CMB621: Cytoskeleton. Also known as How the cell plays with LEGOs to ensure order, not chaos, is temporally and spatially achieved

Transcription

1 CMB621: Cytoskeleton Also known as How the cell plays with LEGOs to ensure order, not chaos, is temporally and spatially achieved

2 Lecture(s) Overview Lecture 1: What is the cytoskeleton? Membrane interaction and general biological functions of cytoskeleton Lecture 1: Cytoskeletal protein biochemistry and polymer formation Actin, Tubulin and Intermediate filaments Lecture 1: Regulation of polymerization Lecture 2: Actin and Contraction Lecture 2: Motility and Cell Symmetry

3 Lecture Goals What is the cytoskeleton role in cells? What are the proteins that make up the cytoskeleton system? How are these proteins regulated to act in the cell? Think about how your own area of interest may be involved with the cytoskeleton Make the next step from Student to PI

4 Cytoskeleton Function Cells have many factors that need to be controlled The cell is not static during this time! Resist deformation Transport Cargo Change shape during movement or division Three major functions within the cell Organize content Connect the cell to its environment Generate forces for movement

5 Why is this Important? All cells are not created equal Cells utilize proteins, specifically the cytoskeleton, to create morphological structures What does this do to the membrane? Create of unique domains within the cell

6 Cell Migration Homeostasis shape Chemical induction Alters receptor organization Morphological changes

7 Cell Migration

8 What do we define as the Cytoskeleton? Three protein structures are what classically define the cytoskeleton Actin Filaments = microfilaments; play a role in membrane dynamics and muscle contraction

9 What do we define as the Cytoskeleton? Three protein structures are what classically define the cytoskeleton Actin Filaments = microfilaments; play a role in membrane dynamics and muscle contraction Microtubules; vesicular trafficking, cell movement, and mitotic spindle

10 What do we define as the Cytoskeleton? Three protein structures are what classically define the cytoskeleton Actin Filaments = microfilaments; play a role in membrane dynamics and muscle contraction Microtubules; vesicular trafficking, cell movement, and mitotic spindle Intermediate filaments; maintenance of cell shape and tension

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12 Actin Protein Compact, 42 kda ATPase Abundant in cells High affinity Mg/Ca binding site and an ATP binding site Minimal conformational differences G-actin/F-actin have different ATPase rates Hydrophobic plus end mediates protein-protein contact Hydrophobic cleft

13 Actin Filaments

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15 Cell Polarity

16 Cell Function Normal cell movement

17 Cell Function Normal cell movement Cell division: Actin polymerization Mitotic spindle

18 Cell Function Normal cell movement Cell division: Actin polymerization Mitotic spindle Cell division: Microtubules separate chromosomes Actin/myosin driven split

19 Cell Function Normal cell movement Cell division: Actin polymerization Mitotic spindle Cell division: Microtubules separate chromosomes Actin/myosin driven split Normal cell movement

20 Morphology When considering the cytoskeleton one should always think dynamic Cytoskeleton is required for stable morphology; yet the structures undergo subunit exchange

21 F-Actin formation (-) End (+) End Growth on the (+) end with ATP-Actin is ~ 10x faster than the (-) end

22 F-Actin formation (-) End (+) End Growth on the (+) end with ATP-Actin is ~ 10x faster than the (-) end Dissociation rate is about the same at both ends, however, this all depends on the concentration of actin monomers

23 F-Actin formation The actin pool is made up of monomers, soluble oligomers, and F-actin Equilibrium concentration is defined as the critical concentration (C c ); measure of actin polymerization

24 F-Actin formation! " = $ %&& $ %' = ( ) The actin pool is made up of monomers, soluble oligomers, and F-actin Equilibrium concentration is defined as the critical concentration (C c ); measure of actin polymerization When above the C C actin will form F-actin, however, at below all F- actin will dissemble

25 Polymerization of actin is unfavorable and slow; only becoming rapid after a nucleation site is created. Follows Mechalis-Menton kinetics and is driven by concentration and time Polymerization can be accelerated by the interaction of nucleation factors or by seeding a solution of monomers with nucleated actin.

26 Treadmilling

27 Why could this be essential?

28 Rapid Dynamic Changes

29 Localization of Nucleation Factors

30 Dendritic Nucleation Model

31 MF involvement in Cells Actin forms MF which are highly involved in cells How?

32 MF involvement in Cells Actin forms MF which are highly involved in cells How? Actin forms two types: bundles and weblike Formation depends on ABP

33 MF Structures Actin forms two types: bundles and weblike Formation depends on ABP Bundle proteins crosslink actin Gel proteins hold actin filaments at a large angle

34 Actin Binding Proteins Nucleation Actin polymerization is mediated by ABPs Initiate polymerization of new MFs Sequestering Bind to ATP/ADP-G-actin to keep them in a holding pattern Severing Sever F-actin, which result in shorter fragments, as well as generating new fragments for nucleation Crosslinkers Create networks, bundle or meshwork can dictate cytoplasmic environment Anchors Anchor MF to other parts

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36 Actin Nucleation Factors Actin-related proteins (Arps) Arp complex cap (-) end for nucleation and binds to F-actin at an angle nucleate branch network near PM...generating lamellipodia

37 Actin Nucleation Factors Formins Single polypeptide chain with multiple domains Dimeric protein that can bind 2 actin monomers Initiates unbranched polymerization at the (+) end Recruited to the PM after activation by Rho GTPase

38 Actin Nucleation Factors Formins Single polypeptide chain with multiple domains Dimeric protein that can bind 2 actin monomers Initiates unbranched polymerization at the (+) end Recruited to the PM after activation by Rho GTPase Seems to have other actin related properties

39 Sequestering Proteins Thymosin Regulates F assembly at (+) end by sequestering G-actin-ATP Abundant; preventing interaction of G- to F- actin; binding dependent on ph, Ca or PIP2 Profilin G-actin nucleotide exchange factor; Actin- ADP exchanged to ATP; supplying G-actin- ATP Promotes rapid F-actin assembly by directing bound G-actin to the (+) end

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43 Microtubules Formed by the protein tubulin (α, β, and ϒ) Typically forms a non-covalent heterodimer of α- and β-tubulin Each tubulin has a single GTP binding site GTP bound at the α/β interface is does not turn over

44 Microtubules

45 Dynamic Instability At this concentration it is possible to have T form growth (+ end) and D form shrink (- end) Growth could change if the T converts to a D even with a constant concentration of G-actin Recover of the filament may occur at some point Both MF and MT display dynamic instability

46 Microtubule Assembly

47 MTOC and Centrosomes Microtubules nucleate from a specific location called the microtubule-organizing center (MTOC) (-) End (+) End ϒ-tubulin serves to nucleate at the MTOC in a ring formation Cells have a defined MTOC called the centrosome Cytoplasmic microtubules emanate from this point Centrosome matrix that houses the centrioles and is composed of ϒ-TuRC

48 MTOC and Centrosomes Microtubules nucleate from a specific location called the microtubule-organizing center (MTOC) (-) End (+) End ϒ-tubulin serves to nucleate at the MTOC in a ring formation Cells have a defined MTOC called the centrosome Cytoplasmic microtubules emanate from this point Centrosome matrix that houses the centrioles and is composed of ϒ-TuRC

49 MTOC: Centrioles Cylindrical structures arraigned at right angles Become basal bodies of cilia and flagella in motile cells Composed of nine sets of triplet microtubule units that organize the centrosome matrix

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51 Association is regulated by phosphorylation Microtubule associated proteins (MAPs) MAPs stabilize microtubule bundles and mediate MT interactions Prominent in neurons; stabilize MT form the core of the axons and dendrites Have one domain bound to the MT and another projecting outwards MAP2 keep MT widely spaced; tau holds MT closely packed

52 MT capture and catastrophe factors (-) is stabilized by the centrosome, (+) end explore and probe the entire cell space Plus-end tracking proteins (+TIPs) accumulate at the active ends Modulate the growth and shrinkage of MT ends as well as positioning

53 MT capture and catastrophe factors

54 Neurodegenerative tauopathies Small, 352 to 441 amino acid, protein abundantly expressed in neurons Binds and stabilizes MT and promotes MT polymerization 79 potential phosphorylation sites; 30 of which have been shown to be functional Hyperphosphorylation leads to filamentous neuronal tau inclusions

55 Neurofibrillary Tangles

56 Intermediate Filaments Elongated polypeptides with central α-helical domain that form coiled-coli with other monomers. Parallel dimers associate to form a staggered tetramer Unusual family of cytoskeleton proteins: Large diversity in individual proteins (~ 60 genes) Non-polar; unable to bind nucleotides; regulated by phosphorylation Function to provide mechanical strength and providing protein-membrane anchoring sites

57 Intermediate Filaments Elongated polypeptides with central α-helical domain that form coiledcoli with other monomers. Parallel dimers associate to form a staggered tetramer Unusual family of cytoskeleton proteins: Large diversity in individual proteins (~ 60 genes) Non-polar; unable to bind nucleotides; regulated by phosphorylation Function to provide mechanical strength and providing proteinmembrane anchoring sites

58 Types of Intermediate Filaments

59 Most diverse IF family: 20 found in human epithelial cells Keratins Made up of an equal mixture of type I (acidic) and type II (neutral/basic) Disulfide bonds allow for formation of tough coverings for animals Epithelial cells produce multiple types of kertains to form a complex network

60 Cell Junctions

61 Epidermolysis Bullosa Simplex (EBS) Mutations in IF are associated with weaken cells Mutation in Keratins, typically K5 and K12, found in basal keratinocytes Severity depends on the location of mutation

62 IF: Neurofilaments

63 Regulation of the Cytoskeleton Each protein component is highly dynamic in cells Length, stability, number, geometry, etc Control is granted via protein-protein contact How can a cell regulated this interaction? Direct covalent modifications; accessory proteins!