Lecture 20: Cytoskeleton Flashcards
cytoskeleton
is a series of long, filamentous protein fibres that are formed within the cell.
Intermediate filaments
tough, ropelike fibers
- made of a variety of
related proteins
Microtubules
- hollow, rigid
cylindrical tubes - made from tubulin
subunits
Microfilaments
solid, thinner structures
- made of actin
Describe the various functions of the cytoskeleton
provides structural support
intracellular transport of organelles and vesicles
as a force generating apparatus
provides an internal framework
essential component of the apparatus that
divides chromosomes during mitosis and meiosis
Describe how the cytoskeleton provides structural support
The cytoskeleton provides structural support that can determine the shape of the cell and resist forces that can deform the cell
• A cell’s shape is closely associated with its function • Cytoskeleton can resist mechanical stresses • Cytoskeleton can rearrange itself to change the shape of cells
All the cell-cell connections are connected to the inside of the cell via the cytoskeleton, providing support in cells and tissues
Describe how the cytoskeleton functions in
intracellular transport
The intracellular transport of organelles and vesicles occurs via the cytoskeleton
• Machinery needed to move materials &
organelles within cell; network of highways
directs movement
• Movement of mRNA molecules to specific
parts of cell
• Movement of vesicles from ER to Golgi
complex
• Movement of neurotransmitter-containing
vesicles from synthesis site to axon terminal
• Transport of peroxisomes over rails of
microtubules; the two are closely associated
(microtubules in red, peroxisomes in green)
Some tracts are really long… some neurons
extend from the spinal cord to the end of your
fingers! Vesicles are carried along microtubules
in the axon. It can take weeks for transport!
Describe how the cytoskeleton functions as a a force generating apparatus
Cytoskeletal elements can function as a force generating apparatus
that moves cells from one place to another (if they’re mobile!)
Single-celled organisms move by crawling
over surface or propelled by protruding
cilia & flagella
Multicellular organisms have variety of
cells capable of independent locomotion - sperm, white blood cells, fibroblasts,
highly motile tip of growing axon
(movement like crawling white blood cell)
Describe how the cytoskeleton provides an internal framework
The cytoskeleton provides an internal framework for positioning the
various organelles within the interior of the cell.
Intracellular organization is
disrupted by drugs or mutations
that interfere with normal
cytoskeleton structures
The location of cellular components isn’t
random… they’re all anchored to and
moved around the cytoplasm by the
cytoskeleton
Describe what role the cytoskeleton plays in mitosis/ meiosis
is an essential component of the apparatus that
divides chromosomes during mitosis and meiosis
the spindle apparatus made of
microtubules
Describe microtubule structure
a. Explain why microtubules have polarity
• Basic unit is a tubulin dimer (microtubule subunit) composed of
alpha (α) and beta (β) tubulin subunits (heterodimer)
polymerize to form a cylinder… the dimers are
not covalently attached to each other
microtubules are therefore
asymmetric & polarized (β end is plus; α end is
minus)
The plus ends have higher rates of tubulin
addition (thus grow faster); the minus ends
are slower growing
What is a protofilament?
• One ‘strand’ of the tube is called a
protofilament
all protofilaments in single MT have same
polarity - microtubules are therefore
asymmetric & polarized (β end is plus; α end is
minus)
β -tubulin can have a _________or
_____ bound
β -tubulin can have a swappable GDP or
GTP bound
α-tubulin ____
has____ bound
a- tublin permanenlty has a GTP bound
Describe how microtubules assemble
• initial polymerization is slow – requires
nucleation (coming together to build starting point) of 13 dimers, with the help of
another tubulin: γ (gamma) tubulin and
other proteins
• γ tubulin dissociates from microtubule
after polymerization has progressed
- subsequent subunit addition is faster (as dimers keep getting added affinity increases
- depolymerization also occurs spontaneously, but at a slower rate.
microtubules are in constant flux,
polymerizing and depolymerizing
• one end of the MT is slower growing (-) while the other is faster (+) giving each microtubule a polarity
What determines rate of assembly and disassembly?
Microtubules may vary in their rate of assembly and disassembly
Tubulin half life is nearly a full day, however, the half life of a given microtubule may be only 10 minutes.
- rate of assembly is dependent on how many tubulin dimers are floating around freely in the cytoplasm • relative rates of assembly and disassembly determine their growth – dependent on local tubulin concentration
γ (gamma) tubulin
dissociates from microtubule
after polymerization has progressed
helps with initial polymerization
What controls microtubule instability?
microtubule’s instability is controlled by hydrolysis of GTP bound by tubulin.
- tubulin dimers bind GTP before binding microtubule
- the microtubule dimer hydrolyzes the GTP to GDP.
- this reduces the affinity of the tubulin for its neighbor, thus leading to disassembly. (destabilize)
• To retain some stability, the GDP- dimers can be regularly replaced
with more GTP-dimers
What other thing also affects disassembly rates?
Proteins called +TIPs can also affect disassembly rates.
List Factors that affecting microtubule stability
- Rate of growth: more growth stabilizes the microtubule because added tubulin dimers have GTP bound (forming a
GTP cap)
• The rate of growth is largely dependent on the concentration of tubulin
dimers available in the vicinity of the growing end - Rate of GDP to GTP exchange: as the dimers hydrolyze GTP further down the MT (thus destabilizing the microtubule), new GTP can be enzymatically swapped in to stabilize the microtubule
- Association with microtubule associated proteins,
some of which help stabilize the microtubule
Describe how chemotherapy drugs target microtubules
• cancer cells, with their high rate of
division, require faster cycling of tubulin
– drugs can prevent that
• Drugs that interfere with MT dynamics
will interfere with the spindle apparatus
that divides chromosomes in mitosis
• Normal cells have a ‘mitotic checkpoint’
that will stop mitosis if something is
wrong. Cancer cells often lack these
checkpoints… thus will try to divide even
with a ‘poisoned’ spindle. This causes
uneven distribution of chromosomes and
will often kill the cancer cell.
List drugs which affect assmbly/disassembly
- colchicine, nocodazole make microtubules
disassemble - Taxol prevents disassembly
- Vinblastine prevents assembly
MTOCs; Microtubule organizing centers
initiate microtubule polymerization
The best understood is are centrosomes and basal bodies.
These are actually interchangeable… the basal body from a sperm becomes the
centrosome of the newly fertilized egg!
Describe centrosome structure
is comprised of two centrioles surrounded by a cloud of molecules
known as the pericentriolar material (or centrosome matrix)
• In centrosome, centrioles usually in pairs, perpendicular to each other
Centrosomes
are usually close to
the nucleus, and microtubules
extend from them.
Basal bodies
assemble the microtubules that make up cilia and
flagella.
What is an example of MTOC in animal cells?
centrosome
• Centrioles
are cylindrical structures 0.2 um in diameter and about 0.4 um long
are not actually involved in microtubule nucleation, but “recruit” the
molecules that do (gamma tubulin and other proteins)
Do plants have centrosomes?
plants lack centrosomes – their MTOCs are typically embedded in the nuclear membrane
. List the functions of microtubule associated proteins
• stabilize microtubules
• alter assembly/disassembly
rates
• crosslink adjacent MTs
MAP activity controlled by addition & removal of phosphate groups on particular amino acid residues by protein kinases & phosphatases, respectively
How may maps be associated with Alzheimer’s disease?
• Abnormally high phosphorylation of a
MAP is implicated in fatal
neurodegenerative diseases like AD
• hyperphosphorylated tau protein sticks
together into neurofibrillary tangles in
neurons; causes microtubules to
disintegrate;
• short microtubules prevent normal
intracellular transport and therefore kill
nerve cells, resulting in brain
deterioration
This happens in addition to the amyloid plaques that are misfolded. So lots can get messed up in neurons in Alzheimer’s patients.
What are some possible AD therapies?
specific kinase
inhibitors
Motor proteins
convert chemical energy stored in ATP into mechanical energy that is used to generate force or to move cellular cargo attached to the motor
B. A single cell may contain dozens of different motor proteins, each specialized
for moving a particular type of cargo in a particular cell region
- Describe the three families of motor proteins
- Kinesins move toward (+) end of
microtubules - Dyneins move toward (-) end of MTs
They can attach to different adapter proteins
that then attach to proteins imbedded in
vesicle membranes (Rabs), the cell membrane,
or adjacent microtubules to generate
movement
Microfilament motor proteins:
3. Myosins
(There are no known motor proteins that
use intermediate filaments as tracts)
Types of cellular cargo transported by these molecular motors include:
ribonucleoprotein particles, vesicles, organelles (mitochondria, lysosomes,
chloroplasts), chromosomes & other cytoskeletal filaments
i
Motion is a bit like rope-climbing or swimming: alternate between power strokes and
recovery strokes:
Note that the ‘cargo’ can have different
motor proteins attached to them at once
Kinesin
move cargo to the + end of a microtubule
are tetramer proteins constructed of 2 identical heavy chains & 2 identical light chains
They`re the smallest of motor proteins (but still big!)
• There are approx 45 different kinesins in humans, each binds a
different subset of cargo
Describe Kinesin structure
A pair of globular heads that bind a MT & generate force by hydrolyzing ATP (thus called the motor domain)
Each head is connected to a neck
The rest of the protein is a rod-like
stalk & a fan-shaped tail that binds
cargo to be hauled
How does Kinesin move?
• Kinesin movements mediated by ATP hydrolysis : 1 ATP per
“step”
• Moves along single MT protofilament (rate proportional to
[ATP]; up to 800 nm/sec)
• Move in distinct steps (1 dimer at time; 8 nm); usually toward plasma membrane (e.g from Golgi to plasma membrane)
attaches to one of the vesicle’s integral proteins (often via Rab proteins; not
shown in this figure).
Movement is prossessive because at least one head is attached to the microtubule at all times. It thus can walk and carry cargo micrometers down a MT
Describe how kinesin hydrolyse ATP to walk along MTs
before attaching to a MT, both heads have ADP bound)
- The front head binds to a microtubule and
releases it’s ADP. - ATP binds to the front motor domain and
causes a conformational change (the neck
region associates with the head region). This
causes the back head to swing forward and
bind the MT. - ADP is released from the (now) forward head.
The (now) back head hydrolyses the ATP to ADP.
The energy released dissociates the head from
the microtubule.
How does kinesin function in neuronal transport?
• Kinesin I was first isolated in 1985
from squid giant axons
• It is responsible for axonal flow, in which vesicles (e.g., precursors of synaptic vesicles formed in the Golgi) are carried along microtubules from the cell body to axon endings.
• Axon endings have an extensive actin cytoskeleton. Myosin V may take over to transport vesicles along actin filaments to near the plasma membrane at the synapse.
Cytoplasmic dyneins
carry cargo to – MT ends
ubiquitous eukaryotic motor protein, only 2 versions in humans
- huge protein (1500 kDa)
- two identical heavy chains: head generates force
• variety of intermediate & light chains (each with different cargo
recognition domains)
• cargo attached by dynactin
Describe the various dynein functions
- chromosome movement in mitosis
- positioning Golgi complex
• movement of vesicles & organelles
through cytoplasm
• involved in axonal retrograde organelle
movement
• in fibroblasts & endothelial cells move
endosomes, lysosomes, Golgi-derived vesicles toward cell center
• also ‘carries’ HIV to the nucleus
Endothelium cells
line the interior surface of blood vessels and lymphatic vessels
Fibroblast
a cell in connective tissue that produces collagen and other fibers
Does GTP or GDP stabilize the MT?
GTP stabilizes GDP detsabilizes
Compare a growing Mt vs a shrinking MT
Growing;
GTP - tubulin dimers add to growing end of MT
addition proceeds faster than GTP hydrolysis by dimer
Shrinking
- GTP hydrolysis is faster than addition of new GTP bound tubulin dimers (GTP cap is lost)
- portofilaments containing GDP peel away
- GDP- tubulin gets released into cytosol
