Microfilaments II (Lecture 19) Flashcards
What is the rate limiting step in actin filament assembly?
formation of an initial actin nucleus from which a filament can grow
What is the rate limiting step used for?
Used as control point to determine where filaments are assembled and what types of actin structures are generated
______ are controlled by signal transduction pathways.
Actin-nucleating proteins
______ are long filaments and ______ are a branched network of actin0nucleationg proteins.
Formin protein family
Arp2/3
How does actin nucleation by formin work?
- The FH2 dimer binds two actin subunits to nucleate formation of a new filament (bypasses formation of three monomer trimer seed).
- One FH2 domain rocks up far enough to allow assembly of one actin subunit onto a protofilament (+) end
- The other FH2 domain rocks up far enough to allow assembly of one actin subunit onto the other protofilament (+) end
- The cycle repeats to elongate an unbranched filament
CapZ stops it
How does the regulation of formin occur?
Rho-binding domain (RBD) binds to Rho-GTP exposing FH2 to nucleate new filament
Active state: FH1 domain is proline rich and recruits profilin-ATP-G-actin
True or False: Arp2/3 complex must be activated by nucleation promoting factor (NPF).
True
NPFs have WCA domain, what does WCA stand for.
WH2 (binds actin)
Connector
Acidic
What are the four steps to actin nucleation by Arp2/3?
- Two NPFs WH2 (W) domains each bind an actin monomer
- Binding of two NPF-actin complexes bind to the Arp2/3 complex induces a conformational change that activates the Arp2/3 complex
- The activated Arp2/3 complex binds to the side of an existing actin filament and binds the (-) ends of actin subunits transferred from the NPF W domains
- Additional G-actins assemble onto the (+) end of new actin filament, which makes a characteristic 70° angle with the old filament
How does regulation of the Arp2/3 complex work?
WASp =NPF inactive state and WCA domain unavailable
Active state: W domain transfers G-actin to activated Arp2/3
A domain activates Arp2/3 binding to side of filament
How does listeria monocytogenes move in around as a bacterial pathogen?
If (-) end of microfilament is fixed in the cell, objects bound to assembling (+) end can ‘ride’ across the
hijacks normal actin processes
______ bacterial cell-surface protein functions like an NPF to activate Arp2/3 complexes
ActA
In clathrin-mediated endocytosis, a burst (seconds) of ______ actin assembly drives internalized endocytic vesicles away from the plasma membrane
Arp2/3-dependent
True or False: The Arp2/3 complex is not essential for several steps in membrane trafficking, but facilitates transport between different compartments of the secretory and endocytic pathways.
True
______, from fungus, blocks (+) end
______ from sponges, sequesters G-actin
Cytochalasin D, Latrunculin
______ from sponge, enhances nucleation ______ from mushroom, locks subunits together in filaments (prevents depolymerization)
Jasplakinolide, Phalloidin
______ are needed to form microfilaments into bundles or networks
Actin cross-linking proteins
______ is the actin cross linking protein found in microvilli, filopodia, and focal adhesions
Fimbrin
______ is the actin cross linking protein found in the leading edge, stress fibers and filopodia
Filamin
True or False: Microfilaments attached to membrane provides erythrocytes the tensile strength and flexibility necessary to survive fluid dynamic forces of blood flow and squeezing through narrow capillaries.
True
______ molecules link the middle of the spectrin tetramer to the Band 3 integral membrane protein
Ankyrin
______ links the hub actin filaments and spectrin ends to the transmembrane protein ______.
Band 4.1, glycophorin C
______ is activated by phosphorylation and links the actin filaments laterally to the microvillar plasma membrane.
Ezrin
______ move along actin filaments.
Myosins
True or False: Myosins all convert energy from ATP hydrolysis to do work and is involved with muscle contraction, moving vesicular cargo, moving organelles and contribute to cell migration.
True
What is the myosin 2 structure?
Two identical heavy chains, two identical light chains, actin-binding site on each head
True or False: ATP-binding and actin-binding sites are both in the head domain (S1)
True
What class of myosin is responsible for organelle transport and is the longest?
Class V
What class involved with contraction.
Class II
What class is involved with memebrane association and endocytosis.
Class I
Myosin I is encoded by ______ different human myosin genes.
8
______ has two head domains and are encoded bu 3 different human myosin genes/
Myosin 5
What are the 5 steps in the cross-bridge cycle.
- Binds ATP, head released from actin
- Hydrolysis of ATP-ADP-Pi, myosin rotates into cocked state
- Myosin head binds to actin filament
- “Power Stroke” release of Pi elastic energy straightens myosin, moves actin filament left
- ADP released, ATP bound, head released from actin
In myosin 5 movement, each head steps ______ nm and cargo moves ______ nm.
72, 36
True or False: One head remains in contact with the microfilament at all times
true
How do you know when myosin 5 is inactive/
Binds to its own tail domain
True or False: Coordinated activities in different cell regions are needed for cell locomotion, integrated with a directed endocytic cycle
True
What are the 4 steps in cell locomotion?
- An Arp2/3-dependent mechanism extends one or more lamellipodia at the cell leading edge.
- Lamellipodia adhere to the substratum by formation of focal adhesions in which integrin mediates a connection between the actin cytoskeleton and extracellular matrix proteins such as fibronectin and collagen.
- Actin-myosin II-dependent contraction at the rear of the cell propels the bulk of the cytoplasm forward.
- Deadhesion and endocytic recycling at the back of the cell:
