BIOL 362 Cellular Dynamics Module 1-3: Cytoskeletal Dynamics III PDF

BIOL 362 Cellular Dynamics January 16, 2025 Module 1-3: Cytoskeletal Dynamics III Mouse fibroblast cells...

BIOL 362 Cellular Dynamics January 16, 2025 Module 1-3: Cytoskeletal Dynamics III Mouse fibroblast cells Actin Microtubules For people on the waitlist Wittm an n T. Niko n Small Wo rld https://www.nikonsmallworld.com/galleries/2003-photomicrography-competition/filamentous-actin-and-microtubules-structural-proteins-in-mouse-fibroblasts  Lecture Outline 1. What is the cytoskeleton? 2. Common properties of F-actin and microtubule dynamics 3. Actin filament dynamics in vitro: treadmilling 4. Microtubule dynamics in vitro: dynamic instability 5. Accessory proteins that regulate filament dynamics in vivo 6. Drugs that modulate cytoskeletal dynamics Relevant chapters in MBoC (7th edition): Chapter 16 2  Learning Goals 1. To understand how accessory proteins regulate cytoskeletons. 2. To understand how drugs control cytoskeletons. 3 1. Individual microtubule dynamics (review + methodology) 4  n in the equation represents net assembly of filaments 𝒅𝒏 = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Net assembly (n) = Number x length n 5 Clicker Q: Which test tube has higher n? Number Test Test tube 1 tube 2 Length: 10 100 50 Length: 8 20 20 Length: 5 0 1000 A: tube #1, B: tube #2 6  Clicker Q: B is correct Number Test Test tube 1 tube 2 Length: 10 100 50 Length: 8 20 20 Length: 5 0 1000 100*10+8*20 50*10+8*20+5*1000 = 1160 = 5660 A: tube #1, B: tube #2 7  MT dilution assay by Mitchison and Kirschner Number of MTs Experiments by Mitchison and Kirschner (1984) Microtubule density (108 /ml) Tubulin Elongation Reaction mix was 50 µM step diluted: Condition 1 Condition1: [free tubulin] ~ 15 µM Condition 2: [free tubulin] ~ 7.5 µM Condition 2 Time after dilution (min) Cc ~ 14 µM 8 Mitchison and Kirschner Nature (1984)  Surprisingly, some microtubules kept growing after the subunit dilution below Cc Experiments by Mitchison and Kirschner (1984) Before Frequencies of microtubules dilution Tubulin Elongation Dilution 50 µM step Condition1: [free tubulin] ~ 15 µM Condition 2: Condition 2 [free tubulin] ~ 7.5 µM Electron Condition 1 microscopy Microtubule length (µm) Mitchison and Kirschner Nature (1984) Cc ~ 14 µM 9 Length information did not follow biochemical theories learned before Number of MTs Microtubule density (108 /ml) Frequencies of microtubules Before dilution Condition 1: Net increase in Condition 1 mass. Condition 2: Net decrease in Condition 2 mass. Both are consistent with Condition 2 Condition 1 biochemical theories. Microtubule length (µm) Time after dilution (min) Mitchison and Kirschner Nature (1984) 10 Concept: Dynamic instability Loss of GTP cap Regain of GTP cap Mitchison and Kirschner Nature (1984) 11  How we handle mass vs length information in this course Actin filament Microtubules Biochemical data Microscopy data (length x number) (mostly focusing on length) In both cases, nucleation steps will be considered qualitatively rather than quantitatively 12  MT length information is captured using microscopy Movie of single MT Green: protein localized to the growing end Magenta: tubulin Movies are not a convenient form of data Movie of motor proteins to present in scientific in a neuron literature. 13  Timelapse data are often presented in a form of data called kymograph Kymograph of Kymograph of Original single MT motor proteins in a neuron kymograph 14 https://soilshop.net/anime/musclecontraction  Kymograph in cell biology is made from a stack of time series images Individual Movie (single MT) timepoints stacked Kymograph (= dense version of the stack) vertically 15 Extra information: how the kymograph is made 16  Clicker Q: Which is the expected kymograph? A B A line drawn here to make a kymograph time time 17  Clicker Q: A is correct A grass The dog passing the yellow line A line drawn here grass to make a kymograph The dog passing the yellow line time time 18 Summary: Kymograph shows temporal changes in MT length - end + end We are going to use time kymograph later. 3. Accessory proteins that control filament dynamics in vivo 20 Accessory proteins modify filament dynamics in vivo Stress fibres Mitotic spindle 1. End-binding protein 2. Non-end binding protein 3. Monomer-binding protein 21  Many proteins have plus-end or minus-end preference - + Tubulin/end binding protein (EB3) Plus-end binding - + Minus-end binding - + 22 Roostalu et al., eLife (2020)  End-binding proteins modify filament dynamics Minus-end Plus-end Polymerization Nucleation Nucleation Stabilization Stabilization Polymerization Nucleation Nucleation Stabilization Stabilization Catastrophe Nucleation Rescue Stabilization 23 What is stabilization?  Stabilization = less dynamic state time Control polymerization rate After stabilization :this slope depolymerization rate :this slope Catastrophe frequency = the number of catastrophe events per time 24  Capping proteins stabilize the actin plus-end Capping proteins: preferentially bind to the actin plus-end. Without capping protein: Both actin plus and minus-ends are dynamic With capping protein: The plus is stabilized With capping protein: The minus end remains to be dynamic https://www.mbi.nus.edu.sg/mbinfo/what-is-capping-protein/ 25 Clicker Q: Actin elongation rate after addition of capping proteins? Control 𝒅𝒏+ Select the correct statement 𝒅𝒕 A: dn+/dt line becomes flat B: dn+/dt line shifts rightwards 𝒅𝒏− C: dn-/dt line shifts rightwards 𝒅𝒕 D: dn-/dt line becomes flat Cc + Cc - [G-actin] 26 Clicker Q: Actin elongation rate after addition of capping proteins? Control 𝒅𝒏+ Select the correct statement 𝒅𝒕 A: dn+/dt line becomes flat B: dn+/dt line shifts rightwards 𝒅𝒏− C: dn-/dt line shifts rightwards 𝒅𝒕 D: dn-/dt line becomes flat Cc + - Note: Cc+ might change Cc as koff and kon will be suppressed differently [G-actin] 27 Accessory proteins can also regulate more than one functions Formin: actin plus-end binding proteins Plus-end Polymerization Polymerization rate Nucleation rate Goode and Eck Ann. Rev. Biochem. (2007) 28  Kymograph of actin dynamics with formin time Control time +Formin Nucleation Nucleation Polymerization rate Polymerization rate (elongation per time) If the dotted line is… More vertical: rate is low More horizontal: rate is high 29 Clicker Q: Which parameter is modified by formin? Elongation rate: Control 𝒅𝒏 = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 A: kon, B: [subunit], C: koff 30 Clicker Q: Which parameter is modified by formin? Elongation rate: Control 𝒅𝒏 = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 A: kon, B: [subunit], C: koff 31  Bulk actin assembly dynamics with formin Control +Formin 100 100 % subunits in filaments % subunits in filaments Cc Cc Steady state Nucleation elongation time time bigger kon Cc = koff/kon smaller Cc 32 Elongation rate in the presence of formin? Control dn/dt = kon[free subunit]-koff Cc We will deal with this type of question in Case Study I. [free subunit] 33 Example 2: The γ-tubulin ring complex stabilizes MT minus-end and promotes nucleation Centrosome contains γ-tubulin ring complex Nucleation rate Minus-end binding Minus-end stabilized 34  MT growth and catastrophe from the minus end is limited due to stabilization by the γ-tubulin complex in vitro + γ-tubulin complex in vivo Plus-end Rescue Catastrophe Minus-end Akhmanova and Steinmetz JCS (2019) 35 Example 3: Plus-end binding XMAP215 and catastrophe factor promotes microtubule polymerization and catastrophe, respectively XMAP215 Catastrophe factor (e.g., MCAK) Catastrophe frequency Growth rate Catastrophe frequency Nucleation rate 36  Effects of XMAP215 on individual microtubule time Control time + XMAP215 37  Some non-end binding proteins regulate filament stability from the lattice Stabilization Severing Disassembly Severing Stabilization Severing 38 Some non-end binding proteins regulate filament organization Bundling Network formation Bundling and crosslinking 39 Monomer binding proteins regulate availability of monomers ADP-actin ATP-actin “ADP-ATP “Sequestration” exchange” (unable to polymerize) Stathmin “Sequestration” (unable to polymerize) 40  Relationship between binding mode and function Function: Nucleation Binding mode: Polymerization Plus-end binding Catastrophe (MT) Minus-end binding Stabilization Non-end binding Severing Monomer binding Disassembly Organization Sequestration ADP-ATP exchange (Actin) 41 4. Cytoskeletal drugs that control filament dynamics 42  Cytoskeletal drugs were mainly isolated from living organisms Actin-targeting drugs Microtubule-targeting drugs Cytochalasin B Latrunculin Phalloidin Colchicine Nocodazole Taxol Artificial compound Amanitia fungi Sponge Crocus Yew tree mushroom 43 Wikimedia  Microtubule drugs have long been used in human history Colchicine Taxol Ancient Egypt Horticulture Cancer chemotherapy (treatment for gout) (creating different flower patterns) One of the most affordable and best- selling chemotherapy drug. Annual sales: > $1 billion USD Levan A. (1939) The Ebers Papyres (1550 BCE) 44  Microtubule drugs are used to treat cancers Chemotherapy drugs for early and locally advanced breast cancer Pill or IV drug (given by vein Drug (abbreviation) Brand name through an IV) 5-fluorouracil (5FU) Various brand names IV drug DNA/RNA synthesis Capecitabine Xeloda Pill /repair Carboplatin Paraplatin IV drug /duplication Cyclophosphamide (C) Cytoxan Pill or IV drug inhibitors Docetaxel (T) Taxotere IV drug Doxorubicin (A) Adriamycin IV drug Microtubule Epirubicin (E) Ellence IV drug inhibitors Methotrexate (M) Various brand names Pill or IV drug Paclitaxel (T) Taxol IV drug https://www.komen.org/breast-cancer/treatment/type/chemotherapy/drugs/  Mechanism of action of cytoskeletal drugs Phalloidin (Stabilization) Plus-end Inhibits subunit incorporation Cytochalasin B (Inhibits polymerization) Nocodazole Colchicine Latrunculin Taxol (Stabilization) There are some similarity between the mechanisms of action of cytoskeletal drugs and accessory proteins. 46  Summary Kymograph is a useful tool to visualize filament dynamics. There are four categories of accessory proteins. Each accessory protein has specific function. Some of them modulate kon, koff, (therefore Cc). Cytoskeletal drugs control filament dynamics. 47