Ch.17 - Cytoskeleton Flashcards
17A - Intermediate filaments. 17B - Microtubules. 17C - Actin Filaments 17D - Muscle Contraction
Summarize the general structure and function of intermediate filaments (ifils).
Ifils are polymers of proteins—wh vary according to cell type—that combine to form strong, cable-like fils that provide animal cells w mech strength.
- 100+ diff types, e.g. keratin (epithelial cells), vimetins (fibroblasts), and neurofils (neurons)
- Greatest tensile strength/flexibility (comp to mtubs/mfils) → bend instead of rupture.
- Typ anchored to cyto-side of pmem via desmosomes → interconn adj cells → form meshwork of struc support (e.g. nuclear lamina).
- Durable - if cells are treated w salt solutions and nonionic detergents, most of cytoskeleton is destroyed, but ifils survive.
Ifil proteins are fibrous subunits, ea w an elongated central rod domain w distinct unstructured terminal domains at either end. Describe how these features influence their function.
Central rod domains of diff ifils are all similar in size/AA seq → enable close assoc/coiled-coil; by contrast, terminal domains (unstructured) of diff ifils vary greatly in size/AA seq → interact w specific components of cytoplasm.
Ifil proteins are fibrous subunits, ea w an elongated central rod domain w distinct unstructured terminal domains at either end. The central rod domain consists of an extended _______ region that enables pairs of ifil proteins to form stable ______ by wrapping around ea/o into _________ config. Two of these structures run in opp directions and assoc to form a staggered, antiparallel _______ → assoc side-by-side into helical array of __ (#) strands → final rope-like ifil
Ifil proteins are fibrous subunits, ea w an elongated central rod domain w distinct unstructured terminal domains at either end. The central rod domain consists of an extended α-helical region that enables pairs of ifil proteins to form stable dimers by wrapping around ea /o into coiled-coil config. Two of dimers run in opp directions and assoc to form a staggered, antiparallel tetramer → assoc side-by-side into helical array of 8 strands → final rope-like ifil
T/F: due to opposed dimer orientation of ifil proteins, tetramer ends are the same.
True
Tetramer ends are the same (structurally nonpolar) - due to opposed dimer orientation; distinguishing feature comp to mtubs/fils.
Ifil-ifil conns dep solely on noncovalent bonding. How can such weak bonds evoke such high tensile strength characteristic of ifils?
Ifil-ifil conns dep solely on noncovalent bonding → tensile strength fr combo of overlapping lateral interactions along length of ifil.
Central rod domains of diff ifils are all similar in size/AA seq, while terminal domains (unstructured) of diff ifils vary greatly in size/AA seq. How does this impact their role in the cell?
Central rod domains of diff ifils are all similar in size/AA seq → enable close assoc/coiled-coil; by contrast, terminal domains (unstructured) of diff ifils vary greatly in size/AA seq → interact w specific components of cytoplasm.
What are the four classes of ifils? Where are they located, and how are they assembled?
Four classes of ifils; all cytoplasmic except nuclear lamins (nucleus), and all assembled via polymerization of subunits:
- Keratin fils in epithelial cells; most diverse class.
- Vimentin and vimentin-related fils in CT, muscle, and supporting cells of NS (e.g. glial cells).
- Neurofils in nerve cells.
- Nuclear lamins of the nuclear envelope.
Note that ea class incl many subtypes, e.g. humans have 50+ keratin genes.
Many ifils are further stabilized and reinforced by accessory proteins. Describe one such protein.
Plectin - cross-link ifils into bundles → link to mtubs/fils and desmosomes.
- Plectin mutations → epidermolysis bullosa simplex, muscular dystrophy, neurodegen.
- Plectin may not be reqd for initial formation of ifils, but its cross-linking action is reqd to provide cells w necessary strength to handle mech stress.
Which of the following types of cells would you expect to contain a high density of ifils in their cytoplasm? Explain.
a. Amoeba proteus (a free-living amoeba)
b. skin epithelial cell
c. smooth muscle cell in digestive tract
d. Escherichia coli
e. nerve cell in spinal cord
f. sperm cell
G. Plant cell
Cells that migrate rapidly from one place to another, such as amoebae (A) and sperm cells (F), do not in general need intermediate filaments in their cytoplasm, since they do not develop or sustain large tensile forces.
Plant cells (G) are pushed and pulled by the forces of wind and water, but they resist these forces by means of their rigid cell walls rather than by their cytoskeleton.
Epithelial cells (B), smooth muscle cells (C), and the long axons of nerve cells (E) are all rich in cytoplasmic intermediate filaments, which prevent them from rupturing as they are stretched and compressed by the movements of their surrounding tissues.
All of the above eukaryotic cells possess at least intermediate filaments in their nuclear lamina. Bacteria, such as Escherichia coli (D), have none whatsoever
The nuclear envelope is supported by a meshwork of ifils called the ___________, wh is composed of _______.
The nuclear envelope is supported by a meshwork of ifils called the nuclear lamina, wh is composed of lamins.
- Lamins - form 2-D meshwork instead of rope-like strucs like cytoplasmic ifils → comprise nuclear lamina, wh dis/assembles at ea cell division; also provides attachment sites for chromos.
- Caution: don’t confuse w laminin - an ECM protein.
Dis/assembly of nuclear lamina is controlled by de/phos of lamins. Explain.
Lamins are phos by protein kinases → conform change weakens binding b/w lamin tetramers → lamins dissoc.
Lamins dephos by protein phosphatases at end of mitosis → tetramer binding becomes energ favorable → lamins reassoc.
Microtubules (mtubs) are long, straight, hollow cylinders of tubulin protein; typ w one end attached to single mtoc/centrosome. Describe several other features of their structure and function.
- More rigid than mfils/ifils; rupture when stretched.
- Dynamic instability - rapidly dis/assemble.
- Organizing euk cells - radiate out fr mtoc to cell periphery → created system of tracks along wh vesicle/organelles/other components can be xprtd.
- Mitosis - mtubs dis/reassemble into mitotic spindle → helps segregate chromos equally.
-
Form stable core of cilia/flagella - rhythmically beating, hair-like structures; extend fr surface of many euks → swim/sweep fluid over their surface.
- Bac flagella have entirely diff struc → swim by v diff mechanism.
Mtubs are hollow, cylindrical tubes composed of ___________ subunits, ea composed of ____ (2/3/4) v similar globular proteins bound tightly by ___________ (non/covalent) interactions.
Mtubs are hollow, cylindrical tubes composed of tubulin dimer subunits, ea composed of two v similar globular proteins (α- and β-tubulin) bound tightly by noncovalent interactions.
__________ are a linear, alternating chain of tubulin dimers, ___ (#) of wh form a mtub.
Protofilaments are a linear, alternating chain of tubulin dimers (heterodimer/αβ-dimer = .mtub subunit); 13 parallel protofils form mtub
T/F: Ea protofil—and mtub as a whole—has struc polarity
True
Ea protofil—and mtub as a whole—has struc polarity, w α-tubulin exposed at one end (“minus” end), β-tubulin at other (“plus” end).
- Think: Beta = Bigger = plus end.
- Protofils align w same orientation → cross-section of mtub would reveal ring of all α- OR all β-tubulin.
- Caution: struc polarity and +/- ends have nothing to do w elec charge; rather, refers to chem/functionally distinct ends.
- Struc polarity is critical for IC xprt.
In concentrated solution of pure tubulin (in vitro), protofils/mtubs polymerize at both ends, but more rapidly at which end?
Polymerization - in concentrated solution of pure tubulin (in vitro), protofils/mtubs polymerize at both ends, but more rapidly at β/plus end.
The ________ is the major mtub-organizing center in animal cells
The centrosome is the major mtub -organizing center in animal cells.
MTOC (centrosome in animal cells) - specialized organizing centers that control location, #, and orientation of mtubs; typ close to nucleus (except during mitosis).
A centrosome consists of pair of ________ surrounded by a matrix of proteins that incl hundreds of ring-shaped strucs of ________ → ea ring complex serves as a starting point (“____________”) for growth of one mtub. _________ dimers add to ea ring complex in specific orientation → mtub’s _____ (+/-) end is embedded in centrosome and ____ (+/-) end extends into cytoplasm.
A centrosome consists of pair of centrioles surrounded by a matrix of proteins that incl hundreds of ring-shaped strucs of γ-tubulin → ea γ-tubulin ring complex serves as starting point (“nucleation site”) for growth of one mtub. αβ-tubulin dimers add to ea γ-tubulin ring complex in specific orientation → mtub’s minus end embedded in centrosome and plus end extends into cytoplasm.
T/F: centrioles have no role in nucleation of mtubs fr centrosome.
True
Centrioles are strange - ea centriole—sitting perpendicular to its partner—is a cylindrical array of short mtubs, yet centrioles have no role in nucleation of mtubs fr centrosome (γ-tubulin ring complex alone is sufficient).
- Besides serving as organizing centers (basal bodies) for mtubs in cilia/flagella, key function still a mystery.
- Note: plant lack centrioles.
Why do mtubs need nucleating sites such as those provided by γ-tubulin rings in centrosome?
Much harder to start new mtub fr scratch (i.e. new ring of αβ-tubulin dimers) than to add dimers to pre-existing γ-tubulin ring.
Despite spont assembly in vitro (w high concen of free tubulin), free/cytoplasmic αβ-tubulin concen in vivo is too low.
- By contrast, free actin monomers are at high enough concen in vivo to spont assemble.
Once a mtub has been nucleated, typ grows outward fr _________ for many minutes by polymerization of ________ to _____ end.
Once a mtub has been nucleated, typ grows outward fr MTOC/centrosome for many minutes by polymerization of αβ-dimers to β/plus end.
Dynamic instability of mtubs has been likened to a fisherman casting a line. Explain.
Dynamic instability occurs w/o warning; mtub can suddenly shrink rapidly inward (depoly) of β/plus end → suddenly start growing again, or may disappear completely, to be replaced by new mtub that grows fr same γ-tubulin ring complex/nucleation site.
- Centrosome/mtoc continually shoots out new mtubs in diff directions in exploratory fashion → many retract, but can be stabilized by attachment to another molecule (capping protein) or cell struc.
- Like a fisherman casting a line - if no bites (stabilized conns) → reels line (mtub) back in → casts in diff direction (fr same nucleation site).
- Used to position organelles relative to one/an.
Describe how selective stabilization can polarize a cell.
Selective stabilization of mtubs can polarize a cell.
Newly formed mtub will persist only if both ends are protected fr depoly: α/minus end typ anchored in mtoc, but β/plus end initially free → must be stabilized by binding specific proteins, e.g. “capping” proteins in cell cortex.
Dynamic instability is driven by ____ hydrolysis
Dynamic instability is driven by GTP hydrolysis.
GTP is tightly bound to tubulin dimers: GTP hydrolysis ↓ affinity of dimers for their neighbors → promote mtub disassembly.
Ea free αβ-dimer contains one GTP tightly bound to β-tubulin → dimer added to growing mtub → quickly hydrolyzes GTP, and GDP remains tightly bound to β-tubulin.
How does this intrinsic capacity of tubulin dimers to hydrolyze GTP contrib to dynamic stability and formation of GTP or GDP ‘caps’?
GTP cap - β/plus end composed entirely of GTP-dimers → favors growth; occurs when poly proceeds faster than GTP hydrolysis.
- GTP-assoc dimers bind more strongly/eff to ea/o than GDP-dimers → mtub continues to poly/grow.
GDP cap - β/plus end composed of GDP-dimers → favors shrinkage; occurs when dimers ‘randomly’ hydrolyze their GTP before next dimers added, e.g. when growth is too slow.
- Recall: rest of mtub is composed of GDP-tubulin → depoly/shrinkage tends to persist once started → may disappear completely → new mtub started fr same nucleation site.
- GDP-tubulin freed during depoly join pool of free dimers in cytosol → exchange GDP for GTP → ready for poly.
Why do you suppose it is much easier to add tubulin to existing mtubs than to start a new mtub from scratch? Explain how γ-tubulin in centrosome helps to overcome this hurdle.
Two αβ-dimers have a limited # of possible interaction sites → lower affinity for ea/o → unlikely that sequential dimers will assoc for long enough to spont form new mtub.
By contrast, αβ-dimers have many possible interaction sites w end of a mtub: end-to-end or side-to-side w protofil.
The preassembled rings of γ-tubulin (centrosome) are held t/g in much tighter side-to-side interactions than αβ-dimers, so binding additional αβ-dimers is similar to adding to existing mtub; thus γ-tubulin rings can be thought of as permanently preassembled nucleation sites.
Colchicine and Taxol are drugs that assoc w free dimers and mtubs (resp) and result in chromosomes being unable to segregate during mitosis.
Briefly describe how such mechanisms might work.
Colchicine - tightly binds free dimers → prevents poly into mtubs; e.g. cell in mitosis stalls as mitotic spindles rapidly disappear, unable to sep chromos.
- Indicates that mitotic spindle is normally maintained by continuous poly/depoly.
Taxol - opp effect as colchicine; binds tightly to mtubs → prevents depoly, but poly still permitted → same net effect: chromos can’t sep → cell in mitosis stalls.
- Cancer cells divide ‘uncontrollably’ → may be killed preferentially by mtub-de/stabilizing ‘antimitotic’ drugs, e.g. colchicine, Taxol, vincristine, and vinblastine
Cells can modify dynamic instability of mtubs for partic purposes. Describe how this applies to mitosis and differentiated cells.
Cells can modify dynamic instability of mtubs for partic purposes:
E.g. mitosis - mtubs become more dynamic → enables rapid dis/reassembly into mitotic spindle
E.g. differentiated cells - dynamic instability of mtubs often suppressed by proteins that bind/stabilize mtub → maintain specialized function.
T/F: Most differentiated animal cells are polarized
True
Most differentiated animal cells are polarized
- E.g. nerve cells - axons extend fr one end of soma (cell body), dendrites fr other; all mtubs in axon have β/plus end pointed toward axon terminals (away fr soma) → directional xprt of organelles, vesicles, and macros.
- E.g. secretory cells - Golgi positioned toward site of secretion.
Dynamic instability causes mtubs either to grow/shrink rapidly. Consider an individual, shrinking mtub:
a. What must happen at the end of the mtub in order for it to stop shrinking and to start growing again?
b. How would a change in the tubulin concen affect this switch?
c. What would happen if only GDP, but no GTP, were present in the solution?
d. What would happen if the solution contained an analog of GTP that cannot be hydrolyzed?
A. The mtub is shrinking bc it has lost its GTP cap (dimers on end in GDP-bound form). While shrinking, free GTP-loaded dimers fr solution will still add to this end; if added quickly enough to overcome depoly (‘cover up’ GDP-loaded dimers), a new GTP cap can form and regrowth is favored.
B. As free (cytosolic) GTP-dimer concen ↑ → rate of addition to mtub ↑. Creates self-balancing system: mtubs shrink, releasing GDP-dimers into cytosol → dimers swap GDP for GTP, thus ↑ free GTP-dimer concen → rate of addition to mtub ↑; and v-v.
C. If only GDP present, mtubs would continue to shrink and eventually disappear, bc dimers w GDP have v low affinity for ea/o and will not add stably to mtubs.
D. If GTP is present but cannot be hydrolyzed, mtubs will continue to grow until all free dimers have been used up.
Mito/smaller mem-enclosed organelles/vesicles travel in small, jerky steps called _____________, wh is much more sustained and directional than continual, small, Brownian movements caused by random thermal (stochastic) motions.
Mito/smaller mem-enclosed organelles/vesicles travel in small, jerky steps called saltatory motion, wh is much more sustained and directional than continual, small, Brownian movements caused by random thermal (stochastic) motions.
- Saltatory movements can occur unidirectionally along mtubs/fils, both driven by motor proteins that use energy derived fr repeated cycles of ATP hydrolysis.
- Motor proteins attach other cell components → xprt cargo along mtubs/fils.
There are dozens of diff types of motor proteins, wh differ in mtub/fil assoc, cargo, and direction of xprt. One type, kinesins, move along cytoplasmic mtubs, typ fr ____ end to _____end. Similarly, dyneins typ move fr ____ end to ____ end.
There are dozens of diff types of motor proteins, wh differ in mtub/fil assoc, cargo, and direction of xprt. One type, kinesins, move along cytoplasmic mtubs, typ fr α/minus end to β/plus end. Similarly, dyneins typ move fr β/plus end to α/minus end.
- E.g. kinesins typ move fr nerve soma (cell body) to axon terminal; v-v for dyneins.
Describe the typ structure of kinesin and dynein.
Kinesins/dyneins are typ dimers w two globular ATP-binding heads and a single tail.
Heads interact w mtubs in stereospecific manner, i.e. attach in only one direction.
- Globular heads of kinesin/dynein are ATPases (ATP-hydrolyzing) → drives directed series of conform changes thru cycle of binding, releasing, and rebinding mtub → saltatory, unidirectional movement.
- Tails typ bind stably to some cell component (e.g. vesicle, organelle) → det cargo xprtd.
Briefly describe how mtubs and motor proteins position organelles in the cytoplasm, e.g. ER and Golgi.
In most animal cells, tubules of ER reach almost to edge of cell, whereas Golgi is located in cell interior, near the mtoc/centrosome; motor proteins establish and maintain distinct orientation:
- As cell grows, kinesins attach outside of ER mem (via receptor proteins) and pull ER outward along mtubs (away fr nucleus), stretching it like a net.
- Dyneins attached to Golgi mem pull it along mtubs in opp direction, toward nucleus.
If cells are treated w colchicine (causes mtubs to disassemble), describe how ER and Golgi change their location dramatically.
ER, wh is conn to nuclear envelope, collapses around nucleus.
Golgi, wh is not attached to any other organelle, fragments into small vesicles, wh then disperse thru/o cytoplasm.
When colchicine removed → organelles return to their original positions, dragged by motor proteins moving along the re-formed mtubs.
Cilia and flagella contain stable mtubs coved by wh motor protein?
Cilia and flagella contain stable mtubs coved by dynein.
- Recall: mtubs can bind accessory ‘capping’ proteins → stabilized.
- Stable mtubs form stiff supports in many polarized strucs, incl motile cilia/flagella.
- Recall: bending of cilia/flagella core is driven by ciliary dynein.
Ea cilium contains core of stable mtubs, arranged in a bundle, that grow fr a _________. A cilium beats by performing a repetitive cycle of movements, consisting of a ______ stroke followed by a ______ stroke.
Ea cilium contains core of stable mtubs, arranged in a bundle, that grow fr basal body (mtoc/centriole). A cilium beats by performing a repetitive cycle of movements, consisting of apowerstroke followed by arecovery stroke.
Flagella are internally similar to cilia, but typ v much ______ (shorter/longer).
Flagella are internally similar to cilia, but typ v much longer; specialize in moving entire cell, rather fluid across cell.
- Propagate regular waves along their length, propelling attached cell along, e.g. sperm/protozoa.
Mtubs in cilia/flagella are slightly diff fr cytoplasmic mtubs: arranged in distinctive _______ pattern
Mtubs in cilia/flagella are slightly diff fr cytoplasmic mtubs : arranged in distinctive 9 + 2 pattern.
- “9 + 2” array - nine doublet mtubs arranged in ring around pair of single mtubs; characteristic of almost all euk cilia/flagella.
Movement of cilium/flagellum is produced by bending of its ________ as mtubs slide against ea/o.
Movement of cilium/flagellum is produced by bending of its core as mtubs slide against ea/o.
Mtubs bear many accessory proteins that serve as cross-links to hold bundle of mtubs t/g; others generate force that causes cilium to bend, e.g. ciliary dynein.
Describe how ciliary dynein generates the bending motion of cilia/flagella cores.
Ciliary dynein closely resembles/funcs like cytoplasmic dynein: attaches by its tail to one mtub, while its two heads interact w adj mtub to generate sliding force b/w two mtubs.
Multiple links hold adj mtub doublet t/g → sliding force b/w adj mtubs is converted to bending motion in cilium.
Actin/mfils are helical polymers of actin protein. They are flexible and organized into variety of linear bundles, 2-D networks, and 3-D gels; present in all euks, and essential for many cell movements, partic those involving pmem.
Where are mfils concentrated? Describe how bound accessory proteins det their function.
Mfils are dispersed thru/o cell, but concentrated in cell cortex.
Bound accessory protein dets function:
- Stiff/stable structures, e.g. microvilli on epithelial cells lining intestine.
- Small contractile bundles that act like tiny muscles in most animal cells.
- Temporary structures, e.g. dynamic protrusions formed at leading edge of a crawling cell.
- Contractile ring that pinches cytoplasm in two during cytokinesis.
T/F: actin monomers have a structural polarity (+/- end).
True
Actin (mfil) monomers are a twisted chain of two identical globular actin subunits, both wh “point” in same direction along axis of chain → structural polarity (+/- end).
Mfils are typ shorter than mtubs, but form larger structures. Explain.
Many lateral interactions b/w actin threads stabilize the helical monomer structure.
Mfils are thinner, more flexible, and typ shorter than mtubs; also more abundant → form much longer structures.
Unlike ifils/mtubs, mfils rarely occur in isolation; typ found in cross-linked bundles/networks (stronger).
A cleft in the monomer provides binding site for ATP/ADP.
At high concens of free actin monomers (double helices) → mfil polys/grows rapidly at ea end.
At intermediate concens of free actin, the mfil ‘response’ is more complex. Explain.
At intermediate concens of free actin:
Plus end grows faster than bound ATP can be hydrolyzed → plus end grows.
Minus end doesn’t grow as fast as ATP hydrolyzed → ADP-actin destabilizes mfil → minus end depolys/shrinks.
These processes occur simult to create ‘treadmill’ effect: individual monomer moves thru mfil fr plus to minus end.
- If rate of poly = depoly → mfil remains same size.
- Recall: dynamic instability of mtubs involves poly/depoly at only β/plus end → more drastic changes in length
About 5% of total protein in typ animal cell is actin → half assembled into fils, other half is free pool of cytosolic actin monomers → unlike mtubs, free concen is much higher than reqd for purified monomers to spont poly (in vitro).
What keeps actin monomers in living cells fr completely polymerizing into mfils?
Cells contain small accessory/regulating proteins (thymosin; proflin) that bind free actin monomers (double helices) → prevent fr poly; store ‘in reserve’ until reqd.
When more mfils reqd → other actin-binding proteins (formins; actin-related proteins, ‘ARPs’) promote mfil poly.
T/F: Many actin-binding proteins in cells, but most bind free actin monomers.
False
Many actin-binding proteins in cells, but most bind assembled mfils rather than free actin monomers; i.e. regulate behavior of intact fils.
- Actin-bundling proteins hold mfils t/g in parallel bundles in microvilli
- Others cross-link mfils t/g in gel-like meshwork w/i cell cortex.
- Filament-severing proteins fragment mfils into shorter lengths → convert actin gel into more fluid state.
-
Myosin (motor protein) assoc w mfils to form contractile bundles.
- Mfils often form tracks along wh myosin xprt organelles.
Cell crawling involves coordinated changes of many molecules in diff regions of cell; i.e. not a single locomotive organelle like flagella. These mechanisms all involve actin, but in diff ways.
Describe what’s occurring at the leading edge of a crawling cell.
At leading edge of cell, rapid, local actin poly—assisted by actin-related proteins (ARPs)—pushes pmem forward (protrusion) → forms new ‘exploratory’ regions of actin cortex.
- Leading edge of crawling fibroblast in culture regularly extends thin, sheet-like lamellipodia, wh contain dense meshwork of mfils, oriented so that most fils have plus ends close to pmem.
- ARPs form complexes that bind sides of existing mfils → nucleate formation of new mfils, wh grow out at an angle to produce side branches → web continues to assemble at leading edge (and disassemble further back).
Briefly describe the cell crawling mechanism.
Crawling mechanism - coordinated changes of many molecules in diff regions of cell; i.e. not a single locomotive organelle like flagella. Processes all involve actin, but in diff ways.
At leading edge of cell, rapid, local actin poly—assisted by actin-related proteins (ARPs)—pushes pmem forward (protrusion) → forms new ‘exploratory’ regions of actin cortex.
New anchorage points are made b/w bottom of cell and surface (substratum) on wh cell is crawling (attachment).
- E.g. when lamellipodia/filopodia contact favorable surface, xmem proteins (integrins) adhere to molecules either in ECM/substratum as well as bind mfils in cell cortex → anchorage point.
Contraction at rear of cell—mediated by myosin moving along mfils—draws body of cell forward.
New anchorage points are established at front, and old ones are released at the back as cell crawls forward.
Cycle repeats over and over, moving forward in step-wise fashion.
All actin-dep motor proteins belong to myosin family: bind/hydrolyze ____ → provides energy for movement along mfils toward ____ (+/-) end.
All actin-dep motor proteins belong to myosin family: bind/hydrolyze ATP → provides energy for movement along mfils toward plus end.
Myosin-I is present in all types of cells, and much simpler in struc/func than myosin-II subfamily. Describe its structure.
Myosin-I subfamily:
Head domain - binds mfil; has ATP-hydrolyzing motor activity → movement via cycle of binding, detachment, and rebinding; recall: head domain always moves toward plus end of mfil.
Tail - varies among diff types of myosin-I; dets type of cargo, e.g. tail may bind to a partic type of vesicle → propel thru cell along mfil tracks, or may bind pmem → pull into diff shape.
Rho family GTP-binding proteins—t/g w protein kinases and accessory proteins—act like a computational network to control actin org/dynamics. Explain.
Complex of Rho protein/kinases/accessory proteins receive EC signals fr nutrients, growth factors, and contacts w adj cells/ECM as well as IC info about cell’s metabolic state and readiness for division.
Rho network processes inputs → activates IC signal paths that shape actin cytoskeleton.
- E.g. activates formin proteins that promote formation of filopodia, or stimulate ARP complexes at leading edge of cell to generate large lamellipodia.
- E.g. in response to signal fr motor nerve → activates rapid rearrangement of cytoskeletal elements → muscle contraction.
Various types of myosin in cells, but myosin-I and myosin-II subfamilies are most abundant. What makes myosin-II structurally diff fr myosin-I, and where is it used most often?
Myosin-II - dimer w two globular ATPase heads and single coiled-coil tail; incl muscle myosin.
- Myosin-I has only one globular head.
- Myosin-II is specialized for muscle contraction.
Clusters of myosin-II bind ea/o thru their ______ (heads/tails) → form ______ (nonpolar, polar, bipolar) myosin fil → _____ (heads/tails) project outward in opp directions, w bare region in middle of fil.
Clusters of myosin-II bind ea/o thru coiled-coil tails → form bipolar myosin fil → heads project outward in opp directions, w bare region (tails only) in middle of fil.
- Small, bipolar myosin-II fil is like a double-headed arrow, w two sets of heads pointing in opp directions: ea set binds mfils in one orientation → myosin fil slides sets of opp oriented mfils past one/an → mediates contraction of interacting actin/myosin-II fils in both muscle/non-muscle cells.
- If mfils/myosin fils organized t/g in a bundle → generate strong contractile force, e.g. muscle contraction, but also in much smaller contractile bundles of mfils/myosin-II fils that assemble transiently in non-muscle cells, and in contractile ring
T/F: Like myosin-I, myosin-II head group ‘walks’ toward plus end of mfil.
True
Like myosin-I, myosin-II head group ‘walks’ toward plus end of mfil.
______ fils slide against ______ fils during muscle contraction.
Actin fils slide against myosin fils during muscle contraction.
- Small, bipolar myosin-II fil is like a double-headed arrow, w two sets of heads pointing in opp directions: ea set binds mfils in one orientation → myosin fil slides sets of opp oriented mfils (actin) past one/an → mediates contraction of interacting actin/myosin-II fils in both muscle/non-muscle cells.
- Muscle contraction - mfils (actin) slide past myosin-II fils (w/o change in length of either fil) → simult shortening of all sarcomeres in cell.
What are myofibrils and sarcomeres?
Myofibrils - contractile elements of muscle cell; comprise bulk of cytoplasm; composed of chain of sarcomeres.
Sarcomeres - tiny contractile units; highly organized assemblies of two types of fils (mfils and muscle-specific form of myosin-II) → form repeating (striated) pattern.
- Myosin-II fils - thicker; centrally positioned in ea sarcomere.
-
Mfils (actin) - slender; anchored by plus end to ea end of sarcomere (Z disc) → extend inward, w minus ends overlapping globular ends of myosin-II fils.
- Recall: tail-only region in middle of myosin-II; no overlap w actin
Myofibrils are the contractile elements of muscle cell that comprised bulk of cytoplasm (‘sarcoplasm’). Myofibrils are composed of a chain of sarcomeres, wh are tiny contractile units that are highly organized assemblies of two types of fils to form a repeating (striated) pattern. Describe the structure and orientation of mfils (actin) and myosin-II fils w/i a sarcomere.
Myosin-II - thicker; centrally positioned in ea sarcomere.
Actin (mfil) - slender; anchored by plus end to ea end of sarcomere (Z disc) → extend inward, w minus ends overlapping ends of myosin-II fils.
Muscle contraction involves mfils (actin) sliding past myosin-II fils (w/o change in length of either fil) → simult shortening of all sarcomeres in cell. Describe the ATP-dep cycle in wh myosin-II heads walk along mfils to prod conform changes.
Muscle contraction overview: myosin head binds/hydrolyzes one ATP → series of conform changes that move tip of head ~5 nm along mfil (toward plus end; unidirectional) → pulls mfil along myosin fil → repeats → sarcomere contracts → after contraction completed, myosin heads all lose contact w mfils → muscle relaxes.
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Attached - start of cycle; unbound myosin head (no ATP/ADP) is tightly attached to actin in rigor conform.
- In actively contracting muscle, rigor conform is v short-lived → rapidly terminated by binding of ATP to myosin head.
- Released - ATP binds large cleft on ‘back’ of myosin head (furthest fr actin) → immediately causes slight change in conform of domains that comprise actin-binding site → ↓ affinity of myosin head for actin and allows it to move along mfil.
- Cocked - cleft closes like a clam shell around ATP → triggers large conform change → displaces myosin head along actin (toward plus end, ~5 nm) → ATP hydrolysis, but ADP + Pi remain tightly bound to myosin head.
- Force-generating - weak binding of myosin head to new site on mfil → simult release of P group (Pi) → triggers power stroke: force-generating change in shape during wh myosin head releases ADP and regains original conform → ready for new cycle.
T/F: At the start of muscle contraction, a myosin head is ATP-bound and tightly attached to actin/mfil in rigor conformation.
False
At the start of muscle contraction, an unbound myosin head (ATP nor ADP) is tightly attached to actin/mfil in rigor conformation.
- In actively contracting muscle, rigor conform is v short-lived → rapidly terminated by binding of ATP to myosin head.
In what stage of the muscle contraction cycle does ATP bind the myosin head? What immediately occurs?
Released - ATP binds large cleft on ‘back’ of myosin head (away fr actin/Z disc) → immediately causes slight change in conform of domains that comprise actin-binding site → ↓ affinity of myosin head for actin and allows it to move along mfil.
During the cocked stage of muscle contraction, ATP-bound myosin head undergoes a conform change that displaces itself along actin, toward its ____ (plus/minus) end, i.e. ______ (toward/away fr) Z disc. After displacement, hydrolysis of ATP occurs, and ADP + Pi ________ (remain bound to/release fr) myosin head.
During the cocked stage of muscle contraction, ATP-bound myosin head undergoes a conform change that displaces itself along actin, toward its plus end, i.e. toward Z disc. After displacement, hydrolysis of ATP occurs, and ADP + Pi remain bound to myosin head.
- If ADP + Pi were released after ATP hydrolysis and before re-binding actin → actin wouldn’t slide across myosin, i.e. no muscle contraction.
During the force-generating stage of muscle contraction, myosin—w ADP + Pi bound—forms a ____ (weak/strong) bond w a new site on actin/mfil, which simultaneously releases ____ (ADP/Pi) and triggers the _____ stroke, wherein myosin releases ____ (ADP/Pi) → conform change to slide actin’s _____ (plus/minus) end toward myosin’s center and restore myosin head’s position relative to actin.
During the force-generating stage of muscle contraction, myosin—w ADP + Pi bound—forms a weak bond w a new site on actin/mfil, which simultaneously releases Pi and triggers the power stroke, wherein myosin releases ADP → conform change to slide actin’s minus end toward myosin’s center and restore myosin head’s position relative to actin.
T/F: All sarcomeres of a muscle are coupled t/g.
True
All sarcomeres of a muscle are coupled t/g → triggered simult → entire muscle contracts almost instantaneously.
W/i a sarcomere, wh parts/ends of myosin-II fils and actin (mfil) overlap?
Actin’s plus end is anchored to end of sarcomere (Z disc) and extend inward to overlap w myosin-II heads. Middle of sarcomere is composed of myosin-II coiled-coil tails, i.e. no globular heads to attach actin.
Myofibrils are the contractile elements of muscle cell that comprise bulk of cytoplasm (‘sarcoplasm’), composed of chain of sarcomeres (contractile units; actin and myosin-II). ________ are invaginations of pmem around ea myofibril, and the _____________ is a specialized region of ER that surrounds ea myofibril.
Myofibrils are the contractile elements of muscle cell that comprise bulk of cytoplasm (‘sarcoplasm’), composed of chain of sarcomeres (contractile units; actin and myosin-II). Transverse/T-Tubules are invaginations of pmem around ea myofibril, and the Sarcoplasmic Reticulum (SR) is a specialized region of ER that surrounds ea myofibril.
What are troponin and tropomyosin?
Tropomyosin - rigid, rod-shaped; binds in groove of actin helix → prevents myosin heads fr assoc w mfil.
- Every tropomyosin has seven evenly spaced regions of similar AA seq, ea wh is thought to bind an actin monomer of mfil.
Troponin - protein complex, incl Ca2+-sensitive protein assoc w end tropomyosin.
- I.e. when AP causes incr in IC [Ca2+], Ca2+ binds troponin, wh then xmts conform change to tropomyosin, wh also changes conform to allow myosin to bind, thus initiating muscle contraction.
In the contraction initiation mechanism, elec excitation (AP) opens ____-gated ____ channels in T-tubule → ion influx (fr _____ to _____) → opens ____ release channels in SR mem → sharp ion influx (fr ______ to ______) → ↑ ___ (IC/EC) ion concen activates molecular switch made of troponin and tropomyosin: ion binds _______ (troponin/myosin) → changes conform → ________ (troponin/myosin) slightly change conform, exposing ______ binding sites on actin/mfil → myosin heads bind actin/mfil, initiating contraction.
Contraction initiation mechanism:
Elec excitation → opens v-gated Ca2+ channels in T-tubule (pmem invagination) → passive Ca2+ influx (fr lumen of T tubule to cytosol) → opens Ca2+ release channels in SR mem → sharp influx of Ca2+ (fr SR lumen to cytosol) → ↑ IC [Ca2+] activates molecular switch made of troponin and tropomyosin: Ca2+ binds troponin → troponin complex changes conform → tropomyosin slightly change conform, exposing myosin binding sites on actin/mfil → myosin heads bind actin/mfil, initiating contraction.
- Note: v-gated Ca2+ channels in T-tubule mem are mechanically coupled to Ca2+ release channels in SR mem; i.e. opening of the Ca2+ release channels is caused by the conform change of v-gated Ca2+ channels (and not Ca2+ concens in cytosol).
Describe the contraction initiation mechanism, starting w electrical excitation of a muscle cell.
Contraction initiation mechanism:
Elec excitation → opens v-gated Ca2+ channels in T-tubule (pmem invagination) → passive Ca2+ influx (fr lumen of T tubule to cytosol) → opens Ca2+ release channels in SR mem → sharp influx of Ca2+ (fr SR lumen to cytosol) → ↑ IC [Ca2+] activates molecular switch made of troponin and tropomyosin: Ca2+ binds troponin → troponin complex changes conform → tropomyosin slightly change conform, exposing myosin binding sites on actin/mfil → myosin heads bind actin/mfil, initiating contraction.
- Note: v-gated Ca2+ channels in T-tubule mem are mechanically coupled to Ca2+ release channels in SR mem; i.e. opening of the Ca2+ release channels is caused by the conform change of v-gated Ca2+ channels (and not Ca2+ concens in cytosol).
Describe the post-contraction mechanism, i.e. what happens after AP terminated.
Post-contraction mechanism:
After nerve signal (AP) terminates → Ca2+ is rapidly pumped back into SR via abundant Ca2+-pumps in SR mem → IC [Ca2+] returns to resting level → troponin and tropomyosin reconfig to original conform, blocking myosin binding sites on mfils → contraction ends.
