Cellular Cytoskeletal System

Cellular Cytoskeletal System

Cellular Cytoskeletal System

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The Cell Cytoskeleton

  • A dynamic three-dimensional structure that fills the cytosol
  • The cytoskeleton comprises a filamentous network that serves to maintain cell shape and to regulate and implement important dynamic cellular functions
  • Function:
    • Cell shape and orientation
    • Organelle movement
    • Cell division
    • Cell migration
  • Eukaryotic cells contain three main kinds of cytoskeletal filaments
    • Microfilaments, microtubules and intermediate filaments
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Cytoskeletal System: Structure

Composition

  • Protein polymers
    • Contain thousands of identical subunits
    • Allows rapid reorganisation
  • Each filament type is formed by polymerisation of a distinct type of protein subunit
  • Has its own characteristic shape and intracellular distribution
  • Distinct size (diameter)
  • Microtubule - 25nm
  • Actin filament - 7nm
  • Intermediate filament - 8-10nm
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Microfilaments

  • Polymers of the protein actin

Actin filaments:

  • Ubiquitously expressed
  • Give shape to the cell surface
  • Important for motility (ability to move spontaneously and actively, consuming energy in the process)
  • Essential for mobility (ability of an object to be moved) and contraction of cells during cell division
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Actin Filaments

  • Thinnest filament of cytoskeleton
  • Fine, thread-like protein fibres, ~7nm in diameter
  • Comprised of actin subunits
  • Present as either a free monomer called G-actin (globular) or as part of a linear polymer microfilament called F-actin (filamentous)
  • Actin is a family of globular multi-functional proteins that form microfilaments or actin filaments
  • An actin protein's mass is roughly 42kDa
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Actin Filaments: Function

  • Microfilament functions
    • Cell shape and orientation
    • Muscle contraction
    • Movement of organelles
    • Cell division
    • Cell migration
  • Actin function depends on interaction with actin binding proteins (ABPs)
  • Over 150 known ABPs:
    • ~25% of cellular protein
    • Bind to g-actin and regulate polymerisation
    • Bind to f-actin and impart structural diversity
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Acting Binding Proteins Modify Actin Function

  • Bind to g-actin and regulate polymerisation
  • Latrunculin (natural product/toxin producted by certain sponges) sequesters G-actin and prevents F-actin assembly
  • Gelsolin (cytoplasmic, calcium-regulated) is an actin binding protein that is a key regulator of actin filament assembly and disassembly
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Cell Shape

  • Microfilaments interact with each other
  • ABP bind to f-actin and impart structural diversity
  • The association of various actin binding proteins can affect how the actin filaments are organised
  • For example they can be tightly cross linked in parallel polarised arrays to form bundles (microvilli) or loosely associated to form a meshwork (platelet)
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Actin and Cell Migration

  • ABPs remodel the cellular cytoskeleton
    • Impart strength
    • Important for cell migration
  • Cellular migration requires orchestrated movement in cells in particular directions to specific locations
    • Tissue formation during embryonic development (embryogenesis)
    • Wound healing
    • Axon growth
    • Immune responses (phagocytosis)
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Phagocytic Cells

Early infection - microbes that penetrate the skin or mucous membranes encounter phagocytic cells

  • Neutrophils
    • Chemo-attraction to site of infection
    • Amoeboid movement
    • Phagocytic
    • Destroyed during response
  • Monocytes
    • More effective phagocytic defense
    • Circulate than migrate to infected tissue
    • Enlarge and become macrophages
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Phagocytic Cells

  • Macrophages
    • Wander or permanently reside in connective tissue or organs
    • Amoeboid cells that phagocytose microbes and cell debris
  • Phagocytic Cell Migration
    • Phagocytic cells are attracted to damaged tissue by chemical signals (chemokines)
    • Neutrophils followed by monocytes (macrophages)
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Actin and Cell Migration

  • Cellular migration occurs in three stages:
    • Protrusion of leading edge - rapid actin polymerisation at the cell's front edge
    • Attachment of leading edge (provides traction) - actin filaments link the cell to extracellular substrates
    • Contraction (movement of main cell body forward) - myosin
      • Head region interacts with actin
      • Tail region binds to plasma membrane
      • Movement of myosin along actin filament causes membrane and cell contraction
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Actin and Cell Migration

  • Protrusion - rapid growth of actin filaments needed to push membrane forward
    • lamellipodia and filipodia formation
    • Actin and ARPs (actin related proteins)
  • Adhesion - adhesive interaction between membrane and substrate
    • Actin interaction with integrins
  • Contraction - generation of internal contractile force
    • Supports forward movement
    • Interaction of actin filaments with motor proteins (myosin)
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Actin Filament Dysfunction

  • The first case of neutrophil actin dysfunction (NAD) was reported in 1974; a male infant with a severe neutrophil motility disorder and poorly polymerisable actin
  • A genetic disorder
  • Neutrophil Actin Dysfunction is associated with impaired phagocytic uptake
  • Actin targeting drugs
    • Stabilise, depolymerise, polymerise or rearrangement of F-actin filaments - responsible for changes of cellular function
    • Actin targeting drugs are divided into three major classes
      • Cytochalasins, latrunculins and jasplakinolides
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Microtubules

  • Polymers of the proteint tubulin
  • Major components of the cytoskeleton
  • Function:
    • Structural support
    • Intracellular transport
    • DNA segregation
  • Tubulin protein - superfamily of globular proteins
  • alpha and beta tubulins polymerise into microtubules
  • Microtubules are hollow tubes ~25nm in diameter
  • Comprised of tubulin dimers - alpha and beta subunits
  • Tubulin dimers are organised in 13 parallel protofilaments
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Microtubules: Dynamic Instability

  • They have a very dynamic behaviour, binding GTP for polymerisation
  • Dynamic assembly and turnover
    • Allows cell to rapidly reorganise cytoskeleton
    • GTP hydrolysis plays important role
  • To form microtubules, the dimers of alpha and beta tubulin bind to GTP
  • GTP-dimers assemble onto the (+) ends of microtubules
  • Beta tubulin is exposed on the (+) end while the alpha tubulin is exposed on the (-) end
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Microtubules - Dynamic Instability

  • After incorporation of the dimer into the microtubule, GTP on betat-tubulin hydrolyses into GDP
  • Whether beta-tubulin is bound to GTP or GDP influences dimer stability
  • Dimers bound to GTP tend to assemble into microtubules (growing microtubule)
  • Dimers bound to GDP tend to fall apart (shrinking microtubule)
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Microtubules - Formation

  • Microtubule growth requires nucleation
    • event that initiates de novo formation of microtubules
  • Occurs at distinct structures, e.g. Centrosome
    • is an organelle that serves as the main microtubule organising centre (MTOC)
    • the structure from which microtubules energy 
  • MTOC functions:
    • Regulator or cell-cycle progression
    • Organisation of eukaryotic flagella and cilia
    • Organisation of the mitotic and meiotic spindle apparatus - separate the chromosomes during cell division
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Microtubles - Centrosome

  • Centrosome (microtubule organisating centre) - made up of centrioles (perpendicular rings; 9 triplets of tubulin)
    • Centriole
    • Mother centriole
    • Distal ends
    • Distal appendages
    • Subdistal appendages
    • Proximal ends
    • Microtubule triplets
    • Interconnecting fibres
    • Microtubules
    • Pericentriolar material
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Microtubles - Centrosome

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Microtubules: Function

  • Provide a set of 'tracks' for organelles and vesicles
    • Interaction with motor proteins
  • Provide structual support
  • Form the spindle fibres for separating chromosomes during mitosis
  • Arranged inside cilia to enable locomotion
  • Active transport of organelles within cytoplasm
    • Endocytosis
    • Exocytosis
    • Mitochondria movement
    • Protein transport between endoplasmic reticulum and Golgi appratus
  • Organelle transport and cell division - requires interaction with motor proteins
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Microtubules and Motor Proteins

  • Motor proteins generate force and 'walk' along microtubules
    • Kinesins - move toward (+) end
    • Dynesins - move toward (-) end
  • 'head' domain binds microtubule
  • 'tail' domain binds membrane or cargo
  • Motor proteins function in concert with microtubules
    • Provide transport for organelles and vesicles
  • Kinesins move along microtubule filaments, and are powered by the hydrolysis of adenosine triphosphate (ATP)
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Microtubule Drugs

  • Microtubules are involved in various cellular processes, cell division, cell cycle and cell proliferation
  • Crucial in the development and maintenance of cell shape, in the transport of vesicles, mitochondria and other components throughout cells, in cell signalling and in cell division and mitosis
  • Two classes:
    • inhibit tubulin polymerisation
    • promote tubulin polymerisation
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Microtubule-Disrupting Drugs

  • Tubulin-binding drugs kill cancerous cells by inhibitng microtubule dynamics, which are required for DNA segregation and therefore cell division
  • Agents which stabilise microtubules:
    • Taxanes (e.g. Paclitaxel (Taxol))
    • Ovarian and breast cancers
      • Prevent dissociation of tubulin subunits
      • Block mitosis (stabilise GDP-bound tubulin)
  • Agents which destabilise microtubules
    • Colchicine
    • Inflammatory conditions (arthritis, gout) promote disassembly of microtubles - dramatic change in organelle location, blocks migration of white blood cells - reduction of inflammation
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Intermediate Filaments

  • Composed of various proteins, depending on the type of cell in which they are found
  • Averaging 10nm in diameter
  • More stable (strongly bound) than actin filaments
  • Heterogeneous component of the cytoskeleton
  • Formed by heterogeneous class of proteins
  • Diameter ~10nm
  • Frequently found:
    • anchored to plasma membrane at cell-cell junctions
    • within nucleus
  • Impart resistance to stress
  • An intermediate filament is a strong fibre composed of intermediate filament protein subunits - not dynamic structures
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Intermediate Filament Assembly

  • The central rod domains of two polypeptides wind around each other in a coiled structure to form dimers
  • Dimers then associate in a staggered antiparallel fashion to form tetramers
  • Tetramers associate end to end to form protofilaments and laterally to form filaments
  • Each filament contains approximately eight protofilaments wound around each other in a ropelike structure
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Intermediate Filaments

  • Toughest of cytoskeletal components
  • Further stabilised through interaction with accessory proteins
    • plectin (links cytoskeleton to plasma membrane, connects cells)
  • Impart resistance to stress
  • Like actin filaments, they function in the maintenance of cell shape by bearing tension (during mitosis and during the positioning of the centrosome)
  • By contrast to microtubules, they resist compression
  • Organise the internal 3D cell structure
  • Anchoring of organelles
  • Structural components of the nuclear lamina
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Intermediate Filaments

  • Participate in some cell-cell connections
    • in combination with proteins and desmosomes
  • Anchor cell-matrix junctionsAllows the cell to communicate to adjust structures of the tissue based on signals from the cell environment
    • used in messaging between cells
  • Whereas actin filaments and microtubules are polymers of single types of proteins (actin and tubulin), intermediate filaments are composed of a variety of proteins that are expressed in different types of cells
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Intermediate Filaments

  • Categorised into four classes:
    • Type 1: keratins
      • hair and nails
    • Type 2: vimentin
      • cellular membranes
      • connective tissue and muscle
    • Type 3: neurofilaments
    • Type 4: nuclear lamins
      • coats lumen of nucleus
      • involved in dissolution of nuclear membrane in mitosis
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Intermediate Filaments

  • Abnormalities of neurofilaments result in disease of motor neurons, particularly amyotrophic lateral sclerosis (ALS)
  • ALS (Lou Gehrig's disease; motor neurone disease)
    • Stephen Hawking
  • Progressive loss of motor neurons
    • Leads to muscle atrophy, paralysis and eventual death
  • ALS and other types of motor neuron disease are characterised by the accumulation and abnormal assembly of neurofilaments
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