Molecular Cell Biology

Transcription

1 Molecular Cell Biology Biophysics of Helical Polymers Intermediate Filaments Michael Greenberg Department of Biochemistry and Molecular Biophysics

2 Intermediate Filaments

3 Diversity of Filaments Actin Filaments Microtubules Intermediate Filaments Adapted from Alberts et al., 2002

4 IntroducBon Filaments 10 nm Wide => intermediate Present in Metazoa / Animals i.e. Not Plants or Unicellular Organisms Complex Gene Superfamily 70 in Human Genome Specific Expression at Different Times and Places

5 Intermediate Filament PotenBal FuncBons In Vivo Mechanical Strength of Cytoplasm Help Epithelial Cells Resist Shear Stress - Filaments Connect to Cell- cell JuncBons Holding the Nucleus in Center of Cell Signaling Organizers (scaffolds) Anchoring AcBn Filaments to Focal Adhesions During Cell MigraBon

6 ConnecBons Between Filaments in Neurons Cortical Region Deeper Axoplasma Green = actin filaments Red = microtubules Yellow = intermediate filaments Cyan = crosslinking proteins (plectin) Klymkowsky 1999

7 Intermediate Filament Structure & Assembly Coiled-coils Nuclear lamina Highly conserved coiled- coil domain with variable tails.

8 Intermediate Filament Structure & Assembly Can form heterodimers and heterotetramers Basic subunit Unit Length Filament (ULF) Very little subunit exchange in larger filaments Filaments are apolar (no motors)

9 Intermediate Filaments Physical ProperBes Intermediate Filaments Adapted from Alberts et al., 2002

10 Intermediate Filaments by EM: Filament Unraveling

11 Intermediate Filaments Physical ProperBes Microtubules are strong but bri_le AcBn filaments are weak Intermediate filaments are strong and do not break easily Response of a network to sheer forces * Denotes failure of the network Adapted from Alberts et al., 2002

12 RegulaBon of IF Assembly Notoriously Stable No NucleoBdes Filaments Move Li_le Precursors Move More Disassemble Somewhat During Mitosis PhosphorylaBon by Cyclin- Dependent Kinases

13 Classes of Intermediate Filaments

14 VimenBn All Cells in Early Development Cage Around Nucleus Interacts with MTs

15 KeraBns Expressed in Epithelia KeraBn Filaments Connect to Desmosome and Hemidesmosomes DifferenBaBon of Epidermis includes ProducBon of Massive Amounts of KeraBn Provides Outer ProtecBon of Skin Composes Hair, Nails, Feathers, etc. Ericksson et al., 2009

16 FRAP of VimenBn vs. KeraBn in One Cell Left: Vimentin (Green) Right: Keratin (Red) 10-min time intervals

17 Filaments are Stable but Precursors Are Dynamic Keratin precursors moving 18 microns over 10 minutes

18 KeraBn MutaBons are Basis for Epidermolysis Bullosa Simplex Point MutaBon in Tail Domains Leads to a Failure to Assemble Tears between basal lamina and plasma membrane Wild-type Mutant Adapted from Alberts et al., 2002

19 Desmin Expressed in Muscle ElasBc Elements to Prevent Over- stretching Connects / Aligns Z lines MutaBons Lead to Myopathies Goldfarb et al., 2009

20 Neurofilaments Neurofilament H, M, L Copolymer Prevent Axon Breakage Diseases with Clumps of Neurofilaments Superoxide dismutase model for ALS Clumps are secondary, not causabve Immunostain for neurofilaments showing clumps (green arrows)

21 Lamins Square Lafce on Inner Surface of Nuclear Membrane Present in Metazoans (Animals, not Plants or unicellular organisms) Mitosis Breakdown PhosphorylaBon of A & C by cyclin- dependent kinase B remains with membrane MutaBons Cause Accelerated Aging Diseases Progerias dominant mutabons Important Roles in Mechanosensing 17 Years Old

22 Cytoskeletal Polymer Dynamics

23 Cytoskeletal Polymers Self Assemble In Vitro Actin Microtubules

24 General principles of macromolecular assembly 1. Large structures assemble from subunits 2. Specificity comes from multiple weak bonds on complementary surfaces 3. Subunits in symmetrical structures have identical or quasiequivalent bonds between subunits Helices Icosahedrons 4. New properties emerge along assembly pathways 5. Regulation is imposed at multiple steps along pathways

25 Advantages of assembling large structures from small protein subunits Allows synthesis of error-free components; errors in protein synthesis occur ~1/3000 residues, so most small subunits are free of errors Quality control: defective subunits can be discarded Subunits are easy to recycle Multiple modes of regulation are available

26 Thermodynamics of PolymerizaBon Add/Lose Subunits Only at Ends ON Rate = k + c 1 N OFF Rate = k - N c 1 = ConcentraBon of Monomers N = ConcentraBon of Filament End If the OFF Rate > ON Rate, get disassembly. If the ON Rate > OFF Rate, get assembly. The transibon from disassembly to assembly occurs when the ON Rate=OFF Rate. This is the cribcal concentrabon, C c. Simple algebra shows that C c = k - / k +

27 Steady- state ConcentraBons of Polymer & Monomer Critical Concentration [Polymer] Amount [Monomer] [Total protein]

28 KineBcs of PolymerizaBon NucleaBon limits polymerizabon NucleaBon occurs with acbn, microtubules, and intermediate filaments (ULF formabon)

29 KineBcs of PolymerizaBon Nucleators (e.g., Arp2/3, gamma- tubulin) reduce the energebc barrier to nucleabon.

30 DirecBonality of PolymerizaBon In this model, there is no preference for adding to/ removing from either end of the filament. In intermediate filaments, the addibon of ULFs is non- direcbonal. In acbn filaments and microtubules, monomers are added direcbonally.

31 PolymerizaBon Occurs DirecBonally in AF and MTs Actin Microtubules DirecBonality is due to conformabonal changes in acbn and microtubules associated with nucleobde hydrolysis.

32 DirecBonality of PolymerizaBon NucleoBde binding and hydrolysis changes the conformabon of the monomer, leading to asymmetric binding interfaces. AddiBon occurs preferenbally at the plus end. The cribcal concentrabon is lower at the plus end than the minus end. Unfavorable (slower) Favorable (faster) Nucleotide binding Dissociation is equally favorable from either end.

33 Treadmilling Can Have Different CriBcal ConcentraBons at the Two Ends Net AddiBon at One End, Net Loss at the Other End

34 Treadmilling: Microtubule Photobleaching Experiment In Vivo Fluorescent Tubulin Microinjected into Cell as Tracer Laser Bleaches a Vertical Stripe

35 NucleoBde Caps If the rate of subunit addibon > hydrolysis rate, get an NTP cap. This occurs during rapid growth. If the rate of subunit addibon is similar to the hydrolysis rate, the cap can disappear, leading to depolymerizabon.

36 Microtubule Catastrophe The hydrolysis of GTP in tubulin is associated with a large conformabonal change that strains the microtubule lafce.

37 Microtubule Catastrophe Loss of the GTP cap leads to the release of strain and microtubule catastrophe. This is dynamic instability.

38 Dynamic Instability of Microtubules GFP-tubulin in Cells Pure proteins in vitro

39 AcBn Filaments Instability In acbn filaments, the conformabonal changes are smaller, meaning that there is less elasbc energy stored in the filament and thus less dramabc instability.

40 Instability Allows for a Dynamic Cytoskeleton Two- Color Speckle Microscopy Microtubules Actin

41 Cells Regulate Polymers Cells Have Unexpectedly High ConcentraBons of Subunits Cells Change their Subunit / Polymer RaBo DramaBcally Filament Lengths in Cells are Short

42 How do Cells Regulate the Level of PolymerizaBon? Total ConcentraBon of Protein Covalent ModificaBon of Subunits (e.g., phosphorylabon of intermediate filaments) Binding of Small Molecules Binding of Another Protein (ABPs, MAPs)

43 AcBn Binding Protein Regulate PolymerizaBon Capping protein Arp2/3 complex binding Profilin Formin WASP alpha-actinin Fimbrin Spectrin Gelsolin- severs and caps ADF/cofilin- severs

44 MAPs Regulate MT PolymerizaBon Protein Activities Stabilizing MAPs Tau Binds side and stabilizes microtubules MAP2, MAP4 Binds side and stabilizes microtubules Destabilizing MAPs Op18/stathmin Binds tubulin dimers & destabilizes MTs Katanin AAA ATPase that severs microtubules XKCM/MCAK Kinesin-related; destabilizes (+) ends End-binding proteins (+TIPs) CLIP170 Links membranes to growing (+) ends EB1, EB3 Bind to (+) ends, reduce catastrophies, link MTs to other cellular structures

45 Small Molecule Regulators of AcBn PolymerizaBon Effects of microinjecting activated Rho-family GTPases (rhodamine-phalloidin) Filipodia Cortical actin Stress fibers

46 AcBn- Binding Drugs Cytochalasin: Binds barbed ends and leads to formation of ADP-actin pool! inhibits polymerization Latrunculin: polymerization Binds monomers (G-actin) and inhibits Phalloidin: Binds and stabilizes F-actin filaments, can be labeled with a fluorescent dye! stain actin in cells

47 Microtubule- Binding Drugs Colchicine: Nocodazole: Taxol: Binds tubulin dimers and inhibits polymerization Binds tubulin dimers and inhibits polymerization Binds to β-tubulin in polymers and stabilizes microtubules Cold and pressure also depolymerize MTs