BIOL 362 Cellular Dynamics Module 1-1: Cytoskeletal Dynamics I PDF

BIOL 362 Cellular Dynamics January 9, 2025 Module 1-1: Cytoskeletal Dynamics I Mouse fibroblast cells...

BIOL 362 Cellular Dynamics January 9, 2025 Module 1-1: Cytoskeletal Dynamics I Mouse fibroblast cells Actin Microtubules 100 million new red blood Slides for people on the waitlist cells are being formed in our body every minute! (source: Cell Biology by the Numbers) 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 (next lecture) 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 shared and different properties of three types of cytoskeletons. 2. To understand the common mechanisms of filament polymerization. 3. To understand the role of nucleotide hydrolysis in filament dynamics. 4. To understand the concept of treadmilling. 3 1. What is the cytoskeleton? 4 Three types of cytoskeletal filaments Actin Intermediate Microtubules filaments filaments  Cytoskeleton is involved in human disease Actin Intermediate Microtubules filaments filaments Myopathy (muscle defects) Cardiac disease Skin disorder Hearing loss Ciliopathies Cataracts (vision impairment) Neuropathy Neuropathy Neuropathy Cancer Kidney disease Cancer Myopathy This module focuses on how they are assembled Actin filament – helical polymers with diverse assembly types Actin filaments Stress fibres Microvilli Helical polymers Muscle  Actin filament — a major component of the cell cortex Actin filaments bottom sideview F-actin plasma membrane C. elegans 6-cell stage embryo Cell cortex is critically important for cell shape change, cell division, migration, etc.  Microtubule — a hollow cylinder made of proteins called tubulin Microtubules Reconstruction by cryo-EM Hollow cylinder TEM Nogales and Kellogg Curr. Opin. Cell Biol. (2017)  Microtubule — localization patterns are diverse Microtubules Mitotic spindle Hollow cylinder Tetrahymena Array Cilia https://www.youtube.com/ watch?v=4AACrVZzqOk  Intermediate filament — rope-like fibres Schooley et al., Chromosoma (20 12) Intermediate filament Nuclear lamina Rope-like filament Keratin (epithelial cells) Intermediate filaments provide mechanical support Dog’s curly hair KRT71 (keratin) mutation Piko Cadieu et al., Science (2009)  Concept: cytoskeleton are polymers of smaller protein subunits Polymer: Actin filament (F-actin) Microtubule Intermediate filament Keratin/Vimentin Subunit: actin (G-actin) α-tubulin/β-tubulin Neurofilament protein Desmin/etc. Globular proteins 13  iClicker Quiz A B C Which type of cytoskeletal filament is not (clearly) found in plant cells? 14  iClicker Quiz A B C Which type of cytoskeletal filament is not (clearly) found in plant cells? However, plants likely have a functional equivalent of nuclear lamina according to recent studies (textbooks have not been updated yet). 15  2. Eight common properties of F-actin and microtubules dynamics 16  1. They are made of small subunits Hypothetical Laser induced-wound in frog oocyte giant protein Wound-healing with actin assembly transportation Slow and Benink and Bement Inefficient Journal of Cell Biol. (2005) Polymerization from a smaller subunit allows efficient regulation of assembly 17 and disassembly  2. They have multiple strands (protofilaments) Hypothetical, single-stranded cytoskeleton Energitically unstable in the (hydrophobic effects) middle and at the end. Multiple strands Energetically stable in the middle yet is dynamic at the end. 18 3. Polymerization/depolymerization occur at the end Tubulin/ end-binding protein  4. They have polarity Minus-end Plus-end: fast-growing Why do they have polarity? 20 A: Transient shape mismatch delays minus-end incorporation conformational change Subunit incorporation to the minus-end is relatively unfavorable. 21  5. Subunits contain nucleotides Actin filament (F-actin) Microtubule Subunit: actin (G-actin) α-tubulin β-tubulin ATP-bound or ADP-bound GTP GTP/GDP 22  6. Subunits are enzymes called nucleotide hydrolase Actin filament (F-actin) Microtubule Subunit: actin (G-actin) α-tubulin β-tubulin ATPase not hydrolyzing GTPase GTP ATP-actin + H2O GTP-tubulin + H2O ADP-actin + Pi GDP-tubulin + Pi 23 Q: Why is ATPase/GTPase activity important? A. It promotes polymerization. B. It promotes depolymerization. 24 7. Nucleotide hydrolysis promotes depolymerization ATP/GTP hydrolysis ATP/GTP ADP/GDP Subunit incorporation rate high low of free monomer Dissociation rate low high of subunit in the filament Because energetically relatively unstable 25  8. Growing actin/microtubule have ATP/GTP cap minus-end plus-end As long as there is a cap structure filament is keep growing 26  Summary: common properties of actin/microtubule dynamics minus T T plus D D T T T T T T D T D T T Yet, dynamics of actin and microtubule are quite a bit different. 27 3. Actin filament dynamics in vitro 28  Actin was discovered 80 years ago Albert Szent-Györgyi’s lab in Hungary Bugyi and Kellermayer J. Muscle Res. Cell Motil. (2020) A novel muscle protein was isolated from muscle tissues, named actin (1942). Actin exists in two different forms, named G-actin and F-actin. They are interconverted upon reversible polymerization in a salt-dependent manner. Actin has ATPase activity. Muscle contraction requires the interaction of actin, myosin, and ATP. Awarded a Nobel Prize in Physiology and Medicine in 1937 for discovery of Vitamin C 29  Actin polymerization can be monitored by fluorescent reporters Pyrene conjugated G-actin + salt Pyrene (small fluorescent molecule) t=0 Perkin-elmer.com Spectrophotometer (also used for measuring DNA/protein) Pyrene-actin signal is 25-fold higher in the polymer form. Kouyama and Mihashi (1981) Eur. J. Biochem. t=0 t=1 t=2 t=3 t=n 30  Reaction curve of actin polymerization is sigmoidal Typical enzyme Actin polymerization reaction Fluorescence [Product] delay time time 31 A sigmoidal curve of actin polymerization explained 32  Concept: Nucleation is a rate-limiting step of polymerization Actin dimer and trimer are unstable # of contacts 1 3 5 7 (hydrophobic interaction) 33 Addition of nucleated actin accelerates the polymerization Growth curve without lag phase 34  How can we quantitively understand filament dynamics? We are going to use simple math You have learned Example: Typical enzyme reaction Michaelis-Menten equation change of [Product] in a short period of time Cytoskeletal dynamics is easier to understand than this. 35  Reminder of Calculus Constant velocity Varying velocity L L (distance (distance traveled) traveled) ΔL ΔL Δt Δt time t1 t2 time t1 t2 Calculated ΔL ΔL dL v = v(t) = lim = velocity Δt Δt 0 Δt dt 36 Concept: Elongation rate can be defined by the simple equation rate constant Incorporation Dissociation Elongation 𝒅𝒏 Rate: = 𝒌𝒐𝒏 𝒇𝒓𝒆𝒆 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 37  Concept: Critical concentration is the free subunit concentration at steady-state Slope of 𝒅𝒏 At steady-state… = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 this curve 𝒅𝒕 𝒅𝒏 =𝟎 𝒅𝒕 kon [subunit] = koff 𝒌𝒐𝒇𝒇 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 = n 𝒌𝒐𝒏 Cc : Critical concentration 38  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 39  Mathematical explanation for the previous slide 𝒅𝒏 [subunit] = Cc = 𝒌𝒐𝒏 𝒔𝒖𝒃𝒖𝒏𝒊𝒕 − 𝒌𝒐𝒇𝒇 𝒅𝒕 𝒅𝒏 𝒌𝒐𝒇𝒇 = 𝒌𝒐𝒏 × − 𝒌𝒐𝒇𝒇 = 𝟎 𝒅𝒕 𝒌𝒐𝒏 [subunit] > Cc (when α > 0) 𝒅𝒏 𝒌𝒐𝒇𝒇 = 𝒌𝒐𝒏 × + 𝜶 − 𝒌𝒐𝒇𝒇 𝒅𝒕 𝒌𝒐𝒏 n = 𝜶𝒌𝒐𝒏 > 𝟎 [subunit] < Cc 𝒅𝒏 𝒌𝒐𝒇𝒇 = 𝒌𝒐𝒏 × − 𝜶 − 𝒌𝒐𝒇𝒇 𝒅𝒕 𝒌𝒐𝒏 40 = −𝜶𝒌𝒐𝒏 < 𝟎  But…actin has polarity! minus end plus end: fast-growing Can we predict filament growth rate for each end? (we can also consider ATP/ADP form differently but will skip that) 41  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 42  Q: Cc- vs Cc+. Which is greater? ATP cap k+on k-on k-off k+off - + 𝒌𝒐𝒇𝒇 - + 𝒌𝒐𝒇𝒇 A 𝑪𝒄 = - B 𝑪𝒄 = + 𝒌𝒐𝒏 𝒌𝒐𝒏 43  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. If 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 44 Treadmilling when Cc+ < [G-actin] < Cc- Labeled G-actin Actin subunits move from the plus end to the minus end 45  Actin treadmilling drives Listeria motility Locomotion of Listeria monocytogenes (foodborne pathogenic bacteria) in a mammalian cell bacteria Subunits inside the filament are moving. (the mechanism will be explained later) Lacayo and Theriot MBoC (2004) We will learn physiological meaning of this phenomenon in Module 3. 46  Summary Three major types of cytoskeleton: actin, microtubules, and intermediate filaments. Common properties of actin/microtubule dynamics: 1. subunit polymerization 2. nucleotide hydrolysis > promotes dissociation 3. plus-end = growing end Critical concentration can predict polymerization dynamics. Filament has nucleation, growing, shrinking, steady-state, and treadmilling states. 47