M4: Cytoskeleton

• BASIC PRINCIPLE

  • Single proteins (monomers) assemble into very long polymers via non-covalentinteractions between subunits
  •  Polymers can assemble and disassemble
  •  Each class of filament has its own set of associated proteins
• MICROTUBULE STRUCTURE AND DYNAMIC

  • Microtubules are unbranched cylinders of 25nm diameter assembled from tubulinheterodimers, hollow tubes, 25nm wide
  •  Alpha and beta tubulin form a stable dimer as soon as they are synthesised
  •  Polar structures:

  1. Plus (+) ends: grow rapidly and Beta tubulin is exposed and are located at the cell periphery
  2. Minus (-) ends: grow slowly, if at all and are located at the cell centre, at the centrosome

  • In a test-tube: Tubulin + GTP + Mg2+ --> (370C) Microtubules
  •  In the cell: tubulin concentration too low for polymerisation to occur spontaneously
• MICROTUBULE STRUCTURE AND DYNAMIC cont.

  • Cells use a template made of gamma-tubulin and other proteins to speed uppolymerisation to produce Gamma tubulin complex
  •  This is called nucleation; this is needed for favourable (fast growth)
  •  Gamma tubulin rings are concentrated on specific structures

  1. Centrosomes (in dividing cell)
  2. Basal bodies (ciliated cell)

  • Microtubules grow at their plus ends from gamma tubulin ring complexes at the centrosome
  •  Ciliated cells have an extra set of microtubules in the cilia which are nucleated by the basal body
• MICROTUBULES ARE DYNAMIC

  • Microtubules are dynamic: each one grows and shrinks independently of itsneighbours
  •  Each microtubule can switch between growing and shrinking = dynamic instability
• ATP AND GTP CONTROLS SHAPE, ACTIVITY AND FUNCTION

  • A slow hydrolysis rate gives a switch activity (ATP and GTP)
  •  Protein shape alters depending on nucleotide bound
  •  Cytosolic pool of unassembled GDP tubulin, GDP tubulin cannot polymerise
  •  Tubulin is a GTPase, GTP tubulin can polymerise
  •  In the microtubule, GTP is gradually hydrolysed to GDP
• DYNAMIC INSTABILITY

  • The protein EB1 binds preferentially to GTP tubulin, so marks growing microtubules
  •  GTP-tubulin dimers bind more tightly to each other than GDP-tubulin dimers becausetheir shape is slightly different
  •  If a GTP cap is present the MT will continue growing
  •  If the GTP cap is lost the MT will depolymerise (= Catastrophe)
  •  If a new GTP cap forms, the MT will start growing again (=Rescue)
• STABILISATION OF MICROTUBULES

  • Tubulin Dimers <- Microtubules
  •  By binding microtubule-associated proteins (MAPs) all along the microtubule
  •  By binding the drug taxol
  •  Neuronal MAPs:

  1. Tau
  2. MAP2

  • Non-neuronal MAPs e.g. MAP4
  •  By capture of MT plus ends by proteins at the cell cortex
  •  Composition of capping complexes is not fully understood
  •  Important in cell migration and establishing cell polarity
• MICROTUBULES CAN BE DEPOLYMERISED EXPERIMENTALLY

  • By putting cells on ice:

  1. Microtubules can depolymerise but can’t grow

  • Using drugs that bind free tubulin dimers, preventing new assembly

  1. Nocodazole
  2. Colcemid
  3. Colchicine
• ACTIN FILAMENTS (MICROFILAMENTS)

  • Actin filaments are a major component of muscle and are found in contractile bundles in almost all other cell types

  1. Stress fibres, contractile ring in dividing animal cells

  • Found in non-contractile bundles in almost all cells

  1. Microvilli, Lamellipodium, Filopodia

  • Structure

  1. Assembled from monomeric actin
  2. Thin, flexible, helical filaments
  3. 7nm diameter
• ACTIN POLYMERISATION

  • Actin filaments have a plus and a minus end
  •  Hydrolyse ATP after assembly (actin is an ATPase)
  •  In the cell, capping proteins usually bind to the minus end, preventing depolymerisation
  •  Disassembly occurs from different ends to microtubules
  •  Actin + ATP + Mg2+ -(370C)-> Actin filaments
  •  Phalloidin stabilises actin filaments
  •  Cytochalasin caps filament ends, preventing actin polymerisation from existing ends
  •  Latrunculin binds to actin monomers, preventing actin polymerisation
• ACTIN POLYMERISATION cont.

  • 5% of the cell’s protein is actin, this concentration of actin would polymerise spontaneously in a test tube (+ATP)
  •  Many proteins bind actin filaments and alter their organisation and dynamics
  •  Actin monomers <--> Monomer sequestering protein (e.g. thymosin)

  1. Some control balance between polymer and monomer

  • Actin monomers <--> Actin filaments

  1. Nucleating proteins promote polymerisation
  2. The cell uses them to control where polymerisation happens

  • Nucleating proteins / Arp2/3 complex: other proteins alter filament length or dynamics
• ACTIN POLYMERISATION cont3.

  
  

  • Nucleating proteins / Arp2/3 complex: other proteins alter filament length or dynamics
  • Some proteins alter filament organisation:

  1. severing proteins
  2. cross-linking proteins
  3. capping proteins

  • Others control or drive movement along actin filaments:

  1. motor protein
  2. side bonding protein
• CONTROL OF ACTIN ORGANISATION

  • Vital for cell function, migration and shape
  • Organisation and dynamics of actin is at the leading edge of migrate cells
  • Actin polymerisation at plus end protrudes lamellipodium
  • Contraction, attachment, further protrusion
• LAMELLIPODIA

  • Arp = actin-related protein
  •  Arp2/3 complex:

  1. Binds to side of existing actin filaments
  2. Nucleates assembly of new actin filaments
  3. Prevents disassembly at the minus end = causes branching

  • Main nucleator of actin filaments in lamellipodia
  •  Actin polymerisation pushes the plasma membrane forward
  •  Distribution/activity of actin associated proteins controls actin dynamics
  • Actin filaments disassemble at the rear of the lamellipodium
• FILOPODIA

  • Filopodia extend by actin polymerisation pushing on the plasma membrane
  •  Formins:

  1. Actin-nucleating proteins attached to the plasma membrane
  2. Add actin monomers to the plus end of actin filaments to form filopedia

  • Filaments don’t slide back because they are anchored by interactions with other actin filaments, via cross-linking proteins
  •  Filopodia play a key role in guiding the migrating cell by probing the environment and establishing new contacts with the surrounding ECM
• MECHANISMS OF ANIMAL CELL MIGRATION

  •  Cell pushes out protrusions at the leading edge of the cell
  •  Protrusions adhere to the surface
  •  Rear of cell is pulled forward

  1. using motor protein II
• PROTUSIONS ADHERE TO SURFACE VIA:

  • Focal contacts containing trans-membrane plasma membrane proteins calledintegrins
  •  Contractile actin bundles (stress fibres) attach to focal contacts
• CELL MIGRATION

  • Localised microtubule stabilisation is important for establishing cell polarity
  •  Cells in the immune system provide excellent examples of cell migration and the importance of chemotaxis in fighting infection
• INTERMEDIATE FILAMENTS

  • Found in animals: not unicellular organisms, plants or fungi
  •  Cytoplasmic- most animals, but not arthropods or hydra

  1. Keratin filaments (epithelial cells)
  2. Vimentin and Vimentin related IFs e.g. desmin (connective tissue cells, muscle cells and glial cells)
  3. Neurofilaments (nerve cells)

  • Nuclear- all animals

  1. Nuclear lamins (all nucleated animal cells)
• NUCLEAR LAMINS

  • Intermediate filaments underlie the nuclear envelope in nucleated animal cells, forming the nuclear lamina
  •  Mutations in nuclear lamins can lead to many diseases, including progeria (premature ageing syndrome)
• INTERMEDIATE FILAMENT PROPERTIES

  • 10nm diameter
  •  Do not bind nucleotides (e.g. ATP, GTP)
  •  Strong, rope-like, durable
  •  Stable (do not grow and shrink rapidly)
  •  Some disassemble during cell division (nuclear lamins, vimentin filaments) andreassemble in telophase
  •  Disassembly is triggered by phosphorylation
  •  IFs provide protection against stretching (epithelial and muscle cells) (strength acrossthe epithelial sheet)
  • Keratin filaments in adjacent cells linked via desmosomes

  1. Keratin mutations cause blistering
• EFFECT OF DESMIN MUTATIONS

  • Desmin is expressed in:

  1. Cardiac, skeletal and smooth muscle

  • Desmin mutations cause muscular dystrophy and cardiac myopathy
  •  Desmin filaments in adjacent muscle cells linked via desmosomes
• NEUROFILAMENTS

  • Strengthen neurons, which can have axons (thin cell processes) > 1 metre long
• INTERMEDIATE FILAMENT ASSEMBLY

  • Alpha helical region of monomer
  •  Lateral association of 8 tetramers
  •  Addition of 8 tetramers to growing filament
  •  Intermediate filaments are symmetrical, not polarised like actin filaments or microtubules due to dimers aligned into pairs in anti-parallel fashion, held together by non-covalent bonds
• INTERACTIONS BETWEEN FILAMENT SYSTEMS

  • Important for cell function and health
  •  Plectin links intermediate filaments, actin filaments,, microtubules and desmosomes
  •  Plectin mutations cause a very severe disease with skin disruption, muscular dystrophy and neurodegeneration
• BASIC PRINCIPLES OF MOTOR PROTEINS

  • Motor proteins use energy from ATP hydrolysis to move along tracks in eukaryoticcells
  •  ATP binding --> ATP hydrolysis creates an irreversible step  release of ADP and Pi
  •  Nucleotide binding and hydrolysis alters motor protein shape
  •  Myosins move along actin filaments, kinesin and dynein family members move alongmicrotubules
  •  Animals including flies and worms
  •  Some family members found in plants, protozoa, fungi and algae
• POLARITY AND MOTORS OF MICROTUBULES

  • Microtubule ends are at the cell periphery, minus ends are at the cell centre (centrosome)
  •  Kinesin moves towards MT plus ends
  •  Dynein moves towards MT minus ends
• KINESINS

  • Many different members
  •  Eg5
  •  Kinesin-1:

  1. Dimer of heavy chains – long coiled coil
• DYNEIN

  • 2 Types of Dynein
  •  Cytoplasmic Dynein
  •  Axonemal Dyneins

  1. Ciliary
  2. Flagellar

  • Cytoplasmic Dynein and its partner dynactin:

  1. Cytoplasmic dynein works with a partner complex, dynactin
  2. There is only one kind of cytoplasmic dynein, which moves many different cargos by binding to different adaptor proteins
• TRANSPORT VIA MICROTUBULE MOTORS

  • Membrane organelles

  1. Membrane traffic between organelles is facilitated by microtubule motors

  • Axonal transport

  1. Cytoplasmic dynein – backward transport (to cell body)
  2. Kinesin – Outward transport (to axon terminal)
  3. Vesicles take ~2 days to move down an axon 1 metre long

  • Transport vesicles move from the golgi apparatus to the plasma membrane along microtubules
• MICROTUBULES AND MOTOR PROTEINS POSITION ORGANELLES IN THE CYTOPLASM:

  • ER mainly moves outward using kinesin but here is also some inward movement driven by cytoplasmic dynein
  • ER moves along MTs in cell extracts (in vitro)
  • Golgi apparatus moves using cytoplasmic dynein
  • Also includes endosomes, lysosomes, mitochondria, nucleus, peroxisomes
  • Viruses are transported from the plasma membrane to the nucleus by cytoplasmic dynein, either within endosomes or as viral capsids
• SPEED OF MEMBRANES

  • A vesicle of 50nm diameter can move at 5um/sec
• MICROTUBULES ARE CARGOS FOR MOTOR

  • Some motors cause sliding:
  •  Anti-parallel sliding – e.g. in mitotic and meiotic spindles
  •  Parallel sliding – e.g. in cilia and flagella

  1. Cells with flagella: vertebrate sperm, some protozoa
  2. Cells with cilia: some protozoa, some epithelia

  --> E.g. airway epithelial cells

  -->Ciliated cells have an extra set of microtubules in the cilia which

  are nucleated by the basal body
• AXONEMES

  • Cilia and flagella contain specialised stable microtubule structures called axonemes
  •  Contains: plasma membrane, dynein arms, single central microtubules, microtubule doublet
  •  Axonemal dynein drives ciliary and flagellar beating
  • In isolated doublet microtubules: dynein produces microtubule sliding
  •  In a normal flagellum: dynein causes microtubule bending
• MYOSINS

  • Myosins move along actin filaments using energy from ATP hydrolysis
  •  Myosin superfamily has many members
• ACTIN FILAMENTS AND MYOSINS

  • Animals’ cells use microtubules for long distance membrane organelle transport
  •  Plants, algae and many fungi use myosins which move along actin filaments
  •  Muscle myosin (myosin II) is the best characterised myosin in animals
  •  Long coiled coil “tail” which can assemble into filaments
  •  Myosin II is found in most animal cell types, not just muscle

  1. Also found in contractile bundles called stress fibres
  2. Stress fibres attach to focal adhesions that adhere to the extracellular matrix
  3. Focal contacts contain integrins that indirectly link ECM to stress fibres
• ACTIN FILAMENTS AND MYOSINS cont.

  • Myosin I has one head

  1. Short distance organelle movement
  2. Myosin I helps reshape the plasma membrane by pulling on the underlying actin filaments
• ACTIN FILAMENTS AND MYOSIN II IN MUSCLE

  • Desmin mutations cause:

  1. Muscular dystrophy
  2. Cardiac myopathy

  • Desmin intermediates form a scaffold that stabilises the muscle Z discs
  •  Desmin IFs maintain organisation in the cell and connect to cell-cell junctions
• SLIDING FILAMENT THEORY OF MUSCLE CONTRACTION `

  • Myosin II walks along actin -> muscle contraction
  • Myosin II cross-bridge cycle:

  1. Attached -> Released -> Cocked -> Force-generating -> Attached