2.4: The Cytoskeleton Flashcards
list the 4 major functions of the cytoskeleton
- structural support: cell shape
- internal organization of cell: organelles, vesicle transport
- cell division: chromosome segregation, divide cell into two
- large scale movements: crawling cell, muscle contraction
list the 3 protein filaments that make up the network of the cytoskeleton
- actin
- microtubules
- intermediate filaments
which of the protein filaments present in the cytoskeleton contribute to the structural support
af, mt, if
which of the protein filaments present in the cytoskeleton contribute to the internal organization of the cell
mt
which of the protein filaments present in the cytoskeleton contribute to cell division
af, mt
which of the protein filaments present in the cytoskeleton contribute to large scale movements
af
compare and contrast the microscopy techniques to look at the cytoskeleton (light microscope, fluorescence microscope, transmission electron microscope)
light microscope
- resolution limit of ~200 nm
- limits from wv of visible light
- cannot resolve cytoskeletal filaments
fluorescence microscope
- reso limit of ~200 nm
- fluorescent labels are added to detect specific proteins eg immunofluorescence
- also makes it seem larger than it is
transmission electron microscope (best, better size accuracy)
- uses beams of e, very short wv
- reso limit of ~1 nm
- reveals detailed structures
state the diameter range of the cytoskeletal filaments
7nm to 25nm
describe the use of immunofluorescence microscopy in in cytoskeletal imaging
- used to determine location of proteins within cell
- cells are fixed (not live imaging) eg by using formaldehyde
- primary antibody used to bind to specific protein of interest
- secondary antibody binds to the primary antibody – covalently tagged to a fluorescent marker
- fluorescence microscope used to excite fluorescent marker and visualize light emitted
- Less amplification effect if you add fluorescence to the primary antibody compared to the second and also it’s more expensive (secondary is cheaper to buy)
order the filaments from smallest to largest
actin < intermediate < microtubules
through what interactions are cytoskeletal filaments held together by
filaments are held together by noncovalent interactions
what are actin filaments, intermediate filaments, and microtubules composed of
actin filaments - actin
intermediate filaments - intermediate filament proteins
microtubules - tubulin
name the two types of IF proteins and their purposes
- cytoplasmic IF: in animal cells subjected to mechanical stress (eg keratin filaments in epithelial cells of the skin), provide mechanical strength (overall examples include presence in connective-tissue cells, muscle cells, glial cells, nerve cells)
- nuclear IF: nuclear lamina (2d meshwork) formed by lamins in all animal cells that have a nucleus (plants have diff ones)
cytoplasmic IF proteins have a conserved _________________ central rod domain and they pack together into rope like filaments
a helical
t/f the N- and C- terminal domains differ in cytoplasmic IF proteins
true
t/f do cytoplasmic intermediate filaments have polarity, and why
no polarity bc no polarity in the tetramers bc the ends are the same
in cytoplasmic intermediate filaments:
2 monomers –> __________________
2 dimers –> _________________
__ (#) tetramers associate side by side and assemble into a _____________
2 monomers –> coiled coil dimer
2 dimers –> staggered antiparallel tetramer
8 tetramers associate side by side and assemble into a filament
in cytoplasmic intermediate filaments:
a) _______ region of monomer
b) _________ _____ dimer
c) ___________ __________ tetramer of b)
a helical, coiled coil, staggered antiparallel tetramer
describe the cytoplasmic intermediate filaments (what adjectives help it not rupture)
tough, flexible, high tensile strength
describe the role of intermediate filaments in an epithelial cell
- keratin filaments in epithelial cells
- form network throughout cytoplasm out to cell periphery
- anchored in each cell at: cell-cell junctions (desmosomes), connect to neighbouring cells
- provides mechanical strength
- epithelium is the sheet of cells covering an external surface or lining an internal body cavity
what are microtubules involved in
- cell organization: vesicle transport, organelle transport and positioning, centrosomes (animal cells)
- mitosis
- structural support: cells, motile structures (flagella, cilia)
name and describe the properties of tubulin
- long, stiff hollow tubes (like cylinders)
- inextensible (= not elastic)
- made of individual subunits of a (-) and b (+) tubulin (closely related globular proteins) = tubulin heterodimer
NOTE THAT THE + - IS NOT BC OF CHARGE - tubulin heterodimer is bound to GTP
- arrangement of a and b subunits = polarity
- 13 protofilaments (a line of heterodimers) = hollow tube
arrange the following into order of organization from smallest to largest (and how many of each if that is known)
protofilament, tubulin dimer, microtubule, tubulin
tubulin, tubulin dimer, protofilament, microtubule
the noncovalent bonds _________ protofilaments are weaker than the bonds ________ each protofilament (options: between, within)
between, within
t/f can growth and disassembly of microtubules occur at both ends
yes
which end of the microtubule is growth more rapid at
plus end
what happens after the protofilament has been there for awhile
After it’s been in a protofilament for a while, beta tubulin will cut GTP to GDP (a phosphate leaves) - and will change it from a t form heterodimer to a d form heterodimer - if we have t form dimers, we are more likely to have microtubule growth and then opposite for d form (shrinking) — any growing or shrinking can only occur at the ends
__ microtubule = __ parallel proton filaments forming the hollow tube. – some cells have bundles of microtubules (bundles of cylinders)
1, 13
in the cell, microtubules grow out from ___________
mtocs (microtubule, organizing centers)
which end of the microtubule is stabilized at mtoc
minus end (alpha end)
describe the growth process of dynamic instability in microtubules
- free ab tubulin dimers bound to GTP are added to growing microtubule at + (beta end)
- shortly after dimer is added to microtubule, b tubulin hydrolyzes gtp to gdp
- rapid addition of ab tubulin dimers: faster than gtp hydrolysis in newly added ab tubulin dimers - leads to formation of gtp cap, stabilizes +
- microtubule continues to grow
what process is the dynamic instability of microtubules necessary for
remodelling
how do the gtp cap on microtubules facilitate growth
Facilitates growth - it’s essentially the t form heterodimers (GTP bound) at the end
which subunit of tubulin hydrolyze gtp into gdp
beta tubulin
how can you identify the new tubulin dimers from old ones
gdp is old, gtp is new
describe the shrinking process in the dynamic instability of microtubules
- free ab tubulin dimers bound to GTP are added to growing microtubule at + (beta end)
- shortly after dimer is added to microtubule, b tubulin hydrolyzes gtp to gdp
- slower addition of ab-tubulin dimers - slower than gtp hydrolysis in newly added ab-tubulin dimers = loss of gtp cap, now gdp tubulin at + end has weaker binding = microtubule disassembles
describe the full dynamic instability mechanism of microtubules
- ab tubulin dimers: addition or loss at +
- stabilized at mtoc
- free ab tubulin dimers (gtp) added to growing microtubule
- b tubulin gtp hydrolyzed to gdp = gdp tubulin dimer
- tightly bound gtp to a tubulin is not hydrolyzed
- gtp cap: straight filaments = stronger bonding, favors growth
- gtp hydrolysis (gdp tubulin dimer): small conformational change = weaker binding, curved filaments = disassembly
t/f tightly bound gtp to a tubulin is not hydrolyzed
true
____ ____________ changes subunit conformation and weakens bond in the protofilaments
gtp hydrolysis
what is the function of mtoc
have nucleating sites for microtubule growth - to start assembling new microtubules
give an example of mtoc in animal cells
centrosome
provide an example of a nucleation site (mtoc) and describe
- y tubulin ring complex: protein complex of y tubulin & accessory proteins
- ring of y tubulin (end) acts as an attachment site for ab tubulin dimers
- end of microtubule at y tubulin ring complex
- end of microtubule grows out
differentiate between the use of dynamic instability needed for remodelling in nondividing and dividing animal cells
non dividing (interphase): most microtubules radiate from one centrosome (mtoc)
dividing: centrosome duplicates to form 2 spindle poles (mtocs), microtubules are reorganized to form a bipolar mitotic spindle - required microtubule dynamics
in order for the cell to make the bipolar mitotic spindles, what does it have to do
The cell has to disassemble the first array to make the bipolar mitotic spindles
microtubule associated proteins are able to: (at least 4)
- nucleate growth of new microtubules
- promote microtubule polymerization
- promote microtubule disassembly
- stabilize microtubules (prevent disassembly): protein bind to sides, plus end linking protein
- branches
give an example of how microtubules can be stabilized to prevent disassembly
cargo transport from the cell body to the axon terminal: how do nt synthesized in the er get to the axon terminals? through motor proteins on microtubules - er and golgi are located in the nerve cell body, these neurons can be a meter long (eg from spine to fingers)
kinesin-1, cytoplasmic dynein are?
dimers
what are the roles of the heads and tails of kinesin-1 and cytoplasmic dynein
heads move along microtubules, use atp hydrolysis for movement
tails transport cargo
which directions do kinesin-1 and cytoplasmic dynein generally move in
kinesin-1 goes to + end (axon terminal)
cytoplasmic dynein goes to - end ((nerve) cell body)
what is included in the cargo of kinesin-1 and cytoplasmic dynein
kinesin-1: cargo of organelles, vesicles, macromolecules
cytoplasmic dynein: worn-out mitochondria and endocytosed material
gamma tubular ring complexes are located in the
centrosome
describe the positioning of organelles by microtubules for er and golgi (+ what dimers are they composed of): eg for microtubules it foes from centrosome (mtoc) to cell periphery
er from nuclear envelope to cell periphery (by kinesin-1 to microtubule + (b)
golgi near centrosome by cytoplasmic dynein (towards microtubule - (a))
t/f are actin filaments present in all eukaryotes
true
actin filaments (aka microfilaments) are made of _________ <– describe it
actin monomers <– flexible, inextensible (can’t stretch)
what are the motor proteins called (actin)
myosins
list the 4 functions of actin filaments
- stiff, stable structures (microvilli)
- contractile activity
- cell motility (crawling)
- cytokinesis (contractile ring)
describe the structure of actin filaments
- helical filament
- composed of a single type of globular protein: actin monomers - with non covalent interactions
0 two protofilaments twisted in a right handed helix
are actin filaments left or right handed helix
right handed helix
do actin filaments have polarity
yes
in which end of actin filaments is growth faster
the + end
actin monomers are all in _________ orientation in each protofilament
the same
describe the growth of actin monomers
- free monomers are bound to atp - bound in the center of the protein
- shortly after actin monomer added to filament: actin hydrolyzes atp to adp = reduces strength of binding between monomers in filament = more likely to dissociate if in adp form in one of the ends
- rapid addition of actin monomers: faster than atp hydrolysis in newly added actin monomers
- actin filaments have an atp cap that promotes growth
to the following properties, identify whether intermediate filaments, microtubules, and/or actin filaments are able to conduct it:
- nucleotide bonding
- overall polarity
- motor proteins
- dynamic properties
- extensibility
- flexibility
- nucleotide bonding: IF no, microtubules have gtp, actin has atp
- overall polarity: IF no, others yes
- motor proteins: IF no, others yes both atp
- dynamic properties: IF is more stable, microtubules have dynamic instability, actin filaments conduct treadmilling
- extensibility: IF yes, others no
- flexibility: IF and actin flexible, microtubules stiff
myosin I and II (actin motor proteins) are + or - end directed
+
cytoplasmic dynein and kinesin-1 (microtubule motor proteins) are + or - end directed
cytoplasmic dynein -
kinesin-1 +
atp hydrolysis is a ___________ change to generate ______
atp hydrolysis is a conformational change to generate force
myosins generally move towards + or - end of actin filaments
+
do myosin heads or tails move along actin filaments, and use atp hydrolysis for movement
heads
differentiate between myosin I and II in terms of organization, tail domain, and role
myosin I: tail domain binds to cargo, used in vesicles (regulated secretion) and plasma membrane (shape)
myosin II: dimer assembles into myosin II filaments (through –>), tails organized in a coiled coil, eg is bipolar myosin II filament: slide actin filaments in opposite directions (+ end of both actin filaments) = generates contractile force
^^Looks like a double headed arrow – heads on the left and right are bound to different actin filaments and move towards each other (blue arrows) – the bipolar myosin moves them tgt and makes the contractile force
bipolar myosin II filament: slide actin filaments in opposite directions (+ or - end of both actin filaments) = generates contractile force
+
name some of the functions that are regulated by actin binding proteins
- sequester actin monomers (prevent polymerization)
- promote nucleation to form filaments
- stabilize actin filaments (capping)
- organize: bundle, cross-link filaments
- severe actin filaments
what is the name of the region for actin filaments and microtubules where there is disassembly
for actin: adp actin
microtubules: gdp ab tubulin dimer
in order to push the leading edge/the cell forward, what must actin filaments rapidly do
rapidly assemble at the leading edge and disassemble further back
explain cell crawling
- dynamic changes in actin filaments
- an example where actin filaments undergo treadmilling
describe actin polymerization in a test tube (in vitro)
hint: there was a graph on the page if that helps job your memory
- actin subunits (monomers) and atp added to a test tube (and some salt to solution to get process going)
nucleation (lag phase): small oligomers form but unstable
elongation (growth phase): some oligomers become more stable = leads to rapid filament elongation (faster at + end)
steady state (equilibrium phase): decrease in actin subunits, rate of subunit addition = rate of subunit disassociation = treadmilling (there is always a % of free subunits in a steady state)
why is growth of actin filaments slow at the beginning
Slow at the beginning bc needs to spontaneously make oligomers (not stable), once gets stable enough it’ll start to elongate
what is the term for the process: loss of actin monomers at the - end (adp actin)
depolymerization
