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128 Cards in this Set
- Front
- Back
The primary component of microfilaments
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Actin
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Actin molecules are what?
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Monomers
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Actin molecules are organized into what?
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A single helical filament
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Non-covalent bonds between actin subunits allows for what?
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rapid turnover of actin monomers
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Polymer dynamics of actin microfilaments is regulated by what?
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ATP hydrolysis
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What is the most abundant intracytoplasmic protein in animal cells (1-10% of total protein)
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Actin
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Actin is _______ during evolution
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highly conserved
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Actin is a globular protein composed of 2 domains that bind ATP/ADP or
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cations such as Mg+2, K+, or Na+
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What will the addition of cations such as Mg+2 have on G-actin
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Induce polymerization of G-actin into F-actin
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Without the addition of _______ a lag period will preceed elongation of the actin filaments.
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an actin trimer nucleus
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The actin cleft faces the plus or minus direction?
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minus
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Actin filament polarity can be demonstrated using what?
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Myosin head decoration and electron microscopy
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In actin myosin head decoration the head points to the ____ end and the barbed end points to the _____ end
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minus; plus
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The cleft on actin subunits points to what end?
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Minus end
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Monomers are preferrentially added to what end of actin filaments?
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plus
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What can you use to prevent assembly at the plus end of actin?
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Capping protein Capz
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What can you use to prevent dissassembly at the negative end of actin?
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Capping protein tropomodulin
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What protein can cap the plus end of actin filaments and sever the filaments?
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Gelsolin with Ca+2 binding to the subunit; it caps the plus end with its second domain preventing assembly
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What end of actin requires a lower concentration (critical concentration) of actin subunits to initiate assembly?
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Plus end
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Treadmilling occurs when for actin filaments?
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When the actin concentration is above Cc for the plus end but below Cc for the minus end
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How does profilin work?
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Profilin enhances treadmilling by binding ADP-G-actin releasing ADP so ATP can bind
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How does cofilin work?
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Cofilin enhances treadmilling by destabilizing ADP-F-actin, binding two actin subunits and twisting it
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How does thymosin-B4 work?
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Thymosin-B4 maintains the large reservoir of actin monomers by binding ATP-G-actin, preventing it addition to either filament end
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Loss of what in actin filaments causes depolymerization?
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Pi from Actin-ADP-Pi at which point cofilin binds
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What are actin-bundling proteins?
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Monomer proteins with two actin-binding sites or dimers of two proteins with single actin binding sites. They induce formation of parallel actin bundles and their size determines the spacing between adjacent filaments
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What is fimbrin?
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An actin-bundling protein monomer inducing tight packaging often located at the leading edge of moving cells
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What is alpha-actin?
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An actin-bundling protein dimer inducing loose packaging often found in stress fibers and focal contacts
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How does Listeria achieve motility?
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It has a surface protein ActA that binds and activates the Arp2/3 complex and also binds VASP
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What are the three important properties of VASP?
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Can bind profilin for more ATP-actin.
Can hold on to the end of the newly formed filament. Can protect the plus end from CapZ capping. |
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What proteins are needed from Listeria mobility?
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ATP-G-Actin, Arp2/3 Complex, CapZ (keeps only filaments near bacterium growing), Cofilin (needed to regenerate fresh actin)
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How do you form actin branches?
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Use Arp2/3 Complex which will bind to the side of an existing filament. WASp activates Arp2/3 so it can act as nucleator for actin filament assembly at a 70 degree angle.
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How does WASp work?
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It is inactive until it binds Cdc42-GTP which then allows it to unfold and activate Arp2/3 for branch assembly.
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Where is Arp2/3 usually concentrated?
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At the leading edge
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What protein in humans is similar to ActA?
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N-WASp
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What is wrong with patients with Wiscott-Aldrich Syndrome?
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A mutated N-WASp protein causing defects in morphology and chemotaxis of platelets and lymphocytes.
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What is special about Shigella?
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It uses N-WASp instead of ActA for motility.
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How is new actin added to growing filament plus ends once it has reached the membrane?
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Thermal fluctuations make space for the subunits which move in resulting in the pushing force
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What does formin compete with?
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Plus end capping proteins
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How does formin work?
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FH2 dimers form a ring which rocks on the plus end of filaments to allow one subunit of actin to bind while holding on to an already bound subunit, processively polymerizing
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De novo assembly of actin filaments is induced by what?
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Formin
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What is formin required for?
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cytokinesis, formation of filopodia, focal adhesion sites, cell-cell junctions
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What is the major actin filament nucleator required for contractile ring assembly and completion of cytokinesis
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Formin
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What protein is required for formin activation?
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Rho-GTP binding
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What is the purpose of the FH1 domain in formin?
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Binds profilin molecules for ADP release of actin in exchange for ATP
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What is one of the only ways of studying an essential gene function?
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Temperature sensitive mutation
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What is the function of microtubules?
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Mechanical support, localization of organelles, cell polarity, cell motility (cilia & flagella), intracellular transport
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What are the monomers of microtubles?
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alpha/beta-tubulin
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The GTP site on alpha-tubulin site is special in what way?
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It is trapped by beta-tubulin and non-hydrolyzable
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The diameter of a microtuble is about how many nm?
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about 24
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What is FtsZ?
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A tubulin-like protein found in prokaryotes and plants. It is required to form the contractile ring of cytokinesis and is similar in structure to tubulins but is very different in sequence.
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What are MTOC's?
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Microtubule-organizing centers. Where all microtubules are nucleated from. Also known as centrosomes in non-mitotic cells, spindle poles in mitotic cells, and basal bodies in the shaft of cilium or flagellum. minus end is always at the MTOC
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What is unique about microtubules in axons?
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They are not continuous and have been released from the MTOC but still have the same polarity.
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What are centrioles?
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Orthogonally arranged structures in the centrosome composed of 9 sets of triplet microtubles similar to basal bodies and surrounded by pericentriolar fluid.
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Microtubule doublets and triplets are composed of what?
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One complete microtuble of 13 protofilaments, and one or two more adjacent microtubles of 10 protofilaments each.
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The beta end of microtubule subunits faces what polarity end?
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Plus
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Where are microtubule doublets usually found?
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cilia and flagella
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Where are microtubule triplets usually found?
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basal bodies, centrioles
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How can microtubule protein be purified?
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By harvesting microtubules from brain, they can then be cooled to 4C so they depolymerize and MAP proteins unbind, then warmed to 37C so they polymerize again.
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What phenomenon do microtubules exhibit that is visualized when they are sheared and broken into shorter segments?
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Dynamic Instability
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How does dynamic instability work?
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Microtubules display alternating phases of growth, stability and shrinkage. They undergo catastrophe where they shrink then rescue where growth resumes. Individual microtubules behave independently. If the beta-tubulin cap is randomly hydrolyzed then protofilaments will curl and dissassemble.
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What is the advantage of dynamic instability?
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Since the protofilaments behind the beta-tubulin cap are constrained the stored energy can do work if a structure such as a chromosome is attached to the disassembling end. It is also used for space searching.
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At what state is dynamic instability at its greatest?
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Steady State
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How does space searching work?
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Assembling microtubules can be stabilized in order to establish polarized transport, such as growing toward the direction of a wound.
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What does colchicine and vinblastine do?
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It sequesters all free microtubule dimers in the cytoplasm resulting in loss (depolymerization) of microtubules due to their natural turnover.
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What does taxol do?
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Stabilizes microtubles against depolymerization by acting as a “glue” that keeps the protofilaments together even when the GTP cap is lost.
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What are destabilizing and stabilizing drugs good for?
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Cancer treatment
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By what method do centrioles replicate?
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Semiconservative Mechanism
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Where is gamma-tubulin localized and what does it do?
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In the pericentriolar material. It associates with itself to form a gamma-tubulin ring complex (gamma-TURC) which binds to tubules of the centrioles to form a nucleating template for new tubule formation.
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What does overexpression of gamma-tubulin do?
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Induces assembly of MTs which are not associated with the centrosome
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What is the function of XMAP15?
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It stabilizes the plus end tip of microtubules by binding two GTP subunits allowing for longer, less dynamic microtubules
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What does kinesin-13 do to microtubules?
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It destabilizes the tip by favoring the formation of curved protofilaments by binding to GTP subunits resulting in shorter, more dynamic microtubules.
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What is the function of +TIP proteins?
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+TIPs promote “capture” of plus ends of microtubules at the plasma membrane and can regulate plus end dynamics, such as EB1 +TIP, increasing the frequency of rescues, promoting polymerization
A number of proteins can “ride” on +TIPs and accumulate at the plus end including APC, formin (actin polymerization near the ends of microtubules). Thus, microtubules indirectly affect actin organization during lamellipodia formation. |
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What is the function of stathmin?
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It sequesters free tubulin enhancing catastrophes and may also bind to curved protofilaments on two microtubule subunits enhancing their disassociation from a microtubule end.
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What is the function of structural MAPs?
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Stabilization of microtubules: suppression of dynamic instability
And the formation of bundles by crosslinking of individual microtubules which can also affect the spacing between microtubules. |
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What effect does the Tau protein have?
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Growth of axon-like projections via microtubules
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What is the unique property of microtubules in dendrites?
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Mixed polarity
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What is unique about microtubule organization in neurons?
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Microtubules of neural extensions are not connected to the MTOC
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What effect does a mutation in spastin have?
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Hereditary spastic paraplegia (degeneration of long spinal neurons) due to loss of microtubule severing function
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What effect does katanin have?
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A microtubule-severing protein of centrosomes, may be responsible for release of microtubules by severing near the minus end. It is an ATPase that binds ATP and hydrolysis of ATP produces a torque that severs the microtubule
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What happens in localized stabilization of microtubules?
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In cells that are polarized some microtubules extending toward the leading edge, they are captured by the plasma membrane and suppression of dynamic instability provides preferred routes for motor-based transport. The centrosome is repositioned to the side where stable microtubules form.
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What are the functions of intermediate filaments?
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structural reinforcement of the plasma membrane and nuclear envelope, organization of cells into tissues, and often associate with cell-cell junctions
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In what cells are intermediate filaments most abundant?
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Very abundant in epithelial and nerve cells
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What are the properties of intermediate filaments?
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They are extremely structurally stable, can be depolymerized using very strong denaturants like urea but remain dynamic after assembly. They do not bind any nucleotides and are not polarized
Some are heteropolymeric, having several isoforms of the monomer which co-polymerize |
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What is keratin?
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An intermediate filament found in epithelial cells
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What is desmin?
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An intermediate filament found in muscle cells
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What is neurofilament?
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Intermediate filaments that are heteropolymers of 3 subunits, NFL-L, NF-M, NF-H: found in neurons and required for radial growth of axons and proper conduction velocity
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What are lamins
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Intermediate filaments that are heteropolymers of lamins A,B,C found in the nuclear envelope
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What is an important structural feature of intermediate filaments?
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They are more resistance to bending than microtubules and microfilaments
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What is the structural composition of intermediate filaments?
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They are made of of coiled dimers that associate anti-parallel into tetramers that interlock into protofilaments. These protofilaments then coil with each other into protofibrils
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What do intermediate filaments co-localize with?
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Microtubules
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What does plectin (a plakin) do?
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Cross-links intermediate filaments with microtubules
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Drugs which depolymerize MTs can cause collapse and aggregation of what?
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Intermediate filament networks
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What is probably involved in distributing IFs along microtubules?
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Kinesin
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Biotin-keratin injections show what about intermediate filaments?
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They are dynamic due to incorporation of keratin into filaments
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What happens with mutations to keratin?
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Skin abnormalities similar to epidermis bullosa simplex disease in humans occurs. Without keratin, mutant cells are more fragile and break as a result of mechanical trauma during movement of limbs
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What can occur with mutations to desmin?
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muscle skeletal/cardiac myopathy
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What disorders can occur with mutations to lamin?
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Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy, premature aging disorder (Hutchison-Gilford progeria syndrome)
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What is myosin?
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A mechanochemical enzyme (motor protein) that hydrolyzes ATP and converts chemical energy into movement along actin
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What is myosin composed of?
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Assembled into bipolar thick filaments of many myosin proteins, each protein is a dimer of a coiled chain tail, and two head groups with a cleft, and light chain regulatory proteins at the base neck region. The head and neck make up the S1 region and the tail S2.
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How does the sliding-filament assay work?
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Myosin heads are attached to glass and a solution of actin filaments are added with ATP causing the heads to walk and cause the filaments to slide minus end forward (due to heads walking toward the plus end)
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What is the function of Myosin II?
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muscle contraction, “amoeboid” movement and cytokinesis
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What is the function of myosin I?
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endocytosis, “amoeboid” movement
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What is the function of myosin V?
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transport of membrane-bound vesicles like smooth ER, and Golgi vesicles, and secretory (synaptic) vesicles, melanosomes and mRNAs
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What is the function of myosin VI?
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transport of vesicles from Golgi to the cell surface, endocytosis, organization of the plasma membrane of stereocilia
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What is the basis of myosin movement?
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Binding and hydrolysis of ATP are translated into large movements of the neck due to conformational changes.
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What is unique about myosin hydrolysis of ATP?
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Myosin is an actin-dependent ATPase, meaning it hydrolyses ATP very slowly, except in the presence of actin
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What determines the step size of myosin?
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The size of the neck region
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What is the rigor state of myosin?
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When myosin is bound strongly to actin in the absence of a nucleotide.
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What triggers an opening of the actin-binding cleft and dissociation from actin?
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Binding of ATP
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What causes a conformational change in the head of myosin, moving it to a new position?
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Hydrolysis of ATP
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What causes the power stroke of myosin?
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Rebinding of actin at a new position and release of Pi
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What causes the return to the rigor state of myosin?
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Release of the ADP
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Is myosin II processive?
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No, it does not stay on an actin filament for extended time
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Is myosin V processive?
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Yes, highly
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What is different about myosin V movement?
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It has a large step size and moves in a hand-over-hand mechanism
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How does myosin II work in cytokinesis?
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Myosin causes sliding of actin filaments of the contractile ring, generating constricting force during late anaphase
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Where is the localization of myosin I and II in amoebas?
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Myosin I is at the leading edge (forward motion) with II at the rear end (retraction of the body)
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What is the structure of muscle?
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It is units of myofiber bundles, each myofiber is composed of myofibrils which are divided into bands between adjacent Z discs within sarcomeres
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What are thick filaments of muscle?
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Myosin II bipolar filaments
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What are thin filaments of muscle?
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Actin filaments
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What do thin and thick filaments of muscle form?
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A hexagonal array
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What are the proteins stabilize actin filaments and associate with Z-discs?
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Actin end-capping protein, CapZ
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How are myosins activated?
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Phosphorylation which causes them to self-assemble into bi-polar filaments
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How do troponin and tropomyosin function?
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Tropomyosin binds along actin filaments with troponin, and Ca+ induces a conformational change in troponin that moves the tropomyosin filament so that actin can bind with myosin.
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What do mutations in genes involved with muscle contraction usually cause?
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Cardiac myopathy due to either deficit or abnormal increase in the sarcomere contractile force
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What is the directionality of most myosins?
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plus-end directed
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What is the directionality of myosin VI and IX?
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minus-end directed
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What is the role of myosin VI in stereocilia?
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It is enriched at a structure at the base of the stereocilia and may be pulling the membrane down (toward the pointed end of actin filament near the base of stereocilia
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