Cells and Cell Systems Review Slides PDF

Cells and Cell Systems Review Slides for the Last Lectures Table of contents 01 02 03 November 15 November 18 November 20 Cytoskeleton for Arteries Smooth muscle function Crawling motility and...

Cells and Cell Systems Review Slides for the Last Lectures Table of contents 01 02 03 November 15 November 18 November 20 Cytoskeleton for Arteries Smooth muscle function Crawling motility and angiogenesis 04 05 06 November 22 December 4 December 6 Cancer Lecture 1 Cancer Lecture 2 Cancer Lecture 3 01 November 15th Cytoskeleton for Arteries Learning Objectives: Students should be able to explain how force is generated on actin filaments through the motor protein myosin. Students should be able to describe optical methods for measuring forces on single molecules AND interpret data from these methods. Students should be able to describe basics of actin/myosin interactions in striated muscle. Students should be able to apply principles from striated muscle to the contraction process in smooth muscle, specifically in arteries. Students should be able to contrast the ways calcium regulates striated and smooth muscle function. Motor Proteins Motor proteins move cargo along a filament. We are focusing on myosin, but it does not hurt to understand structural similarities that allow these proteins to perform their functions Difference: myosin associates w/microfilaments (actin), while kinesin and dynein associates w/microtubules Similarities: 2 force generating heads Filament binding → each have a motor, lever, & stalk Cargo binding → Kinesin and myosin have tails, and dynein and kinesin have light chains. Myosin Function → Myosin proteins are a family responsible for pulling on actin filaments, moving things around cells, and generating the contractile force in muscle cells. The different types of myosin have different functions with shared structures like the force-generating ATP-binding motor domain Structure: Tail region = coiled coil (structurally robust) that connects the filament and cargo binding regions Regulatory subunits that change when the myosin is activated (regulates myosin/actin interactions) Studying Myosin Movement Biochemistry method →S1 fragments of myosin are fixed to a slide, and then fluorescent actin is settled down on the slide along as ATP is added. The myosin then interacts with the actin to generate a force that moves the actin along the slide. This method allows us to quantify the strength and type of force generated as well as estimating the amount of ATP required and the turnover rate. Optical methods = optical tweezers, fluorescent spot tracking, and atomic force microscopy (next slides) Optical Tweezers Optical Tweezers measure the force generated by a single contraction and the distance that the myosin moves (power stroke) How it works: 1. Two 1 µm beads are suspended with an actin filament between them. 2. The filament is lowered onto a bead coated with myosin. When the myosin head attaches to an actin filament, it undergoes a conformational change that pulls on the actin filament which then moves the 1 µm beads a short distance. 3. Photodetectors measure the displacement of the 1 µm beads, which is then used to determine the distance of a power stroke and force generated by a single myosin motor protein with nanometer and piconewton accuracy! Fluorescent Spot Tracking Fluorescent particle tracking measures the distance moved in each step (swing) by labeling a myosin tail with a probe and then tracking its movement down a microfilament. Graph (result) interpretation Swinging motion indicated by 72 µm distance as opposed to a 36 µm which would indicate dragging ○ This makes sense because swinging allows the myosin to cover larger distances Plateaus on the graph indicate that the myosin is stationary between swings ○ This does NOT make sense. It could not move move fast enough for its functions if it was stationary like that! moved 72 nm Therefore, this optical method likely does not give the no movement… whole picture. Atomic Force Microscopy Atomic force microscopy = Visualizes movement in real-time of myosin as it changes its shape during a power stroke. Works by vibrating needles across a surface of a protein fast enough that it detects where the pieces of protein are in time. The results are like a topography of the surface of the protein. This method has many other applications, but it can be helpful to characterize myosin movement because it is suited for the study of highly dynamic proteins. Actin/Myosin Interactions Actin/myosin interaction ------------------------> Striated muscle contraction: When muscle is not contracted, there is a gap between the two thin filaments When the muscle is contracted, the sarcomere (gap) is shortened The structure of the myosin protein keeps it in close proximity to actin filaments Could dynamic instability or treadmilling occur in these actin filaments? NO because accessory proteins keep the actin polymerized Striated vs. Smooth Muscles - Bipolar FIlaments Myosin II in both smooth and striated muscle is a dimer that assembles into a bipolar filament (heads pointing both ways) in its functional form While having filaments that pull in opposite directions seems counterintuitive, it is important generate force in the large scale Actin does not interact with bipolar filaments in an inactive state! Phosphorylation of the light chain by myosin light chain kinase (MLCK) causes spontaneous self assembly into a bipolar filament with other myosin molecules Striated vs. Smooth Muscles - Sarcomere? Striated muscle contraction involves sarcomeres that shorten (gap decreases) as myosin pulls on the actin Smooth muscles do NOT have sarcomeres, but have related structures called dense bodies that are usually relaxed but shorten (move inward) during contraction ○ Dense bodies = intermediate filament structures in smooth muscle cells that anchor actin filaments Striated vs. Smooth Muscles - Calcium Regulation Striated → actin-associated proteins (troponin + tropomyosin) ○ Low Ca2+ → tropomyosin covers myosin binding site ○ High Ca2+ → troponin binds Ca2+ → conformational change pulls the tropomyosin off, allowing the actin to bind to myosin for contraction Does not interact directly with myosin Smooth → calmodulin ○ Low Ca2+ → calmodulin is inactive in the cytoplasm ○ High Ca2+ → calmodulin is activated → activates MLCK to phosphorylate myosin for contraction Phosphorylation causes the tail to be extended, which then causes bipolar filaments to assemble Rigor Mortis Rigor mortis is muscle stiffening after death because there is no ATP produced by the mitochondria. Without ATP, the myosin heads can not detach from the actin filament leaving the muscle locked in a contracted state. Are smooth muscle cells also affected by rigor mortis? YES because while the regulation by calcium is different, the myosin still requires ATP to dissociate from the actin filament in both muscle types 02 November 18th Smooth Muscle Function Learning Objectives: Students should be able to describe the assembly and disassembly of contractile units in smooth muscle and explain the complications of myosin filament disassembly Students should be able to describe how caldesmon solves assembly problems and regulates contraction in smooth muscle. Students should be able to dissect signaling process to identify key elements controlling smooth muscle contraction Students should be able to predict what signals cause opening or closing of capillary beds via smooth muscle sphincters Force generation requires interaction between actin and myosin The basic unit of contraction is the interaction between actin and myosin. Myosin "pulls" on actin filaments through ATP-driven cross-bridge cycling. What is needed? Actin and myosin must be brought into proximity. Myosin must undergo conformational changes to interact with actin. Molecular Conformation Determines Function Proteins like myosin and regulatory enzymes (e.g., MLCK, MLCP) operate by switching between active and inactive conformations, which is often triggered by signals like calcium or phosphorylation. What is needed? Calcium ions act as a universal signal to regulate the activity of calmodulin, MLCK, and MLCP. Phosphorylation of the myosin light chain (MLC) is essential for altering myosin structure, enabling its interaction with actin. Stability and change in filamentous structures Filaments such as actin and myosin are dynamic. Their assembly and disassembly are controlled by physical and biochemical factors. What is needed? Actin filaments require stabilization by proteins (e.g., tropomyosin) to maintain structural integrity for contraction. Myosin filaments need to self-assemble into a functional form to exert force, which depends on the phosphorylation state of MLC. Energy is Required for Both Assembly and Force Generation ATP is the universal energy currency in cells. Its hydrolysis drives both myosin conformational changes (force generation) and filament disassembly (relaxation). What’s needed? Sufficient ATP to fuel cross-bridge cycling during contraction. ATP for phosphorylation and dephosphorylation processes that regulate filament assembly and disassembly.  3. Contraction Assembly From Principles to Process Calcium binds calmodulin → MLCK is activated → MLC is phosphorylated. 1. Signal Detection Myosin forms filaments and interacts Stimuli (e.g., neurotransmitters) cause ion with actin anchored to dense channels to open, increasing intracellular bodies/plaques. [Ca2+] 4. Force Generation RhoA signaling may also activate Myosin heads undergo conformational independently to enhance myosin changes, pulling actin filaments inward, activity. shortening the muscle. 2. Energy Flow 5. Relaxation/Disassembly ATP powers all critical steps: myosin Relaxation signals lower [Ca2+] or activation (via MLCK), filament assembly, activate MLCP, reversing MLC and cross-bridge cycling. phosphorylation. Myosin heads detach, filaments disassemble, and actin is stabilized in a relaxed state. 03 November 20th Crawling motility and angiogenesis Learning Objectives: Students should be able to contrast force generation in crawling motility to smooth muscle contraction. Students should be able to describe how Rac and Rho collaborate to create directional movement. All below are for Angiogenesis: Students should be able to describe roles of pericytes in blood vessels and angiogenesis. Students should be able to contrast pericyte function to endothelial tip growth in vessel formation. Students should be able to compare/contrast signaling processes (HIF1,VGF, Delta/Notch) in tip growth Moving Cells Cell crawling involves several distinct events: (1) extension of a protrusion at the cell’s leading edge (2) attachment of the protrusion to the substrate (3) generation of tension, which pulls the cell forward as its “tail,” or trailing edge, releases its attachments and retracts. Cell Protrusion To crawl, cells must produce specialized extensions called protrusion at their front or leading edge ○ Types of protrusions: A thin sheet of cytosol called a lamellipodium (singular) Thin, fingerlike projections called filopodia (plural) Forward assembly of these protrusions is driven by Arp2/3-dependent branching The small GTPases Rho, Rac, and Cdc42 regulate the types of polymerized actin that cells produce. Activated Rac promotes lamellipodia, whereas Cdc42 promotes filopodi Retrograde flow is fundamental to the dynamics of protrusions Cell Attachment Attachment of the cell to its substrate is also necessary for cell crawling ○ Attachment sites between a cell and its substrate consist of attachment protein called integrins. ○ On the outside of the cell, integrins attach to extracellular matrix proteins. Inside the cell, integrins are connected to actin filaments through linker proteins These integrin-dependent attachments are known as focal adhesions  Cell Contraction and Detachment Contraction at the rear of migrating cells is due to the action of myosin and actin under the control of the protein Rho (a GTPase) ○ Contraction of the cell body also requires detachment of the trailing edge of the cell. Detachment requires breaking adhesive contacts. Force at the Front Control of cell–substratum adhesion at the leading edge of a migrating cell. (A) Actin monomers assemble on the barbed end of actin filaments at the leading edge. Transmembrane integrin proteins (blue) help form focal adhesions that link the cell membrane to the substrate. (B) If there is no interaction between the actin filaments and focal adhesions, the actin filament is driven rearward by newly assembled actin. Myosin motors (green) also contribute to filament movement. (C) Interactions between actin-binding adaptor proteins (brown) and integrins link the actin cytoskeleton to the substratum. Myosin-mediated contractile forces are then transmitted through the focal adhesion to generate traction on the extracellular matrix, and new actin polymerization drives the leading edge forward in a protrusion. Chemotaxis Chemotaxis is a directional movement in response to a graded chemical stimulus ○ When a migrating cell moves toward a greater or lesser concentration of a diffusible chemical, the response is known as chemotaxis The molecule(s) that elicit this response are called chemoattractants (when a cell moves toward higher concentrations of the molecule) or chemorepellants (when a cell moves away from higher concentrations of the molecule). Steps in Cell migration Polarity is intrinsic to a migrating cell ○ (A). Cdc42, along with Par proteins and aPKC, are involved in the generation of polarity. Several additional proteins are implicated in polarity, which results in directed vesicle trafficking toward the leading edge, organization of microtubules (in some cells), and the localization of the MTOC (in some cells) and Golgi apparatus in front of the nucleus. In the presence of a chemotactic agent, PIP3 is produced at the leading edge through the localized action of PI3K, which resides at the leading edge, and PTEN, a PIP3 phosphatase that resides at the cell margins and rear. PTEN and myosin II are implicated in restricting protrusions to the cell front. The migration cycle begins with the formation of a protrusion.  Steps in Cell migration (B). WASP/WAVE proteins are targets of Rac and Cdc42 and other signaling pathways and regulate the formation of actin branches on existing actin filaments by their action on the Arp2/3 complex. Actin polymerization, in turn, is regulated by proteins that control the availability of activated actin monomers (profilin) and debranching and depolymerizing proteins (ADF/cofilin), as well as capping and severing proteins. Protrusions are stabilized by the formation of adhesions. This process requires integrin activation, clustering, and the recruitment of structural and signaling components to nascent adhesions. Integrins are activated by talin binding and through PKC-, Rap1-, and PI3K-mediated pathways. Integrin clustering results from binding to multivalent ligands and is regulated by Rac. At the cell rear, adhesions disassemble as the rear retracts. Steps in Cell migration (C). This process is mediated by several possibly related signaling pathways that include Src/FAK/ERK, Rho, myosin II, calcium, calcineurin, calpain, and the delivery of components by microtubules. Many of these molecules may also regulate the disassembly of adhesions at the cell front, behind the leading edge. Amoeboid Movement Involves Cycles of Gelation and Solation of Actin In an amoeba, as a pseudopodium is extended, more fluid material streams forward in the direction of extension and congeals at the tip of the pseudopodium (this event is often called gelation). Meanwhile, at the rear of the moving cell, gelatinous cytosol changes into a more fluid state and streams toward the pseudopodium (solation). Cytoplasmic streaming: an actomyosin-dependent movement of the cytosol within the cell Angiogenesis Remember: the circulatory system needs to provide nutrients and remove waste from ALL cells in the organism So, under situations like the following, you would need to grow more vasculature: Growth of tissue Repair of tissue Inflammation Try to think of others! Low O2-induced Angiogenesis Normoxia: ○ Cells constantly produce HIF-1a transcription factors, which are then hydroxylated by an enzyme called PHD, which will add oxygen to HIF-1a. ○ When HIF-1a is hydroxylated, ubiquitination will happen (ubiquitination is also a post-translational modification or PTM) ○ The more that ubiquitination occurs, the greater the proteasome degradation ○ Ubiquitination, in this case, is a signal to break down HIF-1a ○ Body constantly produces HIF-1a (a transcription factor) just in case Your body will continue to ubiquitinate it and break it down HIF-1a if there’s enough oxygen Low O2-induced Angiogenesis Hypoxia: ○ HIF-1a will become stabilized and will bind to HIF-1B They can move into nucleus together and bind to HREs (hypoxic responsive elements), which increases or upregulates transcription for a whole host of proteins that respond to low oxygen including generating new blood vessels (including VEGF!) Pericyte Singaling Endothelial cells secrete PDGF-B, that causes pericyte precursor cell proliferation and migration through activation of PDGFR- receptors. Pericytes surround and cover early endothelial tubes. By contrast, endothelial cells in vascular sprouts release VEGF, which in turn mediates suppression of PDGFR-signaling through the induction of VEGFR-2/PDGFR- complexes. This pathway abrogates pericyte coverage of endothelial sprouts leading to vascular instability and regression. Review this for Final! (Pericytes) Pericytes Interact with Endothelial Cells Serve as an intermediary, communicating to the endothelia the goings-on in the surrounding tissue!  Paracrine signal, Delta-Notch Microanatomy of a capillary sprout and tip cell selection. (A) An interstitial gradient for VEGF-A and an endothelial cell gradient for VEGFR2 are shown. Tip cell migration is thought to depend upon the VEGF-A gradient and stalk cell proliferation is thought to be regulated by the VEGF-A concentration. (B) Delta-Notch signaling is critical for tip cell selection. Activation of notch receptors on stalk cells induces proteolytic cleavage and release of the intracellular domain, which enters the nucleus and decreases gene expression of VEGFR2. Delta/Notch Lateral Inhibition Angiogenesis Summary Angiogenesis is a dynamic process that responds to multiple stimuli Uses activated division of pre-existing endothelial cells rather than stem cells Pericytes generally stabilize the endothelial structure and provide information on the perivascular environment Uses signaling to both initiate endothelial growth and termination of the process 04 November 22nd First Cancer Lecture Cancer Cell Evolution Mutations can create new beneficial trait Environmental constraints select for these mutations This allows for different lineages with different fitness levels This can occur because cancer cells divide so rapidly.  Sometimes in multicellular cancers, unicellular functions can be reactivated and upregulated through mutation ○ Undoing a ratchet kindve Multicellular regulators of these unicellular functions are downregulated See on next slide Contact Inhibition This is a form of division regulation in which a cell will undergo a negative regulation mechanism when in contact with another cell Cancer cells have a mutation in this mechanism that causes it not to function properly so cells continue to divide and stack. The two phases of tumor growth: Need some kind of mutation (DMBA) and some of kind of proliferation inducing irritant (croton oil) ○ On next slide Either by themselves does not lead to tumors Cells will continue to mutate and be under selection even after becoming a tumor Two general types of tumour growth: Increased Division Decreased cell death Sometimes both Vascularization: This is how a tumor supplies itself with nutrients Once a tumor stacks cells enough, the cells in the interior will not be exposed to enough nutrients (become hypoxic), so they secrete hormones/ signals that will recruit the formation of new vasculature. Remember the example (on the next slide) where the tumor that was already heavily exposed to oxygen, didn’t cause vascularization/growth , because it didnt have to! Only on a need basis.  Metastasis: Cancer cells invade the bloodstream and are deposited elsewhere in the body. They can likely vascularize in this new spot as well. Not every cell can go everywhere in the body though. Sometimes dependent of function/ where cell came from Researchers also perform artificial microevolution to determine how a cancer can become super metastatic by allowing a cancer to metastasize in an organism, extracting it, and then repeating in another organism until cancer is extremely good at it. Then study differences in those cells and healthy cells. 05 December 4th Second Cancer Lecture Ames Test for Carcinogens This test is used to determine how carcinogenic or how capable it is of causing cancer. They start with a bacteria that lacks a necessary gene to survive (cannot make their own histidine but need it to survive) This bacteria is incubated with this compound If the compound is carcinogenic, then some of the bacteria will undergo mutations that restore the histidine synthesis gene. * Ames Test for Carcinogens Carcinogenic Potency- how effective a compound is at causing cancer. A lower dose that can cause cancer will be more potent than a large dose that causes to cancer. So, we see that the highest potency on the graph is 10^-3 mg/kg/day Mutagenic Potency- how effective a compound is at causing cancer. It is expressed as the amount of the substance needed to produce 100 colonies per plate in the Ames test. The higher number of mutations a compound can make, the easier it will be able to make 100 colonies survive. So, the What compound has the highest compound with highest mutagenic potency will carcinogenic potency? Which one need the lower amount required to do this. has the highest mutagenic potency.? (look in speaker notes) Past Shinkle Question (!!!) To the right is a graph indicating results from an Ames test plotted against results from a mouse carcinogenesis experiment (same graph as in your book). A point represented by which letter (A-E) represents results showing high carcinogenic activity and low mutagenic activity? UVB Radiation The first graph shows how certain mutations are much more likely to cause some types of cancer than others Skin cancer is much likely to be caused by CC->TT mutations than internal cancers are ○ Using this information, we know can narrow down what is causing the cancer (not all carcinogens will do the same mutations in the same frequency) UV radiation often causes those double base mutations Oncogenes Proto-oncogenes are normal genes that regulate essential cellular processes like growth, and division. But when they are mutated they can become oncogenes. Oncogenes are mutated or overexpressed versions of proto-oncogenes that promote uncontrolled cell growth and division, leading to cancer. There are many different ways and Many of these mutations have the opposite cause situations a protoncogen can become (deletion, insertion etc) but all cause either an oncogene and how that oncogene abnormal hyperactive proteins or an excess of a can cause cancer protein Oncogenes Proto-oncogenes are normal genes that regulate essential cellular processes like growth, and division. But when they are mutated they can become oncogenes. Oncogenes are mutated or overexpressed versions of proto-oncogenes that promote uncontrolled cell growth and division, leading to cancer. There are many different ways and Many of these mutations have the opposite cause situations a protoncogen can become (deletion, insertion etc) but all cause either an oncogene and how that oncogene abnormal hyperactive proteins or an excess of a can cause cancer protein Tumor Suppressor Proto-oncogenes are normal genes that regulate essential cellular processes like growth, and division. But when they are mutated they can become oncogenes. They act as the a brake pedal by slowing down or inhibiting uncontrolled cell division. ○ They maintain normal cell function ○ But also are a defense against cancer Fill in the Blank Questions! Fill each blank with either tumor suppressor gene or protooncogene 1. A ________ promotes cell growth and division, while a ________ inhibits cell growth and ensures proper regulation of the cell cycle. 2. A ________ is often referred to as the "accelerator" of cell division, whereas a ________ acts as the "brake" to prevent uncontrolled growth. 3. Mutations in a ________ typically result in a gain of function, while mutations in a ________ usually result in a loss of function. 4. The inactivation of a ________ contributes to cancer development, while the activation of a ________ has the same effect. ` 5. For cancer to develop, a mutation in one copy of a ________ is usually sufficient, whereas both copies of a ________ typically need to be mutated or inactivated. 6. Examples of ________ include genes like p53 and RB, while examples of ________ include genes like RAS. 7. Base Excision Repair (fixes smaller mutations) 1. The first step involves a specific enzyme, called a DNA glycosylase, that identifies and binds to the damaged base. 2. Once the damaged base is recognized, the glycosylase removes it by breaking the bond between the base and the sugar-phosphate backbone. This leaves an empty site in the DNA called an AP site 3. An enzyme known as AP endonuclease then comes in to cut the DNA backbone at the location of the AP site 4. The gap is filled by DNA polymerase, which adds the correct base, using the complementary strand as a guide. a. The complementary strand is the template strand ` and is tagged with methyl groups 5. The final step is performed by DNA ligase, which reattaches the DNA backbone, sealing the repaired strand and restoring the DNA to its original state. Nucleotide Excision Repair (fixes bigger mutations) This figure to the right shows an example of nucleotide excision repair This is done as a response to DNA damage that caused a thymine dimer It involved a cut of a section of DNA rather than just one base ` 06 December 6th Third Cancer Lecture A crude method for finding oncogenes This method combines a cancer cell and a normal cell The hybrid cell has both the normal cell’s and and the cancer cell’s DNA. Meaning that there are working tumor suppressor genes that can suppress the oncogenes of the cancer cell nuclei ○ That's why there is normal growth But after subsequent division, some chromosomes are lost. Meaning that some many of the cells will have enhanced protein activity (oncogene) but no brakes ` Can be used to find tumor suppressor genes too! You just need to determine what genes are missing from cancerous cells. Human Papilloma Virus After DNA damage the p53 pathway is activated as a response. The top most part of the figure shows the normal function of p53-> apoptosis But, HPV makes a protein (E6) that ubiquitinates p53 which marks it for degradation ○ No longer can fix DNA damage or encourage apoptosis It also makes another protein (E7) that binds to Rb and inhibits it from halting cells at restriction point in cell division ○ It can’t act as a brake for cell division anymore ` Retinoblastoma (a tumor suppressor) genetics Hereditary Retinoblastoma: A fertilized egg inherits a RB mutation that is is then present in all cells in the body A second mutation must occur in the other copy of the RB gene in one or more of the retina cells With both copies of RB mutated, Rb function is lost, leading to uncontrolled cell division and tumor formation. ` Retinoblastoma (a tumor suppressor) genetics Nonhereditary Retinoblastoma: The individual is born with two normal copies of the RB gene. There must be two Mutations in the Retinal Cells in each copy of the RB gene. ○ Mutations happen randomly as the cells began to divide ` Good Luck! Don’t hesitate to ask the peer tutors, Dr. Shinkle, or Dr. Gilley for help General Advice This test will follow the same format of previous tests. Think about the study strategies that worked or did not work for you. You got this! You have three hours to complete a regular length exam! Use the time to ensure that you used the correct verbage and that you explicitly answered the question for free response questions. Do NOT memorize every little detail of older content. Focus more on how older concepts are integrated into newer content and things that have come up over and over again. This class primarily focuses on structure/function relationships. Always keep that in mind when you are answering questions. Note from Gilly/Shinkle: Be concise in answering free responses. Sometimes writing more than needed can make your point cloudy/ harder to grade/ give points. Categories of Final Content Cell signaling Protein structure/function** Extracellular matrix Protein modification Blood pressure regulation Membrane transport Cancer Cytoskeleton/motor proteins Multicellularity Transcription factors Endomembrane system Tool box items Note: Peer tutors do not typically hold office hours during reading days or finals week! These are days for everyone to prepare for their exams so we hope that you can extend us this same courtesy! However, some peer tutors may hold additional hours if they do have the time. We will let you know if this happens!