BIOL 362 Cellular Dynamics: Cytoskeletal Dynamics II PDF

BIOL 362 Cellular Dynamics January 14, 2025 Module 1-2: Cytoskeletal Dynamics II Mouse fibroblast cells...

BIOL 362 Cellular Dynamics January 14, 2025 Module 1-2: Cytoskeletal Dynamics II Mouse fibroblast cells Actin Microtubules Slides 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 (review) 4. Microtubule dynamics in vitro: dynamic instability 5. Accessory proteins that control filament dynamics in vivo (next lecture) 6. Drugs that inhibit cytoskeletal dynamics (next lecture) Relevant chapters in MBoC (7th edition): Chapter 16 2  Learning Goals 1. To understand how to employ a simple equation to predict cytoskeletal dynamics. 2. To understand the concept of dynamic instability during microtubule assembly. 3 1. Actin filament dynamics (review) 4 Actin polymerization follows a sigmoidal curve Steady state t=3 t=2 t=0 5 Addition of actin oligomers accelerates the polymerization Growth curve without lag phase 6 Concept: Elongation rate can be defined by the simple equation rate constant Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 7  Four key points to note when using this equation Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 1. What does “n” stand for in real life? 2. Why does the incorporation rate depend on [free subunit]? 3. What is the difference between elongation rate and polymerization rate? 4. Can we apply this equation for nucleation step? Clicker Q: What does “n” stand for in real life? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Original definition of n n in real life? A. Filament length B. Filament number C. Filament mass Clicker Q: What does “n” stand for in real life? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Original definition of n n in real life? A. Filament length B. Filament number C. Filament mass (length x number) Clicker Q: Why does the incorporation rate depend on [free subunit]? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Because [free subunit] determines… A. kon B. The frequency of interactions between filament-ends and free subunits. C. The speed of free subunit movement. Clicker Q: Why does the incorporation rate depend on [free subunit]? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Because [free subunit] determines… A. kon B. The frequency of interactions between filament-ends and free subunits. C. The speed of free subunit movement. What is the difference between elongation rate and polymerization rate? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Select the correct statement: A. Elongation rate can be negative. B. Polymerization rate can be negative. C. Dissociation rate can be negative. What is the difference between elongation rate and polymerization rate? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Select the correct statement: A. Elongation rate can be negative. B. Polymerization rate can be negative. C. Dissociation rate can be negative. Incorporation rate = polymerization rate. Dissociation rate = depolymerization rate  Concept: Critical concentration is the subunit concentration at steady-state Slope of 𝒅𝒏 At steady-state… = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 this curve 𝒅𝒕 𝒅𝒏 =𝟎 𝒅𝒕 kon [subunit] = koff 𝒌𝒐𝒇𝒇 n 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 = 𝒌𝒐𝒏 Cc : Critical concentration Net Incorporation and dissociation 15 are balanced  Discuss with your neighbor: Can we apply this equation for nucleation step? Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 koff koff kon kon kon kon koff koff Clicker Q: Critical concentration Let’s assume we have a test tube containing steady-state actin filaments after an in vitro polymerization assay. After which of the following manipulations will the critical concentration change? A: Adding more free subunits to the test tube. B: Addition of proteins that facilitate subunit incorporation. C: Removing filaments from the test tube. 17 Clicker Q: Critical concentration Let’s assume we have a test tube containing steady-state actin filaments after an in vitro polymerization assay. After which of the following manipulations will the critical concentration change? A: Adding more free subunits to the test tube. B: Addition of proteins that facilitate subunit incorporation. C: Removing filaments from the test tube. B: some accessory proteins can modify kon or koff 18  We can intuitively predict polymer dynamics when Cc is known 𝒅𝒏 = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 When [subunit] = Cc : equilibrium [subunit] > Cc : polymer n will grow [subunit] < Cc : polymer will shrink 19  More intuitive mathematical explanation 𝒅𝒏 = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒚 = 𝒂𝒙 − 𝒃 𝒅𝒕 linear equation y Cc Cc 0 x -b (-koff) [subunit] 20  Critical concentration can be defined for each end to understand polymer dynamics in detail ATP cap k+on k-on k-off k+off Elongation rate Elongation rate at the minus end at the plus end 𝒅𝒏− 𝒅𝒏+ + 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌+ − 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌− = 𝒌on = 𝒌on off 𝒅𝒕 off 𝒅𝒕 At equilibrium At equilibrium - + -𝒌𝒐𝒇𝒇 + 𝒌𝒐𝒇𝒇 𝑪𝒄 = - 𝑪𝒄 = + 𝒌𝒐𝒏 𝒌𝒐𝒏 21  Difference in Cc between plus/minus ends creates a new state: treadmilling Note: Elongation rate indicates net growth or shrink. This is not same as the polymerization rate. When 𝒅𝒏+ [G-actin] < Cc+ 𝒅𝒕 Both ends shrink 𝒅𝒏 𝒅𝒏− [G-actin] > Cc- 𝒅𝒕 𝒅𝒕 Cc + Both ends grow Cc - Cc+ < [G-actin] < Cc- Plus ends grow [G-actin] Minus ends shrink 22 Movie: Actin Treadmilling https://www.youtube.com/watch?v=VVgXDW_8O4U 23 Clicker Q: Steady-state in this graph? Note: Elongation rate indicates net growth or shrink. This is not same as the polymerization rate. 𝒅𝒏+ Which of the following 𝒅𝒕 [G-actin] concentrations achieves steady-state? 𝒅𝒏 𝒅𝒏− 𝒅𝒕 𝒅𝒕 Cc + p q r A. p B. q Cc - C. r [G-actin] 24 Clicker Q: Steady-state in this graph? Note: Elongation rate indicates net growth or shrink. This is not same as the polymerization rate. 𝒅𝒏+ Which of the following 𝒅𝒕 [G-actin] concentrations achieves steady-state? 𝒅𝒏 𝒅𝒏− 𝒅𝒕 𝒅𝒕 Cc + p q r A. p B. q Cc - C. r At q, growth at the plus-end and [G-actin] shrinkage at the minus-end 25 cancel out (= zero net growth).  Discuss with your neighbor: Which is the correct graph? In vitro polymerization assay Time = 0 Steady state A Filament mass (n) There will be no assembly when at steady state initial [G-actin] is less than or equal to Cc. B When [G-actin] > Cc, polymers will be formed. (higher amount of steady state polymers when you add more monomers) 0 Cc Initially added [G-actin] at t = 0 26 Clicker Q: Which is correct? In vitro polymerization assay Time = 0 Steady state A at steady state [G-actin] C B 0 Cc Initially added [G-actin] at t = 0 27  Answer: B In vitro polymerization assay Time = 0 Steady state When initial [G-actin] < Cc, no net growth (no polymer formation). Thus, all subunits remain to be free. at steady state [G-actin] When initial [G-actin] > Cc, remaining free subunit concentration is B equal to Cc. Cc 0 Cc Initially added [G-actin] at t = 0 28 2. Microtubule dynamics 29  Discovery of microtubules Discovery of nucleus (1831) A microtubule inhibitor colchicine was used to Invention of Discovery of Discovery of cell treat gout in ancient microscope “Cell” division (1837) Discovery of Egypt. (1550 BCE) (16th century) (17th century) microtubules (late 19th century) Limitation of naked eye Technology development Identification of Colchicine binding protein: tubulin (1967) 30  Discovery of microtubules Walther Flemming Theodor &ofMarcella Boveri Discovery (1843-1905) (1862-1915; nucleus (1831) 1863-1950) A microtubule inhibitor colchicine was used to Invention of Discovery of Discovery of cell treat gout in ancient microscope “Cell” division (1837) Discovery of Egypt. (1550 BCE) (16th century) (17th century) microtubules (late 19th century) Paweletz N. Nature (2001); Flemming W. Advancements in Maderspacher F. Curr. Biol. (2008); Boveri T. (1899) Die Entwickelung von Limitation of naked eye Zellsubstanz, Kern und Zelltheilung (1882). Ascaris megalocephala mit besonderer Rücksicht auf die Kernverhältnisse technology Identification of Colchicine binding protein: tubulin (1967) 31 Microtubule dynamics in vitro is also predictable when Cc is known When Filament dynamics [free subunit] = Cc : no net growth Cc [free subunit] > Cc : polymers will grow [free subunit] < Cc : polymers will shrink [free subunit] 32 What we have learned so far are theories to predict bulk assembly Biochemical analysis (info of population of cytoskeletons) What people were able to infer was 100 bulk assembly of filaments. (mass = number x length) Filament mass (n) Critical concentration Steady state Visualization by microscopy Nucleation elongation Individual microtubules exhibited unexpected behaviors time 33 3. Individual microtubule dynamics 34  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 35 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 36  What does this information tell about filament mass? 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) 37  A ground-breaking theory of microtubule dynamics proposed in 1984 Some microtubules kept Individual MT is at either growing even when growing or shrinking phases [free tubulin] < Cc Hypothesis: A microtubule keeps growing until randomly transitions into the shrinking phase. Mitchison and Kirschner Nature (1984) 38 Visualization of individual MT dynamics at steady state Time-lapse imaging (steady-state) Tubulin/end binding protein Shrinking Growing Shrinking Time Growing Shrinking Horio and Hotani Nature (1986) Roostalu et al., eLife (2020) 39  GTP hydrolysis serves as a switch between growing and shrinking phases Tubulin/end binding protein WT tubulin GTPase mutant tubulin Roostalu et al., eLife (2020) 40 Concept: Dynamic instability Loss of GTP cap Regain of GTP cap Mitchison and Kirschner Nature (1984) 41  Concept: Catastrophe is triggered by the conformational change of microtubule lattice Growing Shrinking Plus-end Igaev and Grubmuller Plos Comp. Biol. (2020) 42  Summary of filament dynamics in vitro Elongation/steady state Shrinking Nucleation kon, koff: constant High koff Low kon GDP Rescue GDP Catastrophe GDP High koff kon, koff: constant Low kon 43  Summary Understanding of bulk assembly and individual filament assembly are both important to fully understand mechanisms of filament dynamics. Microtubule repeats two different steps: growth and shrinkage. Transition between these states are called catastrophe and rescue, respectively. Catastrophe is induced by the GTP hydrolysis-dependent conformational change of tubulin oligomers. 44 5. Practice question set 45  Question 1 Indicate if each of the following structures is based on actin filaments (A), microtubules (M), or intermediate filaments (I). Your answer would be a four-letter string composed of letters A, M, and I only; e.g. AAMI. ( ) The cell cortex ( ) The mitotic spindle ( ) The nuclear lamina ( ) Thin filaments A: AAIM B: AMMI C: AMIA D: MMIA 46  Answer: C Cell cortex Mitotic spindle Nuclear lamina Thin filament 47  Question 2 Indicate true (T) and false (F) statements below regarding the cell cytoskeleton. Your answer would be a four-letter string composed of letters T and F only; e.g. TTFF. ( ) The three major building blocks of cytoskeletal filaments can bind to and hydrolyze nucleotides. ( ) The building blocks of actin filaments and microtubules are globular proteins, whereas those of intermediate filaments are themselves filamentous proteins. ( ) Intermediate filaments provide mechanical support to the cell. ( ) Plant cells lack microtubules. A: TTFF B: FTTF C: TFTF D: FFTT 48  Answer: B Polymer: Actin filament (F-actin) Microtubule Intermediate filament Keratin/Vimentin Subunit: actin (G-actin) α-tubulin/β-tubulin Neurofilament protein Desmin/etc. Globular proteins 49  Question 3 Which of the following event corresponds to the “lag phase” of the filament growth curve during in vitro polymerization? A. Nucleation B. Reaching steady state C. ATP or GTP hydrolysis D. Treadmilling 50  Answer: A Actin dimer and trimer are unstable # of contacts 1 3 6 8 (hydrophobic interaction) Also true for microtubules 51  Question 4 The time courses of seeded actin polymerization under two different conditions are compared in the following graph. If the overall kon for polymerization is known to be the same under both of these conditions, which curve—(1) or (2)—corresponds to the condition with a higher koff rate constant? Which one corresponds to a higher Cc for polymerization? (1) subunits in filaments Percentage of actin (2) Time after salt addition A. Curve (1); curve (1) B. Curve (1); curve (2) C. Curve (2); curve (1) D. Curve (2); curve (2) E. Curve (2); both curves have the same Cc. 52  Answer: D The time courses of seeded actin polymerization under two different conditions are compared in the following graph. If the overall kon for polymerization is known to be the same under both of these conditions, which curve—(1) or (2)—corresponds to the condition with a higher koff rate constant? Which one corresponds to a higher Cc for polymerization? 100% Cc (1) When koff is high, subunits in filaments Percentage of actin Cc (2) Cc is high because Cc = koff/kon Cc is the unused free subunit concentration at the steady state (shown by arrows). Time after salt addition A. Curve (1); curve (1) B. Curve (1); curve (2) C. Curve (2); curve (1) D. Curve (2); curve (2) E. Curve (2); both curves have the same Cc. D is correct 53  Question 5 If the concentration of free subunits is C, under which condition does the growth of a cytoskeletal filament proceed spontaneously? A. C > 1/kon B. C < 1/koff C. C > kon/koff D. C > koff/kon E. C > Cc×kon/koff 54  Answer: D If the concentration of free subunits is C, under which condition does the growth of a cytoskeletal filament proceed spontaneously? A. C > 1/kon B. C < 1/koff D is correct C. C > kon/koff D. C > koff/kon koff/kon is Cc E. C > Cc×kon/koff 55  Question 6 In the following graph of actin elongation rates under different subunit concentrations, which of the following corresponds to the slope of the line? grow A. kon Elongation rate B. koff C. kon/koff D. koff/kon 0 E. kon – koff shrink Subunit concentration 56  Answer 6 In the following graph of actin elongation rates under different subunit concentrations, which of the following corresponds to the slope of the line? grow A is correct. A. kon Elongation rate B. koff dn/dt C. kon/koff D. koff/kon 0 E. kon – koff shrink Subunit concentration This is a linear equation like 𝒅𝒏 y = ax-b = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 Then “a” is the slope 57  Question 7 According to the following graph, which shows the elongation rate at the plus and minus ends of actin filaments as a function of actin subunit concentration, at what concentration (A to E) does the total length of the filament remain more or less constant with time? grow Elongation rate 0 shrink A BC DE Subunit concentration 58  Answer 7 According to the following graph, which shows the elongation rate at the plus and minus ends of actin filaments as a function of actin subunit concentration, at what concentration (A to E) does the total length of the filament remain more or less constant with time? grow Elongation rate B is correct. 0 shrink A BC DE Subunit concentration 59