exam 3b Lecture 27 Flashcards
What are the three filament systems of the cytoskeleton?
Microtubules (largest diameter), actin filaments (smallest diameter), intermediate filaments
Functions of the cytoskeleton
Movement of cells through liquid, movement of fluids over cells (cilia), movement of organelles in cells, movement within cells (mitotic spindle), movement of cells, cytokinesis (splitting of two cells), cell shaping (microvilli in intestinal epithelial cells example), structural support of cells/tissues (intermediate filaments especially)
Properties of cytoskeleton filaments
The filaments are polymers. They are dynamic. They have polarity.
How are filaments polymers?
Assembled from monomer subunits (polymerized)
How are filaments dynamic?
Signaling – disassembly/reassembly; nucleotide hydrolysis controls assembly of actin, tubulin; protein phosphorylation controls assembly of intermediate filament proteins
What is polarity of filaments?
Structurally different at each end. Have different assembly characteristics. Different in function and structure (not intermediate filaments).
How are cytoskeleteal filaments held together?
By many noncovalent bonds (end to end and side to side) between the subunits
Are single filaments flexible? They are also known as?
Yes. Protofilaments.
How are multiple aligned protofilaments different from a single protofilament?
They are stronger but less flexible.
Why are intermediate filaments strong and flexible?
Due to staggered arrangement of elongated subunits
Functions of actin cytoskeleton
Filament assembly dynamics; actin-binding proteins; cell shaping; generation of force for cell movements
How does actin cytoskeleton generate force for cell movement?
a. myosin motor proteins b. actin assembly generates force
How do chloroplasts move in Elodea leaves?
On actin filaments
What are actins?
Highly conseeved 50 kDa proteins in all eukaryotes
What do actins bind?
ATP that can be hydrolyzed when the monomer is in a polymer
What does G (globular) actin form when it spontaneously polymerizes?
F (filamentous) actin
What is actin also known as?
Microfilaments
How do actins polymerize?
Monomers assemble head-to-tail, resulting in structural polarity
What is an actin polymer?
Right-handed helix of two protofilaments
Diameter of actin polymer?
8-9 nm
What ends does actin polymer have?
Plus and minus ends
How long is actin molecule?
37 nm
Assembly components of actin filaments?
Purified actin + Mg2+ + ATP in physiological salt concentration
What does actin rate of assembly depend on?
Concentration of monomer (KonCc)
What is rate of dissambly independent of monomer concentration?
Koff
Equilibrium equation
konC = koffCc = koff/kon
when does spontaneous actin polymer assembly happen?
If Conc of monomer is above Cc
When does spontaneous actin polymer disassembly happen?
If Conc of monomer is below Cc
Is ATP hydrolysis required for actin polymer assembly?
No. It occurs in the presence of nonhydrolyzable analogs of ATP.
What is Cc?
Concentration of monomers at steady state
Three phases of polymerization of actin?
Nucleation (lag phase), elongation (growth phase), steady state (equilibrium phase)
What does structural polarity of the filament result in?
Assembly polarity.
Where is rate of assembly higher?
At the plus or barbed end. It is lower at minus or pointed end.
How does ATP hydrolysis occur within the polymer? How does it affect the monomer?
Stochastically (randomly). It changes the shape of the monomer, giving it less affinity for the polymer.
With what is the minus end enriched?
ADP actin. ATP hydrolyzation happens because the polymerization rate is slower, allowing for ATP hydrolysis. More ADP on pointed end than barbed end.
How doe Cc for minus and plus end compare? Why?
Cc for minus end is higher than for plus end. Cc = koff/kon = rate of disassembly/rate of assembly. Rate of disassembly is higher for minus end because ADP reduces monomer affinity for polymer.
What happens when the [C] of free monomer is between Cc of plus and minus ends?
The filaments undergo treadmilling.
What is treadmilling?
Assembly at the plus end and disassembly at the minus end results in a flux of monomers through the polymer. There is addition at plus end but overall loss at minus end, so length doesn’t change.
How does cytochalasin D affect actin assembly?
Binds and caps F-actin plus ends; filament disassembles
How does Latrunculin affect actin assembly?
Binds G-actin, preventing assembly
How does Phalloidin affect actin assembly?
Binds and stabilizes F-actin
What sequestering proteins control actin filament assembly?
Thymosin and profilin
How do profiling and thymosin compete?
In binding G-actin
Thymosin?
Binds G-actin (about half of G-actin in a typical cell is sequestered), prevents assembly (keeps monomer concentration high, > Cc for assembly)
Profilin?
Binds G-actin and promotes assembly of F-actin at the plus end.
What does inactive profilin bind?
Phospholipids PI(4,5)P2; release by phospholipase C promotes filament assembly at plasma membrane
Two actin assembly proteins
Arp2/3 and formin
Role of Arp2/Arp3
Nucleates assembly of actin filament network
Where is ARP complex localized?
At minus ends of actin filaments – growth at plus ends
Where does ARP complex bind?
To side of “older” (ADP-actin) filament to form filament network
What is WASp family protein?
Activates inactive ARP complex
Where do networks of short actin filaments assemble? What facilitates this?
Near the plasma membrane. Profilin activated at plasma membrane.
Role of Formin
Promotes assembly of F-actin bundles
How does formin work?
Formin associates with PM. Formin works with profilin; adds actin monomers to the plus ends of actin filaments. Long filaments are bundled.
What proteins regulate the dynamics of actin filaments?
Cofilin, gelsolin, capping protein , tropomyosin
What are actin filament regulatory proteins the target of?
Signaling molecules such as Ca2+
Cofilin
Binds ADP-actin filaments, accelerates disassembly
Gelsolin
Severs filaments and binds to plus end
Capping protein
Prevents assembly and disassembly at plus end
Tropomyosin
Stabilizes filament
Two actin filament cross-linking proteins
Alpha-actinin and fimbrin
How does alpha-actinin cross-link actin filaments?
Contractile bundle in loose packing that allows myosin-II to enter bundle
How does fimbrin cross-link actin filaments?
Parallel bundle in tight packing that prevents myosin-II from entering bundle
What does three-dimensional network of actin filaments form?
Viscous gel required for extension of leading edge in migrating cells
Role of ERM proteins
Link actin filaments to membranes
ERM protein cycle
Inactive folded ERM protein undergoes phosphorylation or PIP2 binding; becomes artive and extended; mediates cross linking between actin filament and transmembrane protein
Three domains of active ERM protein
Membrane-binding domain; alpha-helical domain, actin-binding domain
What are myosin motor proteins?
Mechanochemical enzymes
How do myosin motor proteins work?
ATP binding and hydrolysis result in conformational changes and movement along actin filament – toward the plus end. Myosin motor domains contain the actin binding site and the ATP binding site
What does lever arm length determine for myosins that form dimmers?
Step distance
What kind of cargo do myosins carry?
Membrane-bound vesicles/organelles/RNA molecules
Three different types of myosin domains?
Coiled-coil domain, cargo-binding domain, lever arm domain
How do myosin and actin filaments move?
Myosin moves towards plus end of filament. Filaments move towards minus end.
What kind of filaments does myosin II form?
Bipolar, coiled-coil filaments
How does myosin II work?
It walks only a short distance along actin filament before falling off, but there are a lot of them, so there is constant contact with actin filament track
Where are large myosin filaments found? Smaller myosin filaments?
Skeletal muscle cells/nonmuscle cells
Steps of myosin II activity cycle
Attached (to filament), released (from filament), cocked, force-generating, attached
Rigor configuration
Myosin head lacking a bound nucleotide is locked tightly onto an actin filament. State rapidly terminated by ATP binding.
What happens when ATP binds to attached myosin II?
Causes conformation change of actin-binding site, reducing the affinity of the head of myosin for actin and allowing it to move along filament.
How is myosin II cocked?
Cleft closes like a clam shell around the ATP molecule and triggers a movement in the lever arm that causes head to be displaced along the filament by a distance of about 5 nm. Hydrolysis of ATP occurs, but ADP and inorganic phosphate remain tightly bound to the protein.
What happens during myosin II force generation?
Weaking binding of myosin head to a new site on actin filament causes release of inorganic phosphate, and head binds to actin firmly. Release of phosphate triggers power stroke, a force-generating change in shape during which head regains original conformation. During power stroke, head loses bound ADP and starts new cycle.
Where is head at end of cycle of myosin II?
Tightly locked to actin filament, but has moved to a new position on the actin filament.
Structure of myosin V
Alpha helical coild coil with globular tail, two lever arms and motor domain at end of each lever arm.
What do long lever arms of myosin V allow it to do?
Take long “steps” along actin filaments
How many myosin v genes do mammals have?
Three
What are myosin v motors involved in?
Transporting vesicles in neuronal cells, endocytic vesicles, Golgi vesicles, RNAa
What do mutations in myosin VA lead to in mice?
A “dilute” color because melanosome pigment vesicles are not tethered properly to the cortical actin cytoskeleton.
What does myosin VA do at cortex of melanocytes?
Retains melanosomes to facilitate uptake by keratinocytes
Role of actin filaments in cell shaping
Actin filament bundles shape microvilli in intestinal epithelial cells to increase membrane surface area
How are actin filaments aligned below the microvilli of the SI epithelial cell?
In a circumferential band that is connected to cell-cell adherens junctions that anchor the cells to each other.
What role do intermediate filaments play in SI epithelial cells?
Are anchored to adhesive structures like desmosomes and hemidesmosomes that connect the epithelial cells into a sturdy sheet and attach them to the underlying extracellular matrix.
Role of microtubules in SI epithelial cells?
Run vertically from top of cell to bottom and provide a global coordinate system that enables cell to direct newly synthesized components to their proper location.
Where are desmosomes located?
Inside cells, but between cells.
Where are hemidesmosomes located?
Inside cells, but between cells and basal lamina
Role of actin in stereocilia
Hair cells in the inner ear contain actin bundles.
How do stereocilia work?
Sound vibrations cause stereocilia to bend, opening mechanically gated ion channels to depolarize membrane, propagate action potential. When bundle is tilted, channel is open.
Some actin homologs in prokaryotes
MreB protein filaments and ParM filaments
MreB protein filaments
Facilitate cell wall deposition and cell shape in rode-shaped bacteria like Bacillus subtilis
ParM filaments
Bind to and separate plasmids, ensuring their distribution to both daughter cells
