Cell and Molecular Biol Test 3 Notes
Cell and Molecular Biol Test 3 Notes BIOL 231
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Chapter 15 3/13 Wednesday, March 13, 201310:20 AM • What we are going to be talking about ○ How are proteins moved ○ Where proteins need to go to function Figure 15.5 • Three main categories how proteins are carried • Most mitochondrion proteins made in cytosol • Protein sorting ○ Transport of proteins to proper location ○ 3 types 1. Through nuclear pores: 1) Nuclear pores that penetrate the inner and outer nuclear membranes. a) Selective active large molecule passive small molecules b) B/c of the way they are made, large molecules can't pass through 2. Across membrane 1) Proteins moving from the cytosol into the ER, mitochondria, or chloroplasts are transported across the organelle membrane by protein translocators located in the membrane. Unlike transport through nuclear pores, the transported protein molecule must usually unfold in order to snake through the membrane. 3. By vesicles if going to: 1) Proteins moving from the ER onward and from one compartment of the endomembrane system to another. These proteins are ferried by transport vesicles, which become loaded with a cargo of proteins from the interior space, or lumen, of one compartment, as they pinch off from its membrane. The vesicles subsequently discharge their cargo into a second compartment by fusing with its membrane. In the process, membrane lipids and membrane proteins are also delivered from the first compartment to the second a) Membrane b) Lumen of vesicles to organelles c) Plasma membrane i) To either become part of membrane ii) Excreted outside the cell Table 15-3 Some typical signal sequence • How does a protein know where to go (live)? ○ Signal sequences Amino acid sequence that directs a protein to a specific location in the cell, such as the nucleus or mitochondria. Imports to ER, Retention in Lumen, Mitochondria, nucleus, peroxisomes (all are different) + are red, - are blue, hydrophobic are green Built into the primary structure of the protein ○ Built into the primary structure of the protein ○ Not necessary for the final functioning of the protein, some cases can even inhibit the function, so in cases they are cleaned off after the protein gets where it needs to go but before it becomes functional ○ If something is going to be imported into the ER, it has its SS at the at the amino terminal end, and if it is continuing on into the lumen then it has one at the carboxyl end as well. ○ If a protein doesn't have a SS, then it can't be transported etc. can cause illness Figure 5.6 *Shows that SS is necessary a. Normal a. Taking of sig. sequence of one and putting it on another b. The switched, the new signal when where it needed to go • Experiment w/ recombinant DNA ○ ER Lumen protein remains in cytosol. If sig seq. removed and cytosolic protein sorts to ER if sig. seq. determines sorting • DNA being recombined ○ B/c DNA codes for protein, so if you change the DNA and add some codes, and add additional sequences to that gene, when that gene gets expressed it will have additional amino acids attached ○ In this way you can take the ER gene off and it will not be about to go to the ER anymore(will go to cytosol), you can also take this gene and place on different protein and it will go to the ER Figure 15-8 • Nuclear Pores: complexes of ~ 30 proteins. Wall w/ fibrils + meshwork of fibers in aq. Passage ○ Large structures 1/10 micron ○ Made of at least 30 proteins Some make up The wall Long skinny fibrils that come off wall Involved in anchoring the pore into the envelope Make up bottom portion of the pore Meshwork of fibers Nuclear basket • Anything large is actively transported • In order for a portion to end up in the nucleus it has to have its proper SS, there is a series of AA that signify and local need for a nucleus. ○ A receptor that is involved in binding to the SS on the protein Nuclear local signal □ Typically consists of one or two short sequences containing several positively charged lysines or arginines • Each pore contains water-filled passages through which small water-soluble molecules can pass freely and nonselective between the nucleus and the cytosol. Many of the proteins that line the nuclear pore contain extensive, unstructured regions, which are thought to form a disordered tangle—much like a kelp bed in the ocean. This jumbled meshwork fills the center of the channel, preventing the passage of large molecules but allowing smaller molecules to slip through Figure 15-9 • The nuclear protein, with its bound receptor, is actively transported into the nucleus. For clarity, the basketlike structure of fibrils that extends into the nucleus is not shown. The cutaway view of one nuclear pore complex shows how the unstructured regions of the proteins that line the central pore form a tangled meshwork that blocks the passive diffusion of large macromolecules into and out of the nucleus. • Nuclear local signal recognized by nuclear transport rec. in cytosol. Rec interacts with fibrils; assists entry. Rec/ protein complex actively transported into nucleus • Active Transport (requires energy) • The blue thing is a receptor ○ Its job is to bind to the SS, this is how it recognizes it’s a protein that needs to go into the nucleus • The receptor assists in getting the protein through the fibrils, the pores and the meshwork Figure 15.10 • Active transport by GTP hydrolysis. Nuclear transport rec. associates with RAN-GTP (small GTP-ase) • Process is continuously • How are ATP and GTP different? ○ Differs in particular nucleoside that it has on it ○ Guanidine triphosphate • Where does the energy come from? ○ ATP- between the phosphates, connected to each other, when broken, free energy ○ GTP- has the same bonds, so should release the energy when it hydrolyzes as well • The transporter receptor its self is not a GTP-ASE, so it associates with RAN, which is a small GTP-ase • The Cycle: a. Nuclear transport receptor binds cargo protein 1. When the receptor (b4 it binds) binds to RAN, it has a GTP with it, since its' GTP-ase (which is favorable), it will allow it to hydrolyze, that releases free energy 2. This will allow the receptor to bind the protein that needs to come into the nucleus and the energy to actively import it to the nucleus b. Receptor transports cargo protein to nucleus c. Cargo protein delivered to nucleus d. Empty receptor returns to cytosol ○ A nuclear transport receptor picks up its cargo protein in the cytosol and enters the nucleus. There it encounters a small protein called Ran, which carries a molecule of GTP. This Ran-GTP binds to the nuclear transport receptor, causing it to release its cargo. Having discharged its cargo in the nucleus, the nuclear receptor—still carrying the Ran-GTP—is transported back through the pore to the cytosol. There, an accessory protein triggers Ran to hydrolyze its bound GTP. Ran-GDP falls off the transport receptor, which is then free to bind another protein destined for the nucleus. Video: Nuclear Import Showing, it can be regulating in the cell, something like the increasing of calcium, allowing it to get in cell Increase in calcium can trigger it to allow it in. Figure 15.11 ??Why would mitochondria and chloroplast have a similar way of bringing in proteins?? ○ Have similar functions Provide energy to cell Surrounded by two membranes If the proteins need to live in either the mitochondrial matrix, or the stroma (in chloroplast) they would need to have similar mechanisms to go through both. • The mitochondrial signal sequence is recognized by a receptor in the outer mitochondrial membrane. The complex of receptor and attached protein diffuses laterally in the membrane to a contact site, where the protein is translocated across both the outer and inner membranes by a protein translocator. The signal sequence is cleaved off by a signal peptidase inside the organelle. Proteins are imported into chloroplasts by a similar mechanism. The chaperone proteins help to pull the protein across the membranes. • Mitochondria (mito) and Chloroplast (chloros) • Signal sequence at N term recognized by TM receptor • Proteins translocation in out (TOMS) and inner (TIMS) membrane align • Protein unfolds as crosses both membrane: Tiny TIMS (inter-membrane chaperones) • Signal sequence cleaved and protein refolds (matrix chaperones) Figure 15.12 • ER ○ Serves as an entry point for proteins destined for other organelles, as well as for the ER itself. Proteins destined for the Golgi apparatus, endosomes, and lysosomes, as well as proteins destined for the cell surface, all first enter the ER from the cytosol. Once inside the ER or embedded in the ER membrane, individual proteins will not reenter the cytosol during their onward journey. They will be ferried by transport vesicles from organelle to organelle and, in some cases, from organelle to the plasma membrane or to the exterior of the cell ○ Two kinds of proteins are transferred from the cytosol to the ER: (1) water- soluble proteins are completely translocated across the ER membrane and are released into the ER lumen; (2) prospective transmembrane proteins are only partly translocated across the ER membrane and become embedded in it. The water-soluble proteins are destined either for secretion (by release at the cell surface) or for the lumen of an organelle; the transmembrane proteins are destined to reside in either the ER membrane, the membrane of another organelle, or the plasma membrane. All of these proteins are initially directed to the ER by an ER signal sequence, a segment of eight or more hydrophobic amino acids (see Table 15–3, p. 502) that is also involved in the process of translocation across the membrane • Trans located during synthesis by ribosomes associated with ER. Proteins of ER • Trans located during synthesis by ribosomes associated with ER. Proteins of ER lumen/membrane, Golgi, lysosomes , endosomes, and plasma membrane Figure 15.13 • Cytosol/Rough ER ribosomes common pool in cytosol. N-term sig. seq. for ER, ribosomes attaches to ER. If not, stays in cytosol. ER protein threaded through translocation channel in ER membranes as translation ○ Both types of ribosomes come from the same pool of rib. Subunits, there are 2 subunits • All protein translation starts out in cytosol, but doesn't induct staying in cytosol, whether it stays or goes depends on if it has signals or not. • Ribosomes that are translating cytosolic proteins remain free in the cytosol. For proteins that will be sent to the ER, a signal sequence on the growing polypeptide chain directs the ribosome to the ER membrane. Many ribosomes bind to each mRNA molecule, forming a polyribosome. At the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol. Chapter 15 3/15 Sunday, March 17, 2013 6:36 PM 3.15.2013 Fig. 15-13 Cytosol/ER ribos common pool in cytosol. N-term sig seq for ER, ribo attaches to ER. If not, stays in cytosol. ER protein threaded through translocation channel in ER membrane as translation proceeds. -protein sorting -protein – ER – membrane -ribos – translate mRNA – 2 subunits -ALL proteins translate in cytosol bc ribos exist in pool w/in cytosol -either attach ER or stay in cytosol by sig seq to determine where/ which location -to ER in process of translation -A LOT of proteins to happen for Fig. 15-14 • The SRP binds to the exposed ER signal sequence and to the ribosome, thereby slowing protein synthesis by the ribosome. The SRP–ribosome complex then binds to an SRP receptor in the ER membrane. The SRP is released, passing the ribosome to a translocation channel in the ER membrane. Finally, the translocation channel inserts the polypeptide chain into the membrane and starts to transfer it across the lipid bilayer. • The ER signal sequence is guided to the ER membrane with the aid of at least two protein components: (1) a signal-recognition particle (SRP), present in the cytosol, which binds to the ER signal sequence when it is exposed on the ribosome, and (2) an SRP receptor, embedded in the membrane of the ER, which recognizes the SRP. Binding of an SRP to a signal sequence causes protein synthesis by the ribosome to slow down, until the ribosome and its bound SRP locate an SRP receptor on the ER. After binding to its receptor, the SRP is released and protein synthesis recommences, with the polypeptide now being threaded into the lumen of the ER through a translocation channel in the ER membrane (Figure 15–14). Thus, the SRP and SRP receptor function as molecular matchmakers, connecting ribosomes that are synthesizing proteins containing ER signal sequences to available ER translocation channels • Translating ribos guided to ER membrane by sig recog particle (SRP); binds ER sig seq of polypep (pauses translation). SRP / cargo bind TM receptor; ferried to translocation channel. SRP released, polypep/ ribo passed to channel, translation resumes. Fig. 15-15 • A translocation channel binds the signal sequence and actively transfers the rest of the polypeptide across the lipid bilayer as a loop. At some point during the translocation process, the signal peptide is cleaved from the growing protein by a translocation process, the signal peptide is cleaved from the growing protein by a signal peptidase. This cleaved signal sequence is ejected into the bilayer, where it is degraded, and the translocated polypeptide is released as a soluble protein into the ER lumen. Once the protein has been released, the pore of the translocation channel closes. • Luminal/ secreted proteins trans located thru ER membrane. • Sig peptidase (enzyme) cleaves sig seq, releases mature polypep into lumen. • Vesicles from ER carry proteins to other organelles. • -N-term (translated 1st) • -Amino term made 1st • -some chaperones to fold correctly Fig.15-16 • An N-terminal ER signal sequence (red) initiates transfer as in Figure 15-15. In addition, the protein also contains a second hydrophobic sequence, a stop- transfer sequence (orange). When this sequence enters the translocation channel, the channel discharges the protein sideways into the lipid bilayer. The N-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane (Movie 15.4). Protein synthesis on the cytosolic side continues to completion. • TM proteins remain in membrane single pass TMs: • N-term start sig seq bound by SRP; binds receptor & carried to channel. • Protein threaded through until hydrophobic stop transfer seq threading stops; translation of cytosol portion continues until completion. • Sig seq cleaved & TM released into bilayer. • -single pass protein = happens once Fig. 15-17 • An internal ER signal sequence (red) acts as a start-transfer signal and initiates the transfer of the polypeptide chain. Like the N-terminal ER signal sequence, the internal start-transfer signal is recognized by an SRP that brings the ribosome to the ER membrane (not shown). When a stop-transfer sequence (orange) enters the translocation channel, the channel discharges both sequences into the plane of the lipid bilayer. Neither the start-transfer nor the stop-transfer sequence is cleaved off, and the entire polypeptide chain remains anchored in the membrane as a double-pass transmembrane protein. Proteins that span the membrane more times contain further pairs of stop and start sequences, and the same process is repeated for each pair. • Double pass: hydrophobic internal start sig seq • Multiple TMs: >1 pair of start/stop sig seqs • -starts inside if 2nd pass • -N-term tail sticks out • -protein continues to make until carboxyl stop • -multiple = more than 1 start/stop • -maybe residents of ER & transmembrane • -can also be residents of other membrane if budded off • -can also be residents of other membrane if budded off Fig. 15-18 Vesicular transport: Transport of material between organelles in the eukaryotic cell via membrane-enclosed vesicles. A major outward secretory pathway starts with the synthesis of proteins on the ER membrane and their entry into the ER, and it leads through the Golgi apparatus to the cell surface; at the Golgi apparatus, a side branch leads off through endosomes to lysosomes (Figure 15–18). A major inward endocytic pathway, which is responsible for the ingestion and degradation of extracellular molecules, moves materials from the plasma membrane, through endosomes, to lysosomes Each compartment encloses a space, or lumen, that is topologically equivalent to the outside of the cell (see Figure 11–19). The extracellular space and each of the membrane-enclosed compartments (shaded gray) communicate with one another by means of transport vesicles, as shown. In the outward secretory pathway (red arrows), protein molecules are transported from the ER, through the Golgi apparatus, to the plasma membrane or (via early and late endosomes) to lysosomes. In the inward endocytic pathway (green arrows), extracellular molecules are ingested in vesicles derived from the plasma membrane and are delivered to early endosomes and then (via late endosomes) to lysosomes. -for proteins that DO NOT remain w/ER BUD from ER → golgi → bud → (to one of the following): 1)transport vesicles→ plasma mem →secretion OR become part of mem OR 2)endosome w/vesicle fr/bind to plasma mem = late endosome = lysosome (contain enzymes = degradation) Process 1 = Exocytosis; Process 2 = Endocytosis -2 directions of vesicles Secretory pathway = transport vesicles from ER to Golgi to plasma mem cargo released into extracellular or TM placed in plasma mem -sugars – lumen inside golgi → then becomes outside of me mfr golgi OR -hydrophilic gets dumped out -signals – neurotransmitters Endocytic pathway = plasma mem buds transport vesicles, fuse w/ vesicles fr Golgi to form endosomes & lysosomes, cargo, degraded -degraded – cytosol – can be used = bldg blocks Fig. 15-19a Essential Cell Biology The best-studied vesicles are those that have coats made largely of the protein clathrin . These clathrin-coated vesicles bud from the Golgi apparatus on the outward secretory pathway and from the plasma membrane on the inward endocytic pathway. At the plasma membrane, for example, each vesicle starts off as a clathrin-coated pit. Clathrin molecules assemble into a basketlike network on the cytosolic surface of the membrane, and it is this assembly process that starts shaping the membrane into a vesicle vesicle ) Electron micrographs showing the sequence of events in the formation of a clathrin- coated vesicle from a clathrin-coated pit. The clathrin-coated pits and vesicles shown here are unusually large and are being formed at the plasma membrane of a hen oocyte. They are involved in taking up particles made of lipid and protein into the oocyte to form yolk. (B) Electron micrograph showing numerous clathrin-coated pits and vesicles budding from the inner surface of the plasma membrane of cultured skin cells. Vesicle budding assisted by proteins coating cytosol surface. Shape mem/ trap cargo. Coated vesicles lose coats to fuse w/ other mem -make pit → bud → cargo inside Fig. 15-19b Clathrin coats: vesicles from Golgi & plasma mem Clathrin proteins assemble basket – like network on cytsolic surface of mem; forms pit, enlarges into bud. • Clathrin coats: Vesicles from Golgi and plasma membrane • Clathrin proteins assemble basket-like network on cytosolic surface of membranes; forms pit, enlarges into bud. • Golgi-part of excositc Chapter 15 3/18 Monday, March 18, 2013 9:45 AM Figure 15.20 Cargo receptors, with their bound cargo molecules, are captured by adaptins, which also bind clathrin molecules to the cytosolic surface of the budding vesicle . Dynamin proteins assemble around the neck of budding vesicles; once assembled, the dynamin molecules hydrolyze their bound GTP and, with the help of other proteins recruited to the neck (not shown), pinch off the vesicle. After budding is complete, the coat proteins are removed, and the naked vesicle can fuse with its target membrane. Functionally similar coat proteins are found in other types of coated vesicles • Endocytic • Receptor-mediated transport (cytosis if at membrane) • Membrane receptor (trans membrane) binds signal sequence of cargo ○ Signal sequence that stats it needs to go in ○ Cargo also called the ligand • Adaptins bind loaded cargo rector and clathrins. Forms coated pit, bud • Dynamin encircles neck (motor protein), squeezes to pinch off vesicle • Adaptins/clathrins released ○ Loses coat so it can fuse ○ Vesicle movement from one place to another is not a random event, transported specifically with motors inside skeletal filaments Figure 15.21 A filamentous tethering protein on a membrane binds to a Rab protein on the surface of a vesicle. This interaction allows the vesicle to dock on its target membrane. A v- SNARE on the vesicle then binds to a complementary t-SNARE on the target membrane. Whereas Rab and tethering proteins provide the initial recognition between a vesicle and its target membrane, the pairing of complementary SNAREs also helps ensure that transport vesicles reach their appropriate target membranes. • Fusion of vesicles with membranes ○ Not random or chance event • Filamentous proteins- sticking off vesicle (v snare) ○ Snares trap something • V snare from vesicle; t-snare/tether from target membrane • Tether binds Rab on vesicle surface causing docking at target membrane ○ Rabs' are a family of proteins, related to Rans Rans are responsible for bringing protein into the nucleus Rans are G proteins, it combines and hydrolyzes GTP • Snares intertwine, pull vesicle down causing fusion to target membrane • Everything is recycled and reused Chapter 16 3/18 Monday, March 18, 2013 10:26 AM Fig 16.2 • For any type of communication to occur: ○ Has to have some type of signal has to be made ○ Something to has to receive it ○ For it to mean anything then receiver needs to have a response to it Cell Signaling • Signal produced (cells/environment) • Signal received by target cell (trans membrane or intracellular receptor) • Signal transduced: Extracellular signal converted into intracellular signal (response) Figure 16.3 Cell to Cell Signaling: 1. Endocrine; Circulation hormone (bloodstream/sap) broadcast to all cells. a. Ex: insulin/blood sugar b. In plants go to the SAP c. Target cell has to have a receptor to receive/respond d. Hormones produced in endocrine glands are secreted into the bloodstream and are distributed widely throughout the body 2. Paracrine: local mediator diffuses thru extracellular fluid to nearby cells. a. Ex; PDGF/ wound healing Platelet derived growth factor b. Paracrine signals are released by cells into the extracellular fluid in their neighborhood and act locally. 3. Neuronal: NT diffuses thru synaptic cleft (<100nm) to postsynaptic cell. a. Ex: Ach (acetylcholine)/muscle contraction b. Neuronal signals are transmitted along axons to remote target cells. 4. Contact-dependent: membrane protein signal displayed to membrane receptors on adjacent cell. a. Ex. Delta/neuronal development b. In contact-dependent signaling, a cell-surface-bound signal molecule binds to a receptor protein on an adjacent cell. • The crucial differences lie in the speed and selectivity with which the signals are delivered to their targets. • • (A) A mobile telephone converts a radio signal into a sound signal when receiving (and vice versa, when transmitting). (B) A target cell converts an extracellular signal (molecule A) into an intracellular signal (molecule B). Part of the pancreas, for example, is an endocrine gland that produces the hormone insulin, which regulates glucose uptake in cells all over the body. Figure 16–3 Animal cells can signal to one another in various ways. (A… More » Animal cells can signal to one another in various ways. (A… More » Somewhat less public is the process known as paracrine signaling. In this case, rather than entering the bloodstream, the signal molecules diffuse locally through the extracellular fluid, remaining in the neighborhood of the cell that secretes them. Thus, they act as local mediators on nearby cells (Figure 16–3B). Many of the signal molecules that regulate inflammation at the site of an infection or that control cell proliferation in a healing wound function in this way. In some cases, cells can respond to the local mediators that they themselves produce, a form of paracrine communication called autocrine signaling; cancer cells sometimes promote their own survival or proliferation in this way. Neuronal signaling is a third form of cell communication. Like endocrine cells, nerve cells (neurons) can deliver messages over long distances. In the case of neuronal signaling, however, a message is not broadcast widely but is instead delivered quickly and specifically to individual target cells through private lines. As described in Chapter 12, the axon of a neuron terminates at specialized junctions (synapses) on target cells that can lie far from the neuronal cell body (Figure 16–3C). The axons that extend from the spinal cord to the big toe, for example, can be more than 1 m in length. When activated by signals from the environment or from other nerve cells, a neuron sends electrical impulses racing along its axon at speeds of up to 100 m/sec. On reaching the axon terminal, these electrical signals are converted into a chemical form: each electrical impulse stimulates the nerve terminal to release a pulse of an extracellular signal molecule called a neurotransmitter. The neurotransmitter then diffuses across the narrow (< 100 nm) gap between the axon-terminal membrane and the membrane of the target cell, reaching the target cell receptors in less than 1 msec. A fourth style of signal-mediated cell–cell communication—the most intimate and short- range of all—does not require the release of a secreted molecule. Instead, the cells make direct physical contact through signal molecules lodged in the plasma membrane of the signaling cell and receptor proteins embedded in the plasma membrane of the target cell (Figure 16–3D). During embryonic development, for example, such contact-dependent signaling allows adjacent cells that are initially similar to become specialized to form different cell types (Figure 16–4) Figure 16.4 • Neurogenesis in fly embryos; one epithelial cell w/in group expresses Delta; this cell differentiates into a neuron. Delta binds to notch on adjacent cells which inhibits their differentiation into neurons. The fly nervous system originates in the embryo from a sheet of epithelial cells. Isolated cells in this sheet begin to specialize as neurons, while their neighbors remain non- neuronal and maintain the epithelial structure of the sheet. The signals that control this process are transmitted via direct cell–cell contacts: each future neuron delivers an inhibitory signal to the cells next to it, deterring them from specializing as neurons too. Both the signal molecule (in this case, Delta) and the receptor molecule (called Notch) are transmembrane proteins. The same mechanism, mediated by essentially the same molecules, controls the detailed pattern of specialized cell types in various other tissues, in both vertebrates and invertebrates. In mutant flies in which this signaling mechanism fails, some cell types (such as neurons) are produced in great excess at the expense of other cell types. Chapter 16 3/20 Wednesday, March 20, 2013 9:53 AM 16.5 • Specific rec/ intracellular sigs/machinery determine response to sig. ○ Ex: Ach in heart muscle inhibits contraction, in salivary gland causes secretion. • Same rec/ different intracellular proteins/different responses. Skeletal Muscle different Ach rec; causes contraction Same signal (Acetylcholine in this case) Skeletal has different receptors than the other two When Ligand open channel Sodium goes in, muscle contracts Heart Muscle cell Positive ions leaving or negative ions coming in Heart vs Salivary Gland Both same receptors But different machinery Different cell types are configured to respond to the neurotransmitter acetylcholine in different ways. Acetylcholine binds to similar receptor proteins on heart muscle cells (A) and salivary gland cells (B), but it evokes different responses in each cell type. Skeletal muscle cells (C) produce a different type of receptor protein for the same signal; this receptor generates intracellular signals that differ from those generated by the receptor type on heart muscle cells. (D) For such a versatile molecule, acetylcholine has a fairly simple chemical structure. Figure 16.6 1. Survive a. Given ABC then it will just survive 2. Grow + divide a. However if you add DE, the cell will grow and divide into 2 cells 3. Differentiate a. If you put FG, it differentiates into a different cell type 4. Die a. Given nothing it dies (apoptotiosis) • Most cells variety of receptors; different responses depending on signals ○ Ex: morphogenetic determinants control cell fate during development Every cell type displays a set of receptor proteins that enables it to respond to a specific set of extracellular signal molecules produced by other cells. These signal molecules work in combinations to regulate the behavior of the cell. As shown here, cells may work in combinations to regulate the behavior of the cell. As shown here, cells may require multiple signals (blue arrows) to survive, additional signals (red arrows) to grow and divide, and still other signals (green arrows) to differentiate. If deprived of survival signals, most cells undergo a form of cell suicide known as apoptosis (discussed in Chapter 18). Figure 16.7 • Extracellular signals ○ Slow or fast responses Slow- □ Rec activation alters gene expression, protein synthesis, cytosol machinery (min to hour) Fast- □ Rec activation alters protein function cytosol machinery (sec to min) Certain types of cell responses—such as increased cell growth and division—involve changes in gene expression and the synthesis of new proteins; they therefore occur relatively slowly. Other responses—such as changes in cell movement, secretion, or metabolism—need not involve changes in gene expression and therefore occur more quickly (see Figure 16–5). Figure 16.8 • Hydrophilic ○ Very large hydrophobic signals bind cell surface receptors (or actively transported) to mediate intracellular response. Small hydrophobic signals pass through membrane; bind intracellular recs to induce response. A. Cell surface receptors a. Anything too large b. Is hydrophilic c. Fast process B. Intracellular receptors a. Small b. Hydrophobic c. If going into nucleus it will go through gene expression d. Slow Process (A) Most extracellular signal molecules are large and hydrophilic and are therefore unable to cross the plasma membrane directly; instead, they bind to cell-surface receptors, which in turn generate one or more signaling molecules inside the target cell. (B) Some small, hydrophobic, extracellular signal molecules, by contrast, diffuse across the target cell’s plasma membrane and activate enzymes directly or bind to intracellular receptors in either the cytosol or the nucleus (as shown here). Figure 16.10 • Cortisol (hydrophobic, stress) diffuses through membrane and binds intracellular nuclear rec. Rec/cortisol transported thru nuclear pore, binds DNA, alters expression of target genes and proteome. • Adrenal gland produces when it is stressed • Adrenal gland produces when it is stressed • Slow response • Chronic Cortisol production ○ In bloodstream can inhibit your immune system, t-cells from functioning, it breaks down the proteins that makes the glucose, cause blood sugar levels to be high, retain fluid, break down proteins that make muscle, etc. (worst case for long term stress) Cortisol is one of the hormones produced by the adrenal glands in response to stress. It diffuses directly across the plasma membrane and binds to its receptor protein, which is located in the cytosol. The hormone–receptor complex is then transported into the nucleus via the nuclear pores. Cortisol binding activates the receptor protein, which is then able to bind to specific regulatory sequences in DNA and activate (or repress, not shown) the transcription of specific target genes. Whereas the receptors for cortisol and some other steroid hormones are located in the cytosol, those for other signal molecules of this family are already bound to DNA in the nucleus even in the absence of hormone. Figure 16.11a • Inside blood vessels, what makes them dilate or not • Nitric Oxide (NO, gaseous local med) produced by endothelial cells of inner wall of blood vessels; slips thru mem, causes dilation and increased blood flow. • Paracrine Figure 16.11 b NO produced when Ach rec binds Ach at synapse. NO diffuses into smooth muscle cells/ binds intracellular target. Smooth muscle layer of vessel relaxes. Nitroglycerine converted into NO, treats angina. Angina- chest pain (lack of blood flow to the cardiac cells) (heart muscle cells) Sequence of events leading to dilation of the blood vessel. Acetylcholine is released by nerve terminals in the blood-vessel wall. It then diffuses past the smooth muscle cells and through the basal lamina (not shown) to reach acetylcholine receptors on the surface of the endothelial cells lining the blood vessel. There it stimulates the endothelial cells to make and release NO. NO diffuses out of the endothelial cells and into adjacent smooth muscle cells, where it regulates the activity of specific proteins, causing muscle cells to relax. Figure 16.12 Signaling Cascade • Cell surface recs; activate intracellular signal cascades • Signal cascade= series of intracellular sigs; change effector proteins ○ Intracellular signals = second messengers ○ Extracellular signals = primary messengers The receptor protein activates one or more intracellular signaling pathways, each mediated by a series of intracellular signaling molecules, which can be proteins or small messenger molecules; only one pathway is shown. Some of these signaling molecules interact with specific effector proteins, altering them to change the behavior of the cell in various ways. Chapter 16 3/22 Friday, March 22, 20139:48 AM Figure 16.3 • Signal cascades: ○ Transduce signal to other form ○ Relay signal to target proteins ○ Amplify signal; many second messenger molecules ○ Integrate signals from different pathways ○ Distribute signal to different effector molecules The components of these intracellular signaling pathways perform one or more crucial functions 1. They can simply relay the signal onward and thereby help spread it through the cell. 2. They can amplify the signal received, making it stronger, so that a few extracellular signal molecules are enough to evoke a large intracellular response. 3. They can receive signals from more than one intracellular signaling pathway andintegrate them before relaying a signal onward 4. They can distribute the signal to more than one signaling pathway or effector protein, creating branches in the information flow diagram and evoking a complex response A receptor protein located on the cell surface transduces an extracellular signal into an intracellular signal, which initiates one or more signaling pathways that relay the signal into the cell interior. Each pathway includes intracellular signaling proteins that can function in one of the various ways shown; some, for example, integrate signals from other signaling pathways, as illustrated. Many of the steps in the process can be modulated by other molecules or events in the cell (not shown). We discuss the production and function of small intracellular messenger molecules later in the chapter. Figure 16.14 • 2 common methods of changing protein function during signal cascaded a. Phosphorylation by Protein Kinase/ Dephosphosphorylation by Protein phosphate b. GTP binding/ GTP hydrolysis (A) Some intracellular signaling proteins are activated by the addition of a phosphate group and inactivated by the removal of the phosphate. In some cases, the phosphate is added covalently to the protein by a protein kinase that transfers the terminal phosphate group from ATP to the signaling protein; the phosphate is then removed by a protein phosphatase. (B) In other cases, a GTP- binding signaling protein is induced to exchange its bound GDP for GTP (which, in a sense, adds a phosphate to the protein). This activates the protein; hydrolysis of the bound GTP to GDP then switches the protein off. ****** All cell-surface receptor proteins bind to an extracellular signal molecule and ****** All cell-surface receptor proteins bind to an extracellular signal molecule and transduce its message into one or more intracellular signaling molecules that alter the cell’s behavior. These receptors, however, are divided into three large families that differ in the transduction mechanism they use. (1) Ion-channel–coupled receptors allow a flow of ions across the plasma membrane, which changes the membrane potential and produces an electrical current (Figure 16–15A). (2) G-protein–coupled receptors activate membrane-bound, trimeric GTP-binding proteins (G proteins), which then activate either an enzyme or an ion channel in the plasma membrane, initiating a cascade of other effects (Figure 16–15B). (3) Enzyme-coupled receptors either act as enzymes or associate with enzymes inside the cell (Figure 16–15C); when stimulated, the enzymes activate a variety of intracellular signaling pathways Figure 16.15a • Ligand gated ion channel/ ion- channel- coupled receptor ○ Acyticoline • Ion-channel-couple rec (aka LG ion channel): electric signal by ion flow across membrane upon binding 1⁰ signal. ○ Ex: Ach-gate Na+ channel neuromas junction ○ Cause cell to depolarize An ion-channel–coupled receptor opens (or closes; not shown) in response to binding of its extracellular signal molecule. These channels are also called transmitter-gated ion channels. Figure 16.15b 2. G protein couple (GPCR) receptors; 7-pass TM proteins. 1 deg. Signal binds extracellular domain, conformation change at cytosol domain, activates G protein (LL), activates another protein (usu. Enzyme). When a G-protein–coupled receptor binds its extracellular signal molecule, the activated receptor signals to a G protein on the opposite side of the plasma membrane, which then turns on (or off) an enzyme (or an ion channel; not shown) in the same membrane. For simplicity, the G protein is shown here as a single molecule; as we see later, it is in fact a complex of three protein subunits. Figure 16.17 GPCR use trimetric G-proteins (a, B, gamma, subunits). Inactive: 3 subunits together, alpha binds GDP. Activation Alpha exchanges GDP for GTP, dissociates from beta and gamma Alpha and Gamma are lipid linked Beta is (A) In the unstimulated state, the receptor and the G protein are both inactive. Although they are shown here as separate entities in the plasma membrane, in some cases at least they are associated in a preformed complex. (B) Binding of an extracellular signal to the receptor changes the conformation of the receptor, which in turn alters the conformation of the bound G protein. The alteration of the α subunit of the G protein allows it to exchange its GDP for GTP. This exchange triggers a conformational change that activates both the α subunit and a βγ complex, which conformational change that activates both the α subunit and a βγ complex, which can now interact with their preferred target proteins in the plasma membrane (Movie 16.2). The receptor stays active while the external signal molecule is bound to it, and it can therefore catalyze the activation of many molecules of G protein. Although it was originally thought that activation of a G protein always causes the α subunit and the βγ complex to dissociate physically (as shown here), in some cases the trimer might simply open up, allowing the activated α subunit and the βγ complex to each interact with a target protein. Note that both the α and γ subunits of the G protein have covalently attached lipid molecules (black) that help anchor the subunits to the plasma membrane. They are composed of three protein subunits—α, β, and γ—two of which are tethered to the plasma membrane by short lipid tails. In the unstimulated state, the α subunit has GDP bound to it, and the G protein is idle (Figure 16–17A). When an extracellular ligand binds to its receptor, the altered receptor activates a G protein by causing the α subunit to decrease its affinity for GDP, which is then exchanged for a molecule of GTP. In some cases, this activation is thought to break up the G-protein subunits, so that the activated α subunit, clutching its GTP, detaches from the βγ complex, which is also activated (Figure 16–17B). Regardless of whether they dissociate, the two activated parts of a G protein—the α subunit and the βγ complex—can both interact directly with target proteins in the plasma membrane, which in turn may relay the signal to yet other destinations in the cell. The longer these target proteins have an α or a βγ subunit bound to them, the stronger and more prolonged the relayed signal will be Figure 16.18 • Alpha activates target protein. Hydrolyzed GTP w/I sec (off). Releases target and binds beta/gamma again. (reused) When an activated α subunit binds its target protein, it activates the protein (or in some cases inactivates it; not shown) for as long as the two remain in contact. Within seconds, the α subunit hydrolyzes its bound GTP to GDP. This loss of GTP inactivates the α subunit, which dissociates from its target protein and—if the α subunit had separated from the βγ complex (as shown)—it now reassociates with a βγ complex to re-form an inactive G protein. The G protein is now ready to couple to another activated receptor, as in Figure 16–17B. Both the activated α subunit and the activated βγ complex can interact with target proteins in the plasma membrane Figure 16.19 • Beta/gamma interacts w/ target proteins. ○ Ex: Ach rec in cardiac cells is GPRC. Beta/Gamma opens K+ channels; K+ efflux. Inhibits AP formation/ ceases contractions. (A) Binding of the neurotransmitter acetylcholine to its GPCR on heart muscle cells results in the activation of the G protein, Gi. (B) The activated βγ complex directly opens a K+ channel in the heart cell plasma membrane, allowing K+ to leave the cell, decreasing the cell’s excitability. (C) Inactivation of the α subunit by hydrolysis of its bound GTP returns the G protein to its inactive state, allowing the K+ channel to close. Chapter 16 3/25 Monday, March 25, 201310:00 AM Figure 16.20 • G α activates memory enzyme; produces diffusible 2nd messengers (amplify) ○ GCPR involved ○ Has 3 subunits α,β, and γ ○ Alpha binds to GTP (lipid linked) They create 2nd messengers Becomes activated (Enzyme membrane bound) ○ Beta and Gamma stay together (conjoined twins) Because each activated enzyme generates many molecules of these small messengers, the signal is greatly amplified at this step in the pathway. The signal is relayed onward by the small messenger molecules, which bind to specific signaling proteins in the cell and influence their activity. Figure 16.21 • Cyclic AMP ○ Produced from ATP w/ enzyme adenylyl cyclase ○ Cyclic AMP phospodiesterase breaks down and stops cAMP □ 2nd messenger produced by adenylyl cyclase (AC) □ Phospodiesterase (PDE) degrades cAMP into AMP □ Always active, constitutive Cyclic AMP (also called cAMP) is formed from ATP by a cyclization reaction that removes two phosphate groups from ATP and joins the “free” end of the remaining phosphate group to the sugar part of the ATP molecule. The degradation reaction breaks this second bond, forming AMP. 16.23 • Skeletal Muscle ○ Adrenaline→Activate GPCR→G alpha activated→ activated AC→ATP makes cyclic AMP→ activate PKA Phosphorylate Kinase→activate glycogen phosphortase→glycogen Hydrolosis→glucose ○ Fast Response • ATP→myosin head (cause muscle contraction ○ Need the glucose to be brown down to make ATP The hormone activates a GPCR that turns on a G protein (Gs). . . which activates adenylyl cyclase, boosting the production of cyclic AMP. Cyclic AMP, in turn, activates PKA, which phosphorylates and activates an enzyme called phosphorylase kinase. This kinase activates glycogen phosphorylase, the enzyme that breaks down glycogen. Figure 16.24 • Adrenaline→GPCR→Galpha→AC→cAMP→PKA→transcription regulator→target genes→Transcription→translation→ changing proteome • Slow response Figure 16.25 • Inositol phospholipid ○ Only one on cytosol leaflet ○ Involved in cell signaling ○ IP3 serves as a ligand on channel on ER A lot of calcium comes out; considered second messenger b/c it amplifies the response Calcium binds to PKC (activates it) then binds to DAG, now can go through membrane ○ Galpha→activate phospholipase C→cleaves inositol phospholipid→IP3 and DAG→IP3 opens LG Ca2+ channels (ER)→Increase Ca + cytosol→Ca binds 2+ PKC→recrutied by DAG→phosphory target protiens Two small messenger molecules are produced when a membrane inositol phospholipid is hydrolyzed by activated phospholipase C. Inositol 1,4,5-trisphosphate (IP3) diffuses through the cytosol and triggers the release of Ca2+ from the endoplasmic reticulum by binding to and opening special Ca2+ channels in the endoplasmic reticulum membrane. The large electrochemical gradient for Ca2+ across this membrane causes Ca2+ to rush out into the cytosol Diacylglycerol remains in the plasma membrane and, together with Ca2+, helps to activate the enzyme protein kinase C (PKC), which is recruited from the cytosol to the cytosolic face of the plasma membrane. PKC then phosphorylates its own set of intracellular proteins, further propagating the signal. Figure 16.27 • Ca2+ activates calmodulin ○ Active calmodulin regulates target proteins including CAM-Kinase ○ Mutations CAMKII □ Alzheimer's, Angel man's syndrome, heart arrhythmia (A) Calmodulin has a dumbbell shape, with two globular ends connected by a long, flexible α helix. Each end has two Ca2+-binding domains. (B) Simplified representation of the structure, showing the conformational changes in Ca2+/calmodulin that occur when it binds to a target protein. In this conformation, the α helix jackknifes to surround the target protein (Movie 16.5). Figure 16.15c • Receptor 3 • Receptor 3 ○ Enzyme coupled receptors (ECR) Enzyme domain active when signal bound Most common □ Tyrosine Kinase (RTK) (C) When an enzyme-coupled receptor binds its extracellular signal molecule, an enzyme activity is switched on at the other end of the receptor, inside the cell. Many enzyme-coupled receptors have their own enzyme activity (left), while others rely on an enzyme that becomes associated with the activated receptor (right). Figure 16.30 1. Dimer signals bind to receptor 2. Receptor phosphorylates tyrosine 3. Receptor Dimerize 4. Intracellular signal binds to receptor Typically, the binding of a signal molecule to the extracellular domain of an RTK causes two receptor molecules to associate into a dimer. The signal molecule shown here is itself a dimer and thus can physically cross-link two receptor molecules. In other cases, binding of the signal molecule changes the conformation of the receptor molecules in such a way that they dimerize. Dimer formation brings the kinase domains of each intracellular receptor tail into contact with the other; this activates the kinases and enables them to phosphorylate the adjacent tail on several tyrosines. Each phosphorylated tyrosine serves as a specific binding site for a different intracellular signaling protein, which then helps relay the signal to the cell’s interior; these proteins possess a specialized interaction domain—in this case, a module called an SH2 domain—that recognizes and binds to specific phosphorylated tyrosines on an activated receptor or on another intracellular signaling protein. ***** KINASE ALWAYS PHOSPHORLATES****** Figure 16.31 • Adaptor activates RAS (activating protein); activates Ras (monomeric G-protein); Ras activates Phosphory cascade An adaptor protein docks on a particular phosphotyrosine on the activated receptor (the other signaling proteins that are shown bound to the receptor in Figure 16–30 are omitted for simplicity). The adaptor recruits a Ras-activating protein that stimulates Ras to exchange its bound GDP for GTP. The activated Ras protein can now stimulate several downstream signaling pathways, one of which is shown in Figure 16–32. Note that the Ras protein contains a covalently attached lipid group (black) that helps anchor the protein to the plasma membrane. Chapter 16 3/27 Wednesday, February 27, 2010:00 AM • Three types of receptors ○ Ion Channeled Coupled Receptors ○ G protein couple receptors (GPCR) ○ Enzyme Coupled Receptor (ECR) • Primary→RTK dimerzation→Phosphoralization of Thyrosines→Adaptor binds→Ras activator binds→Ras binds GTP Figure 16.32 • (primary) Mitogen= extracellular signal for cell proliferation (to create more of) ○ Generate mitosis in cell who receives it • MAPK ○ Mitogen activate protein kinase Signal Cascade □ Mitogen→RTK→adaptor→Ras activating protien→Ras • memMAPKKK→Cytosol MAPKKK→MAPK ○ Transcription regulators (slow) and other effector proteins (fast). ○ Mutant RAS in 30% cancer A Ras protein activated by the process shown in Figure 16–31 activates a three-kinase signaling module, which relays the signal. The final kinase in the module, MAP kinase, phosphorylates various downstream signaling or effector proteins. These proteins can include other protein kinases and, most importantly, transcription regulators that control gene expression. The resulting changes in gene expression and protein activity lead to complex changes in cell behaviors such as proliferation and differentiation. 16.42 • DO NOT memorize Chapter 5 3/27 Wednesday, March 27, 2013 10:30 AM Figure 5.2a • Cells store, use and pass genetic information • DNA=nucleotide polymer • Nucleotide made of ○ Phosphate ○ Sugar ○ Base (nitrogen containing) Each nucleotide is composed of a sugar–phosphate covalently linked to a base. Figure 5.2b • 4 types of nucleotides covalently bonded in linear array w/ directionality ○ The nucleotides are covalently linked together into polynucleotide chains, with a sugar–phosphate backbone from which the bases (A, C, G, and T) extend. Figure 5.2c • DNA= double stranded helix ○ Each strand has a backbone of sugar-phosphate; with bases inside ○ Strand are antiparallel and complementary base pairs • A DNA molecule is composed of two polynucleotide chains (DNA strands) held together by hydrogen bonds between the paired bases. The arrows on the DNA strands indicate the polarities of the two strands, which run antiparallel to each other in the DNA molecule. Figure 5.6 • Hydrogen bond b/t complementary base pairs ○ A-T; G-C • Phosphodiester bonds b/t sugar phosphate groups • The shapes and chemical structure of the bases allow hydrogen bonds to form efficiently only between A and T and between G and C, where atoms that are able to form hydrogen bonds can be brought close together without perturbing the double helix. Two hydrogen bonds form between A and T, whereas three form between G and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel—that is, oriented in opposite polarities.. The nucleotides are linked together covalently by phosphodiester bonds through the 3′-hydroxyl (–OH) group of one sugar and the 5′-phosphate (–PO4) of the next. This linkage gives each polynucleotide strand a chemical polarity; that is, its two ends are chemically different. The 3′ end carries an unlinked –OH group attached to the 3′ position on the sugar ring; the 5′ end carries a free phosphate group attached to the 5′ position on the sugar ring. Figure 5.7 • Double helix • Double helix ○ 2nm wide ○ Right handed coil ○ 10 bases per turn ○ Spaces b/t are grooves Major Minor • The two strands of DNA wind around each other to form a right-handed helix with 10 bases per turn. The coiling of the two strands around each other creates two grooves in the double helix. The wider groove is called the major groove and the smaller one the minor groove. The colors of the atoms are: N, blue; O, red; P, yellow; and H, white. Figure 5.8 • Information ○ Linear sequences of bases ○ Language 3 letter words each coding for particular amino acid Chapter 5 4/8 Saturday, April 13, 2010:07 AM Figure 5.9 • DNA partitioned into individual long molecules called chromosomes. ○ Each chromosome contains linear arrays of genes. Genes code for AA's in polypeptide or RNA □ 20 genes smallest □ 100's larger • DNA portioned into individual long molecules called chromosomes ○ Each chromosome contains linear array of genes Genes code for AA's in polypeptides or RNA • As we discuss in Chapter 7, each protein-coding gene is used to produce RNA molecules, which then direct the production of the specific protein molecules. Figure 5.10 • Collect chromosomes from cell, take photos and arrange them ○ Karyotype Mitotic chromosomes "Painted" or stained □ Painting is DNA hybridized to fluorescent cDNA probes • Somatic cells ○ 2 versions of each chromosome "Homologues" ○ Sperm and eggs are the only NON somatic cells • 46 chromosomes, 23 pairs • We are diploid ○ 2 set of chromosomes ○ Gametes are haploid The chromosomes shown here were isolated from a cell undergoing nuclear division (mitosis) and are therefore in a highly compact state. Chromosome painting is carried out by exposing the chromosomes to a collection of human DNA molecules that have been coupled to a combination of fluorescent dyes. For example, DNA molecules derived from Chromosome 1 are labeled with one specific dye combination, those from Chromosome 2 with another, and so on. Because the labeled DNA can form base pairs, or hybridize, only to its chromosome of origin ), each chromosome is differently labeled. For such experiments, the chromosomes are treated so that the double-helical DNA separates into individual strands, to enable base-pairing with the labeled, single- stranded DNA while keeping the chromosome structure relatively intact. The chromosomes as visualized as they originally spilled from the lysed cell. The same chromosomes have been artificially lined up in order. In this so-called karyotype, the homologous chromosomes are numbered and arranged in pairs; the presence of a Y chromosome indicates that the DNA was isolated from a male. Figure 5.11 • Staining Label A:T (Base pairs) ○ Label A:T (Base pairs) ○ Distinctive banding pattern for each chromosome • Chromosomes 1 through 22 are numbered in approximate order of size. A typical human somatic (that is, non-germ) cell contains two of each of these chromosomes plus two sex chromosomes—two X chromosomes in a female, one X and one Y chromosome in a male. The chromosomes used to make these maps were stained at an early stage in mitosis, when the DNA is compacted, but not so heavily compacted as it will be later in mitosis. The horizontal red line represents the position of the centromere, which appears as a constriction on mitotic chromosomes; the knobs (red) on Chromosomes 13, 14, 15, 21, and 22 indicate the positions of genes that code for the large ribosomal RNAs (discussed in Chapter 7). These patterns are obtained by staining chromosomes with Giemsa stain, which produces dark bands in regions rich in A-T nucleotide pairs. Figure 5.12 • Karyotypes detect chromosomal abnormalities • Extra, missing pieces ○ Ataxia 12 has an extra piece chromosomal piece of missing 4 attached ○ Disorder of muscle, trouble walking, motor ability limited • (A) A pair of Chromosomes 12 from a patient with inherited ataxia, a disease characterized by progressive deterioration of motor skills. The patient has one normal Chromosome 12 (left) and one abnormal, longer Chromosome 12. The additional material contained on the abnormal Chromosome 12 was determined from its banding pattern to be a piece of Chromosome 4 that had become inappropriately attached to Chromosome 12. Figure 5.15 • Interphase ○ During interphase, the cell is actively expressing its genes. For part of this time, the DNA is being replicated and the chromosomes are duplicated. Once DNA replication is complete, the cell can enter M phase • M-Phase ○ When mitosis occurs. Mitosis is the division of the nucleus. During this stage, the chromosomes condense, gene expression largely ceases, the nuclear envelope breaks down, and the mitotic spindle forms from microtubules and other proteins. The condensed chromosomes are then captured by the mitotic spindle, and one complete set of chromosomes is pulled to each end of the cell. A nuclear envelope forms around each chromosome set, and in the final step of M phase, the cell divides to produce two daughter cells. • Interphase ○ Chromosome shape change Figure 5.16 • Need to be long enough for replication • Interphase ○ De-condensed (long/skinny) proteins access to replicate each strand of double helix ○ The DNA replicates in interphase, beginning at the origins of replication and proceeding bidirectionally from the origins across the chromosome. • Mitosis Condensed, copies moved by spindle to opposite poles ○ Condensed, copies moved by spindle to opposite poles ○ In M phase, the centromere attaches the duplicated chromosomes to the mitotic spindle so that one copy is distributed to each daughter cell when the cell divides. The centromere also helps to hold the duplicated chromosomes together until they are ready to be pulled apart. The telomeres form special caps at each chromosome end. • Light microscope view mitosis • Each chromosome has multiple origins of replication, one centromere, and two telomeres. The sequence of events that a typical chromosome follows during the cell cycle is shown schematically. Figure 5.17 • Metaphase "In middle" ○ Each duplicate is 1 µm wide • Replicated and condensed chromosome (2) • Chromatid attached at centromere • Because of chromosome replication during interphase, each mitotic chromosome contains two identical daughter DNA molecules. Each of these very long DNA molecules, with its associated proteins, is called a chromatid. Figure 5.1a • Anaphase ○ Pull apart to opposite sides • Interphase ○ Stretched out/decondensed • A) Two adjacent plant cells photographed through a light microscope. DNA is fluorescing brightly with a dye (DAPI) that binds to it. The DNA is present in chromosomes, which become visible as distinct structures in the light microscope only when they condense in preparation for cell division, as shown on the left. The cell on the right, which is not dividing, contains the identical chromosomes; they cannot be distinguished as individual chromosomes in the light microscope at this phase in the cell’s life cycle because the DNA is in a much more extended conformation. Figure 5.18 • Chromosomes still there, just can't see so well • Chromosomes exist in interphase nucleus • DNA probes coupled with differ
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