PFOS-014, 015, 011, 012

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PFOS-014, 015, 011, 012
Foundation Studies

1. Cytoskeleton and cell movement
2. Cellular Organelles
3. Cell membranes
4. Membrane Transport

Prof. K.M. Chan, Rm 513B, BMSB Dept. of
Biochemistry Chinese University Email
kingchan_at_cuhk.edu.hk Tel [phone]
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Objectives Be able to understand

• cellular system for life processes
• defects of the cellular system with clinical
  manifestations
• cells coup with environmental changes or external
  stimuli with membrane proteins and transporters
• basic structure and function of different
  cellular components, including membranes,
  cytoskeletons and organelles
• Malfunctions of and drug actions on cellular
  components

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PFOS-014 THE CYTOSKELETON CELL MOVEMENT

• Proteins are dynamic and movable
• Interact to make up a cell and maintain cell
  shapes they organize the cytoplasm, move
  organelles, etc
• They form major machinery for cell movement in
  response to environmental changes.

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The cytoskeleton composed of three systems

• (1) actin,
• (2) microtubules, and
• (3) intermediate filaments
• Extra-cellular Matrix will be discussed in other
  sections.
• (4) Summary and clinical correlations.

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Extra-cellular Matrix and three types of protein
filaments that form the cytoskeleton
4 Extra-cellular matrix, may restrict cell
movement
Cell membrane
Nucleus
1 Actin Filaments (microfilaments, 7 nm) are
helical polymers of actins
2 Microtubules with centrosome Using tubulin to
form hollow cylinders of 25 nm diameter to move
cargoes around
3 Intermediate Filaments rope like fibers with
10 nm size.
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1. ACTIN FILAMENTS

• Form micro-villi and contractile bundles
• Form sheet-like or fingerlike protrusions
  (pseudopodia) from the leading edge of a moving
  cell
• Many proteins (e.g myosin) bind to actin to
  modify its properties to form contractile
  structures in non-muscle cells and myofibril in
  muscle cells

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1.1 Actin filaments contain two chains of
polymerised actin monomers coiled together.
Thread-like structure
Other proteins attach to actin filaments, e.g.
myosin
7 nm in diameter
Actin Filaments
F-actin
Twisted chain of polymerized globular actin
molecules
Polymerized actin
Actin monomers
G-actin monomers
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1.2 Migrating cells use actin filament to create
podia ( foots)

• Lamelli-podium (thin sheet) and filo-podium
  (thin, point) are supported by actin filaments
  made underneath the cell membrane.
• The podia are for cell crawling.

Podia
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1.3 Skeletal Muscle Cells

• Muscle fiber cells are elongated
• A few cm long, with a diameter of only 50 µm
• Contain numerous myofibrils with actin and myosin
  filaments forming a highly ordered structure

Myosin filaments
Z disc
Z disc
Actin filaments
By moving the mysoin closer to the Z disc
alongside the actin filaments, muscle cells
become contracted
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Myosin molecule walks along actin filament using
ATP energy
Myosin head attached to actin filament
Myosin filament
ATP
Actin filament
ATP hydrolysis releases myosin head from actin
Binding of myosin head to a new site
ADP
Pi
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1.4 The Erythrocyte Cytoskeleton

• In red blood cells (erythrocytes), spectrins bind
  with actin to form spectrin membrane skeleton and
  maintain the biconcave (donut) shape of
  erythrocytes keeping the elasticity as well as
  flexibility of the erythrocytes.
• The actin filament is short and contains only 14
  monomer. It is stabilized by tropomyosin, and
  each binds 6 spectrin tetramers of 2a2ß
  heterodimer.

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To hold up the red blood cell, intracellular
protein network holds up the fluid like layer of
plasma membrane with peripheral proteins.
Erythrocyte Membrane structure
Spectrin
aß
Actin
Tropomyosin
Band 4.1
Ankyrin
Band 3
Transmembrane protein
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• Hereditary spherocytosis
• A genetic disease characterized by anemia,
  jaundice and splenomegaly (enlargement of the
  spleen).
• spherical and fragile red cells due to reduced
  spectrin content the spectrin cannot bind with
  4.1. protein to form the actin-4.1-spectrin
  complex.
• In hereditary elliptocytosis, spectrins form
  dimer instead of tetramer.

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2. MICROTUBULES

• Long, hollow cylinders made of tubulin proteins
• Outer diameter could reach 25 nm
• More rigid than actin or intermediate filaments
• Usually attached to centrosome which is the
  microtubule-organizing centre
• From free tubulin to a polymerized microtubule,
  the formation requires GTP binding
• During cell division, the microtubule framework
  forms mitotic spindle to guide chromosomes to
  move and segregate

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2.1 Microtubules are stiff hollow tubes of
protein monomers of tubulin polymerized together
as proto-filament.
25 nm
Tubulin is heterodimer of aand ßsubunits
Lumen at centre
Protofilament
atubulin
ßtubulin
GTP ( ) is needed to add on tubulin to the
protofilament as a growing microtubule.
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2.2 Properties and function of microtubules

• Dynamic instability tubulin hydrolyzes GTP to
  GDP building different conformations, growing or
  shrinking, to make up the interior of the cell.
• Proto-filaments with GDP are unstable and can
  peel away from the microtubule wall.
• The cells polarity is also created by
  microtubules which move different organelles to
  different ends of the cell. Microtubule has its
  polarity.
• Motor proteins like kinesins and dyneins are
  involved in controlling the two ends ( and -) of
  microtubules.
• Organelles move along the microtubules with the
  help of kinesins to the plus end, and dyneins to
  the minus end.

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Dyneins and kinesins transport cargoes
(organelles or macromolecules) along microtubules.
Dynein
14 nm
microtubules
Kinesin
8 nm
Colchicine makes microtubules disassemble and
the organelles change locations and go all over
the cell.
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2.3 Cancer Drugs kill cells by blocking the
cytoskeleton

• Colchicine blocks tubulin to form mitotic bundle
  and hence breaks tubulin cell division stopped.
• Taxol has an opposite action to hold microtubules
  and arrests dividing cells in mitosis, thus less
  side effects are found.
• Taxol and colchicine are effective anti-cancer
  drugs.

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3. INTERMEDIATE FILAMENTS

• Have great tensile strength to withstand
  mechanical stress.
• Known as intermediate because its diameter is
  around 10 nm, in between actin (7 nm) and
  microtubules (25 nm).
• They are tough and durable filaments to form a
  network in the cytoplasm or anchored to the
  plasma membrane for cell-cell interaction.

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3.1 Rope-like fiber of intermediate filaments
form web like structure to hold up cell
morphology, whereas actin and tubulin are
globular proteins.
C
N
N
C
C
N
N
C
C
N
C
N
C
C
C
N
N
Protofilaments are tetramer of two coiled-coil
dimers.
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3.2 Intermediate filaments can go through
cell-cell junction via desmosome to make cell
linked together, very common in epidermal cells.
Intermediate filaments
Desmosome
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3.3 Intermediate Filaments in various cell types

• Three classes in different cell types
• Keratin filaments in epithelial cells
• Vimentin and vimentin-related filaments in
  connective tissues
• Neurofilaments in neurons NFL, NFM, NFH.

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• Form nuclear lamina in the nucleus to strengthen
  nuclear envelope.
• When phosphorylated, they fall apart to
  facilitate disassembly of the nucleus for cell
  division.
• Chromatin in nucleus is also associated with
  nuclear lamina.
• The proto-filament proteins are called lamins.

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4.1 Clinical correlations (1)

• After a person has died, ATP synthesis stopped
  and thus myosin firmly attached to actin. This is
  why in the corpse, the muscle is stiff and rigid,
  a condition called rigor mortis.
• Integrin connects ECM and cytoskeleton through
  plasma membrane, tumor cells start to migrate
  (metatasize) must first disrupt integrin,
  collagen and other ECM proteins with digestive
  enzymes (matrix metalloproteinases, MMPs). MMP
  inhibitors may be useful in stopping metatasis of
  cancer cells.

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Clinical correlations (2)

• Linker molecules in ECM or actin-anchored
  proteins can act as chemotactic receptors.
• Neutrophils, e.g. respond to N-formylated
  peptides derived from bacteria by reorganizing
  the actin network and form microspikes that
  propel the cell toward the bacterium.

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4.2 The connective tissues control collagen
secreted in a organized way by attachment to
actin filament via fibronectin and integrin.
Collagen
Fibronectin
Integrin going through the membrane
Cell membrane
Adaptor proteins
Actin filament
Collagen from ECM attached to cytoskeleton
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Clinical correlations (3)

• Muscular dystrophy dystrophin is a cytoskeletal
  protein joining the membrane of muscle cells that
  mediates the interactions of extracellular
  matrix.
• It is a progressive myodegenerative disease when
  dystrophin is mutated, and some may die by 20
  with heart and lung failure.

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4.2 Summary

• Cytoskeletons are macromolecules of protein with
  tertiary structures in a highly ordered
  organization.
• ATP energy is required to change the conformation
  of myosin head for actin affiliation, leading to
  movement of cytoskeletons or muscle fibers.
• GTP is required to assemble tubulin monomers into
  filaments of microtubules.
• Anti-cancer drugs control cell division by
  attacking tubulins, e.g. Taxol (avoid GTP
  detachment) and Colchicines (avoid GTP binding
  for formation of filament).
• Mutations of genes causing defects in proteins
  and malfunction of the specialized functions,
  e.g. muscle contraction, anemia or hemolysis.