S2W4 - The Cytoskeleton Flashcards
define the cytoskeleton
a highly dynamic network of proteins with many important functions
four main roles of the cytoskeleton
- structural support (AF, MT, IF) for cell shape
- internal organization of cell (MT) for organelles and vesicle transport
- cell division (AF, MT) for chromosome segregation and division of cell into 2
- large scale movements (AF) - crawling cell and muscle contraction
three components of cytoskeleton
actin filaments (d:~7nm), microtubules (d:~25nm), intermediate filaments (d:~10nm)
range of diameter of cytoskeletal filaments
7-25nm
light microscopy
- resolution limit of ~200nm
- limits from wavelength of visible light
- cannot resolve cytoskeletal filaments
fluorescence microscope
- light microscope with same resolution
- but fluorescent labels are added to detect specific proteins (eg cytoskeletal filaments)
transmission electron microscope
- uses beams of electrons of very short wavelength
- resolution limit of ~1nm
- reveals detailed structures
immunofluorescence microscopy
- used to determine location of proteins within cell
- cells are fixed (not light imagine)
- primary antibody used to bind to specific protein of interest
- secondary antibody binds to the primary antibody covalently tagged to a fluorescence marker
- fluorescence microscope used to excite fluorescent marker and visualise light emitted
draw a simplified diagram of the three types of filaments
filaments are held together by
noncovalent interactions
intermediate filaments
- involved in structural support
- different types of IF proteins
two main types of IFs
cytoplasmic and nuclear
cytoplasmic IFs
- in animal cells subjected to mechanical stress
- provide mechanical strength
nuclear IFs
- nuclear lamina - 2D meshwork formed by lamina in all animal cells
- plants have different lamin-like proteins
do plants need cytoplasmic IFs?
no; the cell wall provides most of the mechanical strength
describe the structure of cytoplasmic intermediate filaments
- Proteins:
- conserved α-helical central rod domain
- N- and C- terminal domains differ - Pack together into rope-like filaments
- 2 monomers → coiled-coil dimer
- 2 dimers → staggered antiparallel tetramer
- 8 tetramers associate side by side and
assemble into filament
- most interactions are noncovalent
- No filament polarity - because no polarity in
tetramer (ends are the same) - Tough, flexible, high tensile strength
Give an example of intermediate filaments
Keratin filaments in epithelial cells
- forms network throughout cytoplasm out to cell periphery
- anchored in each cell at cell-cell junction (desmosomes) and connect to neighbouring cells
- provide mechanical strength
define an epithelium
sheet of cells covering an external surface or lining an internal body cavity
function of microtubules
- cell organization: vesicle transport, organelle transport and positioning, centrosome in animal cells
- mitosis
- structural support for cells and motile structures (flagella, cilia)
structure of microtubules
- Long hollow tubes made of individual subunits of two closely related globular proteins, α-tubulin and β-tubulin
- form a tubulin heterodimer bound to GTP
- This regular arrangement of α & β subunits gives the microtubule polarity (plus end (β) is different from minus end (α))
- 13 parallel protofilaments make up a hollow tube
all bonds between individual subunits of microtubule profilaments are
noncovalent
the bonds between protofilaments are —- than the bonds within each protofilament
weaker
can growth and disassembly of microtubules can occur at both ends?
yes, but is more rapid at plus end
experiment to show that microtubule growth is faster at the plus end
- A bundle of microtubules isolated from a cilium
- Isolated microtubules incubated with a high concentration of tubulin (subunit) and GTP
- Faster growth of microtubules (more heterodimers being added) at the plus end
dynamic instability
plus ends of microtubules grow and shrink, which is needed for remodelling
dynamic instability: growing
- free αβ-tubulin dimers bound to GTP are added to growing microtubule at plus end
(minus end stabilized at MTOC) - Shortly after dimer added to microtubule, β-tubulin hydrolyzes GTP to GDP
- there is rapid addition of αβ-tubulin dimers which is faster than GTP hydrolysis in newly
added αβ-tubulin dimers - this leads to formation of GTP cap which stabilizes plus end
- Microtubule continues to grow
dynamic instability: shrinking
- free αβ-tubulin dimers bound to GTP are added to growing microtubule at plus end
(minus end stabilized at MTOC) - Shortly after dimer added to microtubule, β-tubulin hydrolyzes GTP to GDP
- there is slower addition of αβ-tubulin dimers which is slower than GTP hydrolysis in newly added αβ-tubulin dimers
- this leads to the GTP cap being lost, so now there is GDP-tubulin at plus end which has weaker binding
- Microtubule disassembles
function of an MTOC
have nucleating sites for microtubule growth to start assembling new microtubules
eg centrosome in animal cells
example of a nucleation site
γ-Tubulin Ring Complex (γ-TuRC):
- protein complex of γ-tubulin & accessory proteins
- ring of γ-tubulin (gold) - acts as an attachment site for αβ-tubulin dimers
- minus end of microtubule at γ-TuRC
- plus end of microtubule grows out
does the alpha tubulin or beta bind to y tubulin
alpha
example of the dynamic nature of the MTOC (non dividing animal cells in interphase)
- mos microtubules radiate from one centrosome
example of the dynamic nature of the MTOC (dividing animal cells)
- centrosome duplicates to form two spindle poles (MTOCs)
- microtubules are reorganised to form a bipolar mitotic spindle, which requires microtubule dynamics (disassembly/assembly)
4 functions of microtubule-associated proteins
- nucleate growth of new microtubules
- promote microtubule polymerisation
- promote microtubule disassembly
- stabilize microtubules (prevent disassembly) by binding to the sides and plus-end linking the protein
give an example of how microtubules can be stabilized to prevent disassembly
- how do neurotransmitters synthesized in the ER get to the axon terminals?
- ER and Golgi apparatus are located in the nerve cell body
- these neurons can be a meter long: from your spinal cord to your fingertip
cargo transport from the cell body to the axon is done by motor proteins on microtubule
motor proteins for microtubules
kinesins and dyneins
kinesins
generally move towards plus end of microtubules
eg. kinesin I: towards plus end to axon terminus, cargo of organelles, vesicles, macromolecule
dyneins
generally move towards the minus end of microtubules
eg. cytoplasmic dynein: towards minus end to cell body, cargo of worn-out mitochondria and endocytosed materia
describe the dimeric structure of kinesin-1 and cytoplasmic dynein
- heads move along microtubules, use ATP hydrolysis for movement
- tails - transport cargo
where do microtubules position organelles?
microtubules go from the centrosome (MTOC) to cell periphery
the ER is pulled from the nuclear envelope to the cell periphery by kinesin-1 (towards microtubule plus end)
Golgi is held near the centrosome by cytoplasmic dynein (towards microtubule minus end)
actin filaments are also known as
microfilaments
arre actin filaments present in all eukaryote?
yes
what are actin filaments made of?
- actin monomers
- flexible, extensible
what motor proteins use actin filaments?
myosins
functions of actin filaments
- stiff, stable structures (microvilli)
- contractile activity
- cell motility (crawling)
- cytokinesis
structure of actin filaments
- helical filament composed of a single type of globular protein - actin monomers, which are held together by noncovalent interactions
- an actin filament is made by two protofilaments twisted in a right-handed helix
is an actin filament polar? explain
- plus end is different from minus end
- actin monomers all in the same orientation in each protofilament
- growth is faster at the plus end
what are free actin monomers bound to?
ATP, which is bound in the centre of the protein
how are actin monomers added to the filament?
- actin hydrolyses ATP to ADP
- reduces strength of binding between monomers in filament
- rapid addition of actin monomers
- this is faster than the ATP hydrolysis in newly added actin monomers, causing actin filament to have an ATP cap, stabilising the structure
actin polymerisation in a test tube (in vitro)
Actin subunits (monomers) and
ATP added to a test tube to study actin filament polymerization
Nucleation (lag phase):
* small oligomers form but are
unstable
Elongation (growth phase):
* some oligomers become more
stable, leads to rapid filament
elongation (faster at plus end)
Steady state (equilibrium phase):
* decrease in [actin subunits]
* rate of subunit addition = rate of
subunit disassociation
* length doesn’t change
* Treadmilling
Process of actin filament growth
At the plus end, there is ATP-actin:
* addition of actin monomers - polymerization
* shortly after, actin hydrolyzes ATP → ADP
At the minus end, there is ADP-actin:
* loss of actin monomers - depolymerization
what happens at Treadmilling Concentration?
Actin filament remains the same size and looks “stable” but there is continual exchange of monomers at ends:
* net addition at the plus end
* net loss at the minus end
Actin monomers move through the filament
until they are eventually replaced
- continuous supply of ATP needed
cell crawling
- dynamic changes in actin filaments
- an example where actin filaments undergo treadmilling
- actin filaments must rapidly assemble at the leading edge (red) and disassemble further back to push the leading edge (and cell forward)
compare actin filaments to microtubules
what are the different functions of actin filaments regulated by?
actin binding proteins
6 examples of regulation by actin binding proteins
- sequester actin monomers (prevent polymerization)
- promote nucleation to form filaments
- stabilize actin filaments (capping)
- organize: bundle, cross-link filaments
- sever actin filaments
what do myosins generally do?
move towards plus end of actin filaments. their heads move along actin filaments, use ATP hydrolysis for movement
two types of myosin proteins
myosin I
myosin II
myosin I
tail domain: binds cargo
* e.g. (B) vesicles (regulated secretion)
* e.g. (C) plasma membrane (shape)
myosin II
dimer
* tails: organized in a coiled-coil
* dimers assemble into myosin-II filaments through their coiled-coil tails
* e.g. bipolar myosin-II filament, which slide actin filaments in opposite directions
(plus end of both actin filaments) and generates a contractile force
