S5 - The Cytoskeleton and Cell Motility

general features of cytoskeleton

• cytoskeleton elements are not membrane- bound

• all cytoskeleton elements are polymers

  • microtubules = polymers of tubulin

  • micorfilaments = polymers of actin

  • intermediate filaments = variable

• non-covalent linkages i.e. dynamic elements

cytoskeleton functions

• structural support

• framework to position organelles

• movement of materials

• cell motility

• mitosis and cytokinesis

microtubules

• largest fibres (25nm)

• hollow tubes

• stiff, hollow, inextensible tube that can resist bending when a cell is compressed

• alpha-tubulin & beta-tubulin → heterodimers (noncovalent)

• heterodimers → protofilaments

• 13 profilaments → microtubule

image of microtubules

other structural feature of microtubules

• polar filaments

• plus end (bet subunit)

  • hydrolyze GTP → GDP

• minus end (alpha subunit)

  • only GTP (not exchangeable)

Structural: Microtubule Associated Proteins (MAPs) function and types

• increase stability of microtubules and promote their assembly

• MAP1, MAP2, MAP4, tau

two types of dynampic MAPs

• kinesin

  • move twds outside of cell

  • “+” end directed movement (kind/+)

• dynein

  • moves twds inside of cell (dying/-)

kinesin related proteins

• ~45 different kinesins

• divided between 14 families

• walk twds plus end (anterograde)

kinesin acceptions

kinesin-14 moves towards minus end

kinesin-13 doesnt move at all

kinesin-1

how does kinesin move?

• “hand-over-hand” mechanism

• “ATP Powers MT binding, ATP & ADP

cytoplasmic dynein

dynactin

required to bind cargo

function of microtubule-organizing centers (MTOCs)

• to organize MT-associated structures and organelles

• to orient kinesin-mediated and dynein-mediated transport of organelles, vesicles, and vesicular tubular clusters

location of microtubule-organizing centers (MTOCs)

• often perinuclear (surrounding nucleus)

• center of cell

nucleation

the initiation of growth of MTs

example of a MTOC

centrosome

centrosomes

two perpendicular centrioles (each made of nine triplet microtubules) + perientriolar material (PCM)

what is periecentriolar material (PCM)?

• a diffuse granular matrix surrounding the centrioles

• enriched with Gamma (beta) tubulin

what is PCM critical for?

microtubule nucleation

flagella: axoneme

• “9+2” array

• 9 doublet microtubules

• 2 normal single microtubules

what does bending in cilia and flagella depend on?

crosslinks in the axoneme

basal body

• MTOC for the axoneme

• structurally identical to centrosome

intermediate filaments (IFs)

• ~10-12 nm

• rope-like, not hollow

• unique to animal cells

• tough flexible, extensible, elastic filament

• no role in motility

• very stable, provides mechanical support

• can withstand tensile forces

• chemically heterogenous (at least 70 types, divided between 6 classes)

how is intermediate filament assemly different than that of microtubules and microfillaments?

• new tetramers incorporated into middle throughout the length of the filament, not ends

• neither ATP or GTP involved

• instead relies on phosphorylation

  • phosphatase - remove PO4 cause assembly

  • kinases - add PO4 cause disasembly

nuclear lamina

thin, dense meshwork of fibers that lines the inner surface of the inner surface of the inner nuclear membrane nuclear membrane and helps support the nuclear envelope

plectin

• an intermediate filament associated protein

• will form bridges with IFs, MTs, MFs

keratins

• tethered to the nuclear envelope and the outer edge of the cell

• desmosomes and hemidesmosomes

role of keratin IFs in cell attachments

• desmosomes

  • cell:cell attachment structures

• hemidesmosomes

  • cell:ECM attachment structures

desmosomes

• found in tissues subjected to mechanical stress, such as cardiac muscle, epithelial layers of the skin and uterine cervix

• the network of intermediate filaments provides tensile strength to the entire sheet of cells

hemidesmosomes

• keratin filaments extending outward into the cytoplasm

• keratin mutations can lead to cell adherence disorders

microfilaments

• smallest fibres (6-8nm)

• made of actin protein

• flexible, inextensible helical filament

• as a contractile element a microfilament can generate tension

• important for movement within cell and of the cell itself

microfilament functions

• cell shape (cortex)

• cell migration

• transport of vesicles and organelles (esp in plants)

• cytokinesis

• muscle contraction

Actin structure

• G-actin (globular actin) = monomer

  • lobes - each lobe has two domains, ATP bind in the cleft

• F-actin (filamentous actin)= polymer

  • in mature filament, two F-actins wrap around each other to form a helical structure

binding of actin molecules, what is least and most stable?

binding provides polarity to the molecule

two = weak

three = stable

myosin

• molecular motor of actin

• plus end directed motors (i.e. towards the barbed end)

• head domain → hydrolyses ATP

• conventional (type II) myosins vs unconventional myosins

• myosin II (conventional)

  • first ones discovered

  • 2 heads and long tail

  • no cargo, they twist with eachother

  • form bipolar filaments

• Myosin I or Myosin V (unconventional)

  • smaller

  • myosin I → single head

  • myosin V →two heads

  • no filament formation

  • tail binds vesicles and membrane

thick filament of skeletal muscle

• hundreds of myosin II molecules

• they twist with eachother

• form bipolar filaments

muscle contraction

relaxed = myosin heads not interacting with actin microfilaments

contracted = myosin heads have pulled the actin closer

sacromere

• muscle fiber

• contains actin (thin) and myosin (thick)

how does myosin move?

• ATP dependent process

• like kinesin and dynein, use chemical energy to do work

two types of actin organization

• both found in cell cortex

1. bundles - parallel fibers (often found in filopodia)

2. networks - can be 2D or 3D

what do actin-binding proteins do?

regulate polymerization and length of filaments

nucleating proteins

forms a nucleating center by mimicking the shape of actin subunits so G-actins will start to add

ex Arp2/3

monomer sequesting

controls amount of G-actin available for polymerization

ex thymosin beta4

end blocking (capping)

fillament grows or shrinks

prevent G-actin addition and loss

• CapZ caps “+” end (filament will shirk)

• Tropomodulin caps ‘minus’ end ‘minus’ end (filament will grow)

monomer polymerizing

increases actin filament growth rates

promotes G-actin addition to filament plus ends

ex profilin

depolymerizing proteins

binds the ‘minus’ (pointed) end and causes depolymerization

ex cofilin

cross-linking and bundling proteins

holds filament together

ex. filamin - holds filaments at right angles

ex. villin - holds filaments in parallel

filament severing protein

• breaks up MF network causing the actin gel to soften and liquefy

• caps newly-exposed plus ends to prevent further polymerization

• ex. gelsolin

membrane binding

secures microfilament to the membrane so that the membrane follows actin movement

ex. dystrophin, vinculin