Lecture 4 Cytoskeleton Filaments 2023 PDF

Cytoskeleton I: Filament Proteins 1 3 Types • Actin filaments (AFs, or microfilaments) • Microtubules (MTs) • Intermediate filaments (IFs) 2 Filament Construction • Small subunits form filaments. • Actin and tubulin, compact and globular. • disassembly, diffusion, reassembly 3 Filament Cons...

Cytoskeleton I: Filament Proteins 1 3 Types • Actin filaments (AFs, or microfilaments) • Microtubules (MTs) • Intermediate filaments (IFs) 2 Filament Construction • Small subunits form filaments. • Actin and tubulin, compact and globular. • disassembly, diffusion, reassembly 3 Filament Construction • Protofilaments, e.g. MTs • Weak noncovalent bonds. 4 Nucleation 5 Tubulin • Heterodimer = 2 different proteins, but considered “1 subunit” of tubulin • each binds GTP; hydrolyzed at only 1 site • 13 protofilaments • “plus” and “minus” end 6 Actin • Monomer • ATP • “plus” and “minus” end 7 Treadmilling and Dynamic Instability • Growth / shrinkage of filament proteins. • Actin and tubulin catalyze hydrolysis of ATP or GTP. • “T” or “D” form indicates if triphosphate or diphosphate form exists. • ATP / GTP caps. • Critical concentration (Cc): where subunit addition equals subunit loss. 8 Treadmilling – Actin Filaments Experiment: 1. Filaments added to ATP-actin. 2. [ATP-actin] high, addition occurs at both ends. 3. [ATP-actin] drops, addition greater at plus end. 4. Steady state. Treadmilling Nucleation is not a factor here since pre-formed filaments were added to actin solution 9 Treadmilling – Microtubules 10 Treadmilling • Rate of hydrolysis and Cc • Linear relationship of [subunit] vs. elongation rate There is no net change in length of T or D ends at their respective Cc 11 Treadmilling behaviour – MT Cell injected with rhodamine-labelled tubulin; White arrow, T-end; Red arrow, unlabelled notch 12 Fig. 16-10. Molecular Biology of the Cell Dynamic Instability – MTs • [Tubulin] within critical values • GTP cap • Continuous transitions 13 Dynamic Instability – MTs • Hydrolysis affects conformation Stable Unstable 14 Kinetics Dependent on [tubulin] Blunt tip Tapered tip At high subunit concentration, the T-end of MTs is modified to promote faster kinetics 15 Gardner et al. (2011). Cell 146:582-292. Dynamic Instability – MTs Epithelial cell, Rhodaminelabelled tubulin Dyn. Inst. Growth cone of neuron 16 Treadmilling and Dynamic Instability What is the purpose? Constant ATP consumption. Is it worth it? • Spatial and temporal flexibility with high turnover. • Fastest way to grow filaments without nucleation. • Exploration for attachment sites and remodelling. 17 Intermediate Filaments 18 Intermediate Filaments • (A,B) Dimer formation. • (C) Tetramer antiparallel formation. • (D) Tetramer-tetramer association. 19 Intermediate Filaments • • • • Tetramers packed into array of 16 dimers in cross-section. Rope-like appearance. Formation by spontaneous interaction. Disassembly likely regulated by phosphorylation. 20 “Apocalyptic hagfish spill covers highway in slime” July 2017 21 Storage and deployment of skeins in thread cells of hagfish Weingard et al. (2014) Nat. Comm. 5:3534; Weingard and Fudge (2010) J. Exp. Biol. 213:1235-1240 22 IF Types Epithelial (e.g. keratins) Axonal (e.g. neurofilament) Epithelial (keratins) • Most diverse family • Type I and II keratin chains • Strength in hair, nails 23 IF Types Axonal (neurofilaments) • Found in central and peripheral axons of vertebrate neurons. • Type L, M or H. • Growth, increase axon diameter. Axon Glia Axon (cross-section) 24 Things to Consider... 1. Can you differentiate between the processes of treadmilling and dynamic instability? 2. Think about the differences in assembly, growth and shrinkage between each of the 3 filament proteins that we discussed. 3. Where does ATP or GTP fit in? 25