 and are activated to initiate the events of mitosis and take the cell to metaphase.
00:00:52.17 So the big question that I want to address today is how is it that these
00:00:55.26 Cdks actually drive these cell cycle events?
00:00:59.05 Now obviously, Cdks are protein kinases, which means that the most likely mechanism
00:01:02.24 by which they promote cell cycle events is through the phosphorylation of other proteins
00:01:07.10 which then bring about those events.
00:01:09.15 And so over the past 10 or 12 years or so, we've dedicated quite a lot of effort
00:01:13.18 to identifying the substrates of the cyclin-dependent kinases
00:01:16.20 in the hope that that will lead us to an answer to this question of
00:01:19.17 how the Cdks actually initiate cell cycle events.
00:01:23.23 So in this lecture I'm going to tell you about two methods that we've used
00:01:26.23 to systematically and comprehensively identify Cdk substrates
00:01:30.09 and then in the second half of the lecture we'll go into some interesting ways
00:01:33.20 in which we use those lists of substrates to address some general questions of
00:01:37.19 Cdk function and phospho-regulation.
00:01:41.07 So the first method we use to identify Cdk substrates
00:01:43.22 began about 10 or 12 years ago in a collaboration with Kevan Shokat, a chemist here at UCSF.
00:01:51.00 Now, Kevan came up with an idea whereby it would be possible to label the specific targets of
00:01:57.04 a protein kinase in a crude cell mixture and this slide attempts to explain that basic method.
00:02:02.09 On the left. let's focus on the left first.
00:02:04.08 On the left is a wild type regular protein kinase like Cdk1 with its cyclin regulatory partner.
00:02:10.08 And typically when one wants to label the targets of a protein kinase, like Cdk1,
00:02:14.28 you simply provide that kinase with a version of ATP in which the gamma phosphate
00:02:20.00 is labeled with a radioactive tag.
00:02:22.28 And then when that protein kinase uses that ATP it will then transfer
00:02:27.24 that P-32 onto its substrates.
00:02:31.11 Now, unfortunately this cannot be used to identify unknown substrates
00:02:34.24 of Cdks because if you take a pure Cdk and some gamma labeled P-32-ATP
00:02:40.16 and put that into a crude cell lysate, you will get not only the labeling of the Cdk's targets
00:02:46.12 but the labeling of all the other protein kinase targets in that lysate because
00:02:49.15 that ATP can be used by any kinase.
00:02:52.22 And so Kevan Shokat's idea was to avoid this problem by using
00:02:56.15 so called analog-sensitive protein kinases.
00:02:58.29 And the strategy is based on the fact that protein kinases
00:03:02.07 tend to contain a large hydrophobic residue in the wall
00:03:05.19 of the adenine binding pocket of their active site.
00:03:08.21 And so the basic strategy is to mutate that large hydrophobic side chain there
00:03:12.28 to a glycine residue resulting in the formation of an extra pocket in
00:03:16.18 the side of that ATP binding site. And as a result
00:03:20.00 this mutant kinase can now use a bulky ATP analog in which extra moieties
00:03:24.25 have been added to the adenine base of the ATP.
00:03:27.28 And so for example, N6-benzyl-ATP can be used by the analog sensitive Cdk1 kinase
00:03:32.28 but cannot be used by a wild type kinase because that bulky ATP analog
00:03:37.04 can't fit into the wild type active site.
00:03:41.14 And so, of course, if you put a radio-label on the gamma phosphate
00:03:44.05 of this bulky ATP analog and then add this kinase to a crude cell lysate
00:03:48.19 what you hope to get is the specific labeling of just the direct targets of
00:03:52.23 that protein kinase and no other kinases in the cell lysate
00:03:56.00 because those other kinases can't use this bulky ATP analog.
00:04:00.12 So this method. we developed this method in collaboration with Kevan Shokat
00:04:04.04 a number of years ago and applied it to the yeast Cdk1 kinase
00:04:08.28 and the results with that are shown in the next slide.
00:04:12.09 So this slide shows an autoradiograph of a protein gel
00:04:15.14 in which we've separated the reaction products
00:04:16.28 from three different reactions, two of which are control reactions
00:04:20.03 and the third of which is the experimental reaction.
00:04:23.09 In the first lane what you see is what happens when you add this radiolabeled N6-benzyl-ATP
00:04:28.16 this bulky ATP to a crude cell extract made from yeast.
00:04:33.05 And the result is that you get very little labeling of anything in that cell extract
00:04:36.14 because that ATP analog cannot be used by the protein kinases in that crude cell extract.
00:04:42.14 The next lane is another control in which we're mixing the purified protein kinase Cdk1-as1
00:04:48.23 together with a cyclin partner and then adding that to some N6-benzyl-ATP
00:04:54.01 that's radio-labeled on its gamma position and the result then is that you
00:04:57.10 see auto-phosphorylation of the cyclin subunit of the Cdk-cyclin complex.
00:05:01.21 And so that results in a background band in the experimental lane over here.
00:05:07.03 But the third lane is really the crucial lane in which all three components
00:05:09.22 have been added. And so we're adding a purified kinase. analog sensitive kinase
00:05:13.14 with the bulky ATP analog and the cell extract and the result
00:05:17.21 is that you see a whole raft of different proteins being radio-labeled
00:05:22.06 in the cell extract and those proteins are presumably the direct targets of
00:05:27.00 Cdk1-cyclin complexes in that lysate.
00:05:31.12 So we obtained this result a number of years ago and then
00:05:33.29 dedicated quite a lot of effort to identifying
00:05:36.03 these various radiolabeled bands in this cell lysate.
00:05:38.05 And to make very long story short, we ended up using proteomic libraries
00:05:42.24 to individually identify substrates and in the end
00:05:45.27 we came up with a list of about 181 proteins in cell extracts that
00:05:50.04 are rapidly modified by Cdk1-cyclin complexes.
00:05:53.27 And so this list of proteins provided us with our initial list of Cdk substrates.
00:05:59.23 These substrates are involved in a wide range of different cellular processes
00:06:02.25 many of which are known to be connected to the cell cycle in some way
00:06:05.20 and are likely to represent important targets of Cdk1 throughout the cell cycle.
00:06:11.01 But for various reasons we decided that this list of substrates was
00:06:14.02 incomplete and also, because it was done in vitro we wanted to get
00:06:18.03 another approach that would allow us to identify, comprehensively, a larger number
00:06:23.09 of Cdk substrates that were modified in vivo by Cdk1.
00:06:26.23 And so the second method we've been using more recently has been to use
00:06:30.17 quantitative mass spectrometry approaches to identify all the phosphorylation
00:06:34.10 sites in the cell that are dependent on Cdk1.
00:06:37.18 In other words, phosphorylation sites whose levels decrease abruptly
00:06:40.18 when you inhibit the protein kinase activity of Cdk1.
00:06:45.12 And that method begins again with the analog sensitive Cdk1 mutant.
00:06:49.17 Now, another advantage of these analog sensitive mutants is that not only do they bind
00:06:53.24 bulky ATP analogs, but they also bind bulky inhibitors that can only fit into the active site
00:06:59.24 of the analog sensitive kinase but not the active site of a wild type kinase.
00:07:05.02 And so, for example, this inhibitor here 1-NM-PP1 binds with extremely high affinity to
00:07:10.05 analog sensitive Cdk1 but has essentially no affinity for the wild type kinase
00:07:14.06 or for any other kinase in the yeast cell.
00:07:16.23 And so we could use this analog sensitive Cdk1 to make a yeast strain in which
00:07:22.02 we can inhibit Cdk1 in vivo rapidly and specifically.
00:07:26.22 And so we did that a number of years ago. We created a yeast strain in which
00:07:29.12 the endogenous Cdk1 protein is replaced with the analog sensitive protein
00:07:33.23 and in that yeast strain it is now possible to almost completely and specifically inhibit
00:07:38.05 Cdk1 activity within minutes by the addition of 1-NM-PP1 to the culture medium.
00:07:44.19 So we used that strain in
00:07:46.08 this quantitative mass spectrometry approach that I want to tell you about
00:07:49.16 which was done in a collaboration with Judith Villen and Steve Gygi of Harvard University.
00:07:55.11 And the basic approach that we used is illustrated in this slide and in the next two slides as well.
00:08:01.11 It begins, as I said, with the analog sensitive yeast strain cdk1-as cells
00:08:05.06 in which Cdk1 can be inhibited specifically with the 1NM-PP1 inhibitor.
00:08:10.21 And what we do is we grow two parallel cultures of this yeast strain.
00:08:14.14 One culture, the so called light culture, is grown in regular lysine and arginine
00:08:19.21 whereas the so called heavy culture is grown in a different form of lysine and arginine
00:08:23.11 in which carbon-13 and nitrogen-15 have replaced the usual carbon-12 and nitrogen-14.
00:08:29.21 And so, as a result, after growth in this medium for some time, all the proteins in these cells
00:08:34.03 have been labeled with slightly heavier than average
00:08:37.04 lysine and arginine residues which means that all
00:08:39.10 the peptides derived from this culture will have a slightly higher mass
00:08:43.01 in the eventual mass spectrometry analysis
00:08:45.21 and that will allow us to identify the peptides coming from these two lysates.
00:08:50.22 So we treat the heavy culture with the inhibitor 1-NM-PP1 for a brief period, 15 minutes.
00:08:56.03 And then we harvest these cells after the inhibitor treatment.
00:09:00.08 Harvest the cells, mix them together, lyse them, break them open,
00:09:04.03 and then treat all the resulting proteins in those cell lysates
00:09:08.18 with trypsin to break them all down into tryptic peptides.
00:09:12.06 And then Judit Villen in the Gygi lab has developed a wide range of
00:09:15.27 powerful methods for purifying the phospho-peptides out of that tryptic peptide mixture.
00:09:20.20 And then we then subject those phospho-peptides
00:09:22.25 to mass spectrometry as shown in the next slide.
00:09:26.23 There are two basic forms of mass spectrometry that are applied to these phospho-peptide mixtures.
00:09:31.07 The first, on top, is to use conventional tandem mass spectrometry methods
00:09:35.15 to actually fragment these peptides and use those fragments
00:09:38.07 to determine their sequence.
00:09:40.02 And so, by this approach we can determine the sequence of all the phospho-peptides
00:09:44.04 coming out of these yeast lysates and just as importantly, we can identify
00:09:47.10 the precise site of the phosphorylation on those peptides.
00:09:50.29 And so by doing this, Judit was able to produce a list
00:09:54.12 of about 10,000 phosphorylation sites on 2,000 different proteins in the yeast lysate.
00:10:00.25 And then, in addition to determining sequence, we also quantify all the peptides
00:10:06.12 and determine the relative amount of the so called light and heavy peptides.
00:10:10.19 What this means is that every peptide coming out of these phospho-peptide mixtures
00:10:15.15 comes in both a light form which originally came from the light medium culture
00:10:19.13 and a heavy form that originally came from the inhibitor-treated heavy culture.
00:10:23.17 And they can be distinguished based on this slight
00:10:25.08 mass difference of their lysines and arginines.
00:10:28.12 And what we're looking for, of course, are peptides that look like this:
00:10:30.29 where the heavy peptide is much less abundant than the light peptide.
00:10:34.16 And that means that that peptide's abundance was inhibited
00:10:37.23 or decreased as a result of Cdk1 inhibition and therefore
00:10:41.02 that phosphorylation site on that peptide represents a
00:10:43.23 Cdk1-dependent phosphorylation site in vivo.
00:10:47.23 And so by applying this approach to the many phosphorylation sites identified here
00:10:52.18 we came up with a list of about 547 phosphorylation sites on about 308 proteins
00:10:58.10 that were clearly Cdk1-dependent and represent likely candidates for Cdk1 targets in vivo.
00:11:04.13 This list of targets included many of the same proteins
00:11:07.02 we had identified in our previous screen in vitro
00:11:09.12 and so for those proteins at least we have very good evidence that these
00:11:11.29 proteins are kinase substrates both in vitro and in vivo.
00:11:17.11 Now the list of substrates includes a wide range of proteins involved in a wide range of processes.
00:11:23.20 I'm not expecting you to see or read any of the gene names on these lists here.
00:11:26.27 This slide is simply meant to illustrate that we have lists of proteins involved in
00:11:30.14 a wide range of interesting processes. Some of these processes are totally expected.
00:11:35.00 For example, DNA replication, spindle behavior, kinetochores and cytokinesis are all
00:11:40.08 processes in which we expect Cdks to be involved in regulating some aspect of those processes.
00:11:45.10 There's also a few surprises here as well.
00:11:47.27 Protein translation, chromatin structure, and nuclear transport and the secretory pathway
00:11:53.09 all have a number of Cdk substrates involved
00:11:56.08 in those processes and so one might imagine
00:11:58.15 that this will lead to some new understanding of how Cdks might control those processes
00:12:02.17 as well as the more conventional cell cycle regulated processes.
00:12:07.14 But for the rest of this lecture today, I'm not going to talk in detail about
00:12:10.21 any specific substrates or processes, but instead I'm
00:12:13.14 going to tell you how we used our lists of substrates
00:12:16.02 to address some interesting general questions on how cell cycle progression is
00:12:20.10 controlled by Cdks in general.
00:12:23.13 And so we're going to address two questions in the remainder of this lecture.
00:12:26.14 The first one of which is shown on this next slide.
00:12:30.15 And that question is this one: How do different cyclins trigger different cell cycle events?
00:12:35.10 So I told you at the beginning of the lecture that
00:12:37.02 S-phase cyclin Cdk complexes initiate S-phase and mitotic Cdk-cyclin complexes
00:12:42.00 initiate M-Phase and there's good evidence from yeast genetics and elsewhere
00:12:46.00 that S-phase Cdk-cyclin complexes have a better intrinsic ability to
00:12:49.21 initiate S-phase than a mitotic cyclin-Cdk complex.
00:12:54.01 So there's something different about cyclin-Cdk complexes that are activated at S-phase
00:12:58.28 that allows them to more effectively activate the onset of S-phase.
00:13:02.05 And so, what is that difference?
00:13:04.12 Well, one obvious possibility is that the cyclin that associates with the Cdk
00:13:07.21 helps determine the substrate specificity of that Cdk.
00:13:11.20 So in budding yeast, for example, where there's only a single Cdk
00:13:14.20 associating with all these different cyclins,
00:13:16.08 one can imagine that associating with an S-phase cyclin
00:13:19.20 at the beginning of S-phase might target that Cdk for specific substrates involved in S-phase.
00:13:26.01 And so we decided we could address this question on a more global level
00:13:29.15 by actually analyzing the relative phosphorylation rate of
00:13:32.09 a wide range of Cdk substrates using purified S-phase Cdks and M-phase Cdks.
00:13:38.15 And specifically we carried out these studies using the S-phase cyclin Clb5 from budding yeast
00:13:44.08 and the M-phase cyclin Clb2 from budding yeast.
00:13:47.15 And Mart Loog, a post-doc in the lab, basically purified these two kinases
00:13:51.14 and then tested their activity towards about 150 different Cdk substrates
00:13:55.17 to look for substrates that were highly specific for one or the other.
00:13:59.21 Some of his early results are shown in this next slide
00:14:02.07 which gives you an illustration of the sort of thing we found.
00:14:06.03 Here we're looking at autoradiographs of protein gels in which three different proteins
00:14:11.00 listed across the top--Mcm3, Orc2, and Orc6
00:14:14.00 have been treated with either the mitotic cyclin-Cdk complex on the left
00:14:18.14 or the S-phase complex on the right.
00:14:20.22 And you can see, quite clearly, that these three proteins are all phosphorylated
00:14:24.08 much more rapidly by the S-phase Cdk-cyclin complex Clb5.
00:14:29.21 So Mart went ahead and did this exact same reaction with about 150 proteins as I said
00:14:35.06 and the results from those experiments are shown on this slide.
00:14:37.24 So this slide summarizes everything that he found.
00:14:40.15 What we're looking at here is a plot of about 150 proteins
00:14:44.06 each one of which is represented by these little circles on this plot.
00:14:47.10 And these circles are plotted according to the rate of their phosphorylation
00:14:52.02 by Clb2 on that axis and Clb5 on this axis.
00:14:55.22 And so most of the proteins are falling along the diagonal of this plot
00:14:59.21 indicating that they are equally well phosphorylated by both kinases.
00:15:02.24 In other words, they're not cyclin specific targets.
00:15:05.09 However, we found a quite large number of proteins over here on the right
00:15:09.17 especially these red circles here that represent proteins that
00:15:12.25 are far more rapidly phosphorylated by Clb5-Cdk1 than they are by Clb2-Cdk1.
00:15:19.18 So these proteins, and note by the way that this is a log phase scale here
00:15:23.03 so some of these proteins are 10 or over 100 or even 1000 fold more rapidly phosphorylated by
00:15:28.02 Clb5-Cdk1 than by Clb2-Cdk1. So these clearly represent proteins that are highly Clb5 specific.
00:15:35.05 That the cyclin is somehow determining or increasing
00:15:37.29 the rate of phosphorylation of these proteins.
00:15:40.14 So what are these proteins? Well, we were satisfied to see that at least five of them
00:15:46.07 are proteins known to be involved in DNA replication, especially Sld2 here.
00:15:50.07 Sld2 is a protein whose phosphorylation is known
00:15:52.25 to be crucial for the initiation of DNA replication.
00:15:55.16 And so these proteins make perfect sense as Clb5 specific targets because those are
00:16:00.04 the proteins that we need to phosphorylate early in S-phase
00:16:02.22 to help drive progression through chromosome duplication.
00:16:07.27 So what this list of cyclin-specific substrates in hand, we next addressed
00:16:12.10 the mechanism underlying this cyclin specificity.
00:16:14.25 Why is it that Clb5-Cdk1 phosphorylates these proteins
00:16:18.06 so much more rapidly than Clb2-Cdk1?
00:16:21.13 Through kinetic studies we discovered that the reason for this higher rate of phosphorylation was
00:16:26.15 that these substrates have a much higher affinity for the Cdk-cyclin complex
00:16:30.05 when Clb5 is associated, suggesting that they might associate with that cyclin subunit.
00:16:36.16 In fact, there's previous suggestions of what
00:16:38.04 the mechanism for this association might be.
00:16:40.25 And those are based on the known crystal structures of Cdk-cyclin complexes from human cells.
00:16:46.06 So this shows the crystal structure of a Cdk-cyclin complex from humans
00:16:50.20 that illustrates very nicely the basic parts of the Cdk-cyclin complex
00:16:55.08 and where cyclin substrates typically associate with this complex.
00:16:59.01 Over on the left is the Cdk catalytic subunit and between these two lobes here
00:17:02.29 is an active site cleft in which you can see this ATP molecule binding right here.
00:17:08.19 Typically a protein substrate would bind along the surface of this protein kinase right here
00:17:12.29 in a way that the serine or threonine hydroxyl would be positioned in such a way
00:17:17.25 to allow the transfer of phosphate from that ATP onto the hydroxyl residue.
00:17:23.10 So, the primary site of substrate association with the Cdk-cyclin complex
00:17:27.00 is of course the active site, the place where that serine or threonine
00:17:30.21 associates with its sequence contacts to be phosphorylated.
00:17:35.16 However, this is probably not the only site of substrate association in a Cdk-cyclin complex.
00:17:39.28 There is considerable evidence from mammals and from yeast as well
00:17:43.13 that there is a docking site on this cyclin itself
00:17:46.20 that can also associate to some extent with parts of the substrate.
00:17:50.10 And this docking site is mostly composed of this large alpha-helix here
00:17:54.18 that contains a number of hydrophobic residues
00:17:57.06 that are together called the hydrophobic patch.
00:17:59.16 It is involved in associating with certain substrates
00:18:01.23 and enhancing activity towards those substrates.
00:18:07.02 So, we obviously hypothesized that perhaps this docking site on Clb5
00:18:10.16 exists on Clb5 and that this docking site is required
00:18:13.26 for the cyclin-specific phosphorylation that we saw in our experiments.
00:18:18.03 And so to test that the obvious approach was to mutagenize this docking site
00:18:21.09 through a number of single point mutations and then test whether that
00:18:25.08 has any impact on cyclin specificity and that is shown in this slide.
00:18:28.20 And the answer was a definite yes, that mutation of that docking site
00:18:32.27 completely abolishes the Clb5 specificity that we had seen.
00:18:35.23 So here again we're looking at autoradiographs of protein phosphorylation
00:18:39.27 by purified Clb2 on the left two lanes and Clb5 on the right.
00:18:43.20 And we're looking at the phosphorylation of 5 highly Clb5 specific proteins.
00:18:47.20 And you can see that the wild type Clb2, the wt here, phosphorylates these proteins
00:18:52.13 rather poorly whereas wild type Clb5 phosphorylates them extremely well.
00:18:57.18 Once again, indicating how specific these proteins are for Clb5.
00:19:01.03 However, if you mutate the hydrophobic patch or the docking site
00:19:04.15 on Clb5 you find that that specific phosphorylation is almost completely lost
00:19:09.12 indicating that that site is really required for the increased affinity
00:19:12.25 that Clb5-Cdk1 has for these substrates.
00:19:18.20 So we conclude that an interaction, probably simultaneous between
00:19:22.01 this docking site and the active site, allows specific Clb5 substrates to interact
00:19:26.19 with the Clb5-Cdk complex in a high affinity fashion
00:19:30.10 that allows more rapid phosphorylation of those proteins.
00:19:34.20 And so that leads us to at least a partial answer for the question that I first posed
00:19:39.04 which is: How do different cyclins drive different cell cycle events?
00:19:43.07 Well, part of the answer appears to be that the associated cyclin that associates
00:19:47.01 with the Cdk helps target that Cdk to specific substrates.
00:19:51.01 And so S-phase Cdk-cyclin complexes when they're activated
00:19:54.14 at the end of G1 tend to phosphorylate more rapidly the proteins
00:19:57.25 that are most important for initiating S-phase.
00:20:02.06 OK, now I want to turn to an entirely different sort of general question
00:20:05.05 that we also used our substrate lists to address.
00:20:09.13 And in particular, we used our recent mass spectrometry analysis
00:20:12.11 and our 547 Cdk1-dependent phosphorylation sites to address this question.
00:20:17.20 And this is a much more general question that just. that goes beyond
00:20:21.28 issues of simple cell cycle control but reaches into areas involved in
00:20:25.26 the general issues of phospho-regulation. And the question is this one:
00:20:30.10 How is it that phosphorylation changes the function of a protein?
00:20:33.10 How is it that the addition of a phosphate group to a protein changes that protein's function
00:20:37.20 in a way that allows it to initiate cell cycle events or do other things?
00:20:41.27 And there are typically a couple of different approaches
00:20:44.09 or different mechanisms that are thought to be involved in changing protein function.
00:20:48.03 And the first and possibly most commonly imagined mechanism is this one here
00:20:51.25 the so-called allosteric switch. And the idea with this mechanism is that the placement
00:20:56.13 of a phosphate on a protein in a very specific location
00:20:59.06 causes a precise conformational change in that protein that then
00:21:02.24 initiates some change in its function, its enzymatic activity or its association with something.
00:21:08.21 Now, this mechanism, of course, requires that the position of that phosphorylation site
00:21:11.29 is extremely precise and conserved. In other words,
00:21:15.10 you can't put a phosphate just anywhere on a protein and
00:21:17.23 cause this very precise conformational change.
00:21:20.08 It has to be extremely well positioned
00:21:21.21 and because of that itâ€™s very difficult to evolve that sort of phospho-regulation.
00:21:26.23 That kind of phosphorylation cannot appear randomly very easily
00:21:31.22 and achieve the kind of regulation that is required.
00:21:35.08 So the alternative mechanism is this one--which I call bulk electrostatics.
00:21:40.08 And this mechanism suggests that the position of the phosphorylation
00:21:43.14 does not require such precise position of the phosphorylation.
00:21:47.27 The basic idea here is that the placement of clusters of phosphorylation sites
00:21:51.22 on the surface of the protein, typically on a loop or a disordered region on the surface
00:21:56.01 can result in interesting regulation such as interference with association with another protein
00:22:01.16 or for that matter, promotion of association with phosphate binding proteins.
00:22:06.04 And so this very simple mechanism of phospho-regulation
00:22:08.18 can occur by the placement of clusters of phosphates in a general region
00:22:13.19 of a protein but the exact position of each of those phosphates is not absolutely important,
00:22:18.06 not critical and therefore the position of those phosphates can shift during evolution
00:22:22.05 in different proteins. And so for that reason this mechanism is much more easily evolved.
00:22:28.10 Itâ€™s very easy to imagine that random mutations
00:22:30.09 could result in the appearance of phosphorylation sites
00:22:32.19 on the surface of certain proteins where they interact with other proteins
00:22:35.17 and that can result in regulatory possibilities that could be selected for.
00:22:41.08 And so, both of these mechanisms are known to be important in different cases.
00:22:46.12 There are examples of proteins that are regulated in both of these ways.
00:22:49.17 But we thought that perhaps our giant list of Cdk substrates would allow us to
00:22:53.11 address the relative importance of these two mechanisms more generally.
00:22:58.06 And so what we did is we took those 547 Cdk1-dependent phosphorylation sites
00:23:03.06 and aligned them with homologous sequences from other species
00:23:07.17 to see how well these phosphorylation sites are actually conserved.
00:23:10.27 And the basic idea here was that if we found that
00:23:13.11 sites are generally, extremely well preserved that might argue for this sort of mechanism,
00:23:17.24 but sites that drift during evolution might argue for this sort of mechanism.
00:23:23.23 So the next slide gives you an illustration of the sort of thing that we found.
00:23:27.14 Now, here we are aligning a bunch of different protein sequences
00:23:29.29 and I don't expect you to actually read these sequences.
00:23:32.17 The important thing is that there are these little yellow boxes that represent
00:23:35.15 a SP or TP di-peptide motifs that are the consensus sequences for Cdk phosphorylation.
00:23:42.09 And along the very top here is the sequence of a part of a protein
00:23:45.27 called Shp1 that we identified two phosphorylation sites in our mass spectrometry experiments.
00:23:51.24 Those sites are site A and site B.
00:23:53.26 One site is over here in a region of the protein that is known
00:23:57.05 to form into a globular domain and anther site is here in a region
00:24:00.21 that's predicted to form a disordered domain.
00:24:03.16 And these other sequences that lie below this top sequence from budding yeast
00:24:06.26 are the sequences of orthologous proteins from various yeast species
00:24:11.25 whose genomes have been sequenced
00:24:13.20 starting with the most closely related yeast here and
00:24:15.25 moving all the way down to the most distantly related yeast at the bottom.
00:24:20.03 And so these protein alignments tell us some very simple things.
00:24:23.19 First of all, site A here is very well conserved in evolution
00:24:27.10 and you can see that almost all of the orthologs of this protein in all these other yeast species
00:24:31.14 contain a likely Cdk consensus site at the exact same position in this highly conserved region.
00:24:38.25 So site A appears to represent an example of the kind of site I mentioned
00:24:42.09 in the left side of the previous slide, a site that is highly conserved in evolution.
00:24:46.27 But site B is not. Site B is very poorly conserved and disappears essentially
00:24:51.15 after a few species and is no longer found at that position in other orthologs.
00:24:57.24 However, if you look in this region of these other species' proteins, you find that
00:25:01.21 SP and TP di-peptide motifs appear scattered throughout this region
00:25:05.08 in a larger number of these yeast homologous proteins,
00:25:08.20 suggesting that even though the initial position here in budding yeast has not been preserved,
00:25:14.09 the Cdk phosphorylation of this region has been conserved in evolution
00:25:18.21 but the exact position of the phosphorylation sites
00:25:20.23 has been shifting dramatically over evolution.
00:25:23.27 So this is obviously consistent with the second idea,
00:25:26.13 that precise phosphorylation site positioning is not
00:25:29.16 required here because these sites might be involved in some more simple, general
00:25:34.03 regulatory mechanism involving association with phosphate binding proteins
00:25:38.03 or interference with protein binding.
00:25:41.11 So we did this exact same alignment for all 547 of our phosphorylation sites
00:25:46.09 and then in the next slide what I'm going to show you
00:25:47.28 is a somewhat complex graphic that illustrates the results that we found from that.
00:25:53.14 And this was done in collaboration with Brian Tuch, a graduate student
00:25:56.22 working in the laboratory of Sandy Johnson at UCSF.
00:26:00.09 And this top plot here represents a hierarchically clustered
00:26:02.27 clustergram as we call it that illustrates the conservation of the precise position of
00:26:08.26 phosphorylation sites in orthologs of the proteins we identified.
00:26:13.29 And so what we're looking at here is a graphic in which there are 547 columns
00:26:18.04 in this graphic, each one of which represents a single Cdk1 dependent phosphorylation site
00:26:23.14 that we identified by mass spectrometry.
00:26:26.17 And then each row in this graphic represents
00:26:29.22 how that phosphorylation site aligns with its orthologs in other species,
00:26:34.06 the same yeast species that I showed in the previous slide
00:26:36.23 starting with the most closely related yeast species
00:26:39.03 and then working to the most distantly related ones.
00:26:41.28 And in each column a yellow box indicates that that phosphorylation site
00:26:47.06 is precisely conserved in its position in that orthologous sequence.
00:26:51.03 In other words, over here on the left this yellow box at the top moves down
00:26:55.16 for a few species and then disappears indicating that this site is only precise.
00:26:59.24 these columns here, these phosphorylation sites
00:27:02.12 are conserved only in the closely related
00:27:04.21 yeasts species but then are lost in all distantly related species.
00:27:10.03 And so by looking at this graph you can see that there's only
00:27:12.10 a small group of phosphorylation sites, these ones in here especially
00:27:16.06 and note particularly these ones here that are conserved
00:27:19.04 throughout all the yeast homologs that we identified.
00:27:21.23 And so this small number of phosphorylation sites, perhaps 30 or 40 of them are
00:27:26.12 at most, are preserved in large numbers of yeast species, indicating that
00:27:31.29 the precise position of phosphorylation
00:27:33.17 has been conserved in a relatively small number of cases.
00:27:37.16 OK, so how do we then test the possibility that instead of precise positioning
00:27:44.01 during evolution that we're looking at drifting phosphorylation site positioning?
00:27:48.28 And that required the development of another graphic which is shown below here
00:27:52.17 in which I'll take you through slowly because itâ€™s a little bit complicated.
00:27:56.16 So in this case, once again, itâ€™s another hierarchically clustered graphic in which
00:28:00.22 there are 547 columns, each representing a different phosphorylation site
00:28:05.15 identified in our analyses. But in this case.
00:28:08.17 and once again, the rows represent alignments with orthologs, orthologous proteins
00:28:13.00 from other yeast species. But in this case the yellow box
00:28:16.02 doesn't indicate precise positioning of phosphorylation site, but instead
00:28:19.18 indicates that the ortholog of that particular protein in these other yeast species
00:28:24.03 has a statistically enriched frequency of Cdk consensus sites, SP and TP motifs.
00:28:30.28 In other words, these yellow boxes represent proteins
00:28:33.21 in which the frequency of SP and TP motifs is far greater than that expected by chance.
00:28:39.00 And so these large numbers of proteins here represent proteins in which
00:28:43.08 even though the precise site of phosphorylation is not conserved
00:28:46.19 as shown up here, these proteins do contain a high frequency of
00:28:50.12 Cdk consensus sites whose positions are clearly drifting during evolution.
00:28:56.04 And so these large number of proteins over here on the right side of this clustergram
00:28:59.24 may represent proteins in which the precise position of phosphorylation
00:29:03.21 does not matter but the regulation of those proteins by phosphorylation
00:29:07.17 is conserved despite that. And so clearly we'd like to think that that evidence
00:29:14.00 tends to suggest that this mechanism on the right here
00:29:16.03 these easily evolved bulk electrostatic mechanism
00:29:19.05 is a major mechanism by which phospho-regulation can easily be evolved
00:29:23.10 and that drifting phosphorylation sites especially clusters of phosphorylation sites
00:29:26.19 on disordered regions is really crucial for
00:29:30.10 the regulation of many different Cdk substrates.
00:29:34.28 So with that, I want to leave us with the question that we started out this whole lecture with
00:29:39.20 and that is: How do Cdk's drive cell cycle events?
00:29:42.04 Well, clearly our list of Cdk substrates, the ones I'm showing here and
00:29:45.21 the many that aren't shown here, probably contain the answer to this question.
00:29:50.06 Clearly through the detailed analysis of large numbers of these substrates
00:29:53.03 and that will lead us to a much better understanding of how Cdks
00:29:57.17 drive the events of cell cycle and how they alter all these different processes
00:30:02.04 in the cell to initiate cell cycle events.
The Earth Has a Pulse—A 27.5-Million-Year Cycle of Geological Activity
Geologic activity on Earth appears to follow a 27.5-million-year cycle, giving the planet a “pulse,” according to a new study published in the journal Geoscience Frontiers.
“Many geologists believe that geological events are random over time. But our study provides statistical evidence for a common cycle, suggesting that these geologic events are correlated and not random,” said Michael Rampino, a geologist and professor in New York University’s Department of Biology, as well as the study’s lead author.
Over the past five decades, researchers have proposed cycles of major geological events—including volcanic activity and mass extinctions on land and sea—ranging from roughly 26 to 36 million years. But early work on these correlations in the geological record was hampered by limitations in the age-dating of geologic events, which prevented scientists from conducting quantitative investigations.
However, there have been significant improvements in radio-isotopic dating techniques and changes in the geologic timescale, leading to new data on the timing of past events. Using the latest age-dating data available, Rampino and his colleagues compiled updated records of major geological events over the last 260 million years and conducted new analyses.
The team analyzed the ages of 89 well-dated major geological events of the last 260 million years. These events include marine and land extinctions, major volcanic outpourings of lava called flood-basalt eruptions, events when oceans were depleted of oxygen, sea-level fluctuations, and changes or reorganization in the Earth’s tectonic plates.
They found that these global geologic events are generally clustered at 10 different timepoints over the 260 million years, grouped in peaks or pulses of roughly 27.5 million years apart. The most recent cluster of geological events was approximately 7 million years ago, suggesting that the next pulse of major geological activity is more than 20 million years in the future.
The researchers posit that these pulses may be a function of cycles of activity in the Earth's interior—geophysical processes related to the dynamics of plate tectonics and climate. However, similar cycles in the Earth’s orbit in space might also be pacing these events.
“Whatever the origins of these cyclical episodes, our findings support the case for a largely periodic, coordinated, and intermittently catastrophic geologic record, which is a departure from the views held by many geologists,” explained Rampino.
In addition to Rampino, study authors include Yuhong Zhu of NYU’s Center for Data Science and Ken Caldeira of the Carnegie Institution for Science.
© 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.
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The Cardiovascular System
The cardiac cycle is vital to proper cardiovascular system function. Comprised of the heart and circulatory system, the cardiovascular system transports nutrients to and removes gaseous waste from the cells of the body. The cardiac cycle provides the "muscle" needed to pump blood throughout the body. Blood vessels act as pathways that transport blood to various destinations.
The driving force behind the cardiac cycle is the electrical system known as cardiac conduction. This powers the cardiovascular system. Specialized tissues called heart nodes send nerve impulses that disperse throughout the heart wall to make the heart muscle contract.