Lecture 19: Cytoskeleton Flashcards
Functions of the cytoskeleton
- Cytoskeleton represents bone of the cell
- important in organization of the cell
- Maintains correctly shaped cells
- Ensures cells are properly structured internally
- Change shape of the cell
- RBCs and the erythrocyte membrane skeleton
- RBCs must be flexible enough to get through tight capillary spaces
- RBCs and the erythrocyte membrane skeleton
- Moves the cell
- Re-arranges the cellular compartment
- Supports the Plasma membrane
- Provide the mechanical strength
- resistance to the stress without being ripped apart
- Pulls chromosomes apart during cell division
- Splits dividing cells during cell division
- Guides intracellular traffic of organelles
- Vesicles move around by using cytoskeleton as a sidewalk
- cells like sperm need to swim-cytoskeleton acts as a motor
- Muscle cell contraction
What are the 3 faimlies of cytoskeletal proteins
- Actin filiaments
- determine the shape of cell’s surface and are necessary for whole-cell locomotion, secretion, endocytosis
- Microtubules
- Form tube like structure
- determine the positions of membrane enclosed organelles and direct the intracellular transport
- make up centrioles and mitotic spindle
- cilia and flagella
- Intermediate filaments
- provide mechanical strength
- strong filament
- resist mechanical stress
Actin filaments
- are two-stranded helical polymers of the protein actin
- actin subunits are compact and globular (G-actin vs. F-actin)
- Flexible structures 5-9 nm in diameter
Microtubules
- Are long hollow cylinder
- are made of tubulin subunits that are compact and globular
- are long and straight
- outer diameter 25nm
- more rigid than actin filaments
- have one end attached to a single microtuble-organizing center called a centrosome
Intermediate filaments
- Are rope-like fiber, diameter 10nm
- Large heterogeneous family
- are made of smaller subunits that are themselves elongated and fibrous
- extend across the cytoplasm to provide mechanical strength (iii)
- span from one cell-cell junction to another to strengthen the epithelial sheet
___ allows fibroblast to crawl but also disassembles so cell can change shape to round prior to division. ____ forms a contractile ring at center of cell (with myosin) to split cells in division
Actin, actin
Microtubel cytoskelton consists of
- long microbtubles that emanate from a single microtubule-organizing center
- (note that the microtubles rearrange to form a bi-polar mitotic spindle)
_____ maintains polarity for intestinal cells- apical surface vs. basolateral surface
The cytoskeleton
____ are attached to desomosomes (adhesive structures) and to hemidesmosomes (cell matrix contact)
Intermediate filaments
___ form tracks to get newly synthesized proteins to proper locations
Microtubules
Cytoskeletal filaments (or polymers) are held together by _______ interactions, which means that their assembly and disassembly can occur rapidly
weak noncovalent interactions
____ are long linear strings of protein subunits joined end to end
protofilaments
(note that protofilaments are thermally unstable- easy to break linear filament that is why you get multiple protofilaments bound side to side)
assembly of actin or tubulin subunits (monomers) into linear polymer
Polymerization
Removal of monomers at the ends of the polymer
Depolymerization
For new large filament to form, subunits must assmble into initial aggregate or nucleus. This initial process is called
nucleation
Formation of an actin nucleus is caused by
random collision of 3 subunits
Course of formation of Cytoskeletal Filaments
- Filament nucleation-lag phase
- a process of formation of initial aggregate, or nucleus
- the rate limiting step
- Filament elongation-growth phase
- Subunits are quickly added onto the ends of nucleated filaments
- Eventually reach steady state in which the rate of monomer addition equals the rate of monomer loss
- Which is the critical concentration called Cc
- Steady state-equilibrium phase
- The rate of addition of new subunits balances the rate of dissociation subunits
- the concentration of free subunits at this point is called critical concentration (Cc)
Tubulin is a hetero-dimer of
- alpha-tubulin and beta-tubulin with non-covalent bonds
- note that both have a binding site for one GTP btu the GTP in the alpha tubulin is never hydrolyzed
1 microtubule is a hollow cylinder structure, consisting of
- 13 protofilamnets aligned in parallel
- longitudinal contact: alpha tubulin -beta tubulin
- Lateral contact: alpha-tubulin - alpha tubulin
Actin monomer contains a binding site for
ATP or ADP
What creates structural polarity of microtubule
- arrangement of alpha and beta tubulins
Actin Filament
- Arranged head-to-tail to generate structural polarity
- consists of 2 protofilaments, held by lateral contacts
- are flexible and easily bent
Plus end
- Fast-growing or shrinking end
- beta-tubulin or refered as barbed end of actin filament
minus end
- Slow-growing or shrinking end
- has alpha-tubulin or the GTP-binding clet on the actin monomer point toward minus end, also referred as pointed end
Elongation proceeds spontaneously when
DeltaG for addition of the monomer is less than zerio, due to the [monomer] exceeds the critical concentration
Treadmilling predominates in ____ filaments
actin
What is a Catastrophe in terms of Dynamic instability
if nucleotide hydrolysis proceeds more rapidly than subunit addition, the cap is lost and the microtubule begins ot shrink
what is a rescue in terms of dynamic instability
- GTP-containing subunits may still add to the shrinking end, and if enough add to form a cap, then microtubule grwoth resumes
Dynamic instability predominates in
microtubules
Construction of intermediate Filaments
- Each monomer is an elongated molecule with an extended central alpha-helical domain (A)
- Monomer forms a parallel coiled-coil dimer with another monomer (B)
- A pair of dimers associates in an antiparallel manner to form a staggered Tetramer (C)
- (note there is no nucleotide binding site and structural polarity)
- Within each tetramer, the two dimers are offset, allowing it to associate with another tetramer (D)
- Eight parallel tetramers (protofilaments) pack together laterally to form the filament (E)
What is the most diverse group fo intermediate filaments
The Keratins
Human genome: ___ distinct keratin genes. about ____ found in human epithelial cells
- 50
- 20
accessory proteins that conrol Assembly and position of cytoskeletal filaments: actin filaments
- Actin subunits
- Formin
- nucleates assembly + remains associated with growing plus end (Remember: for new large actin filaments to form, subunits must assemble into initial aggregate or nucleus (nucleation))
- Tymosin
- Binds subunits, prevents assembly
- Profilin
- binds subunits, speeds elongation
- ARP complex (actin related protein) nucleates assembly to form a web and remains associated with minus end
- Formin
- Actin Filaments
- Cofilin
- binds ADP-actin filaments, accelerates disassembly
- Gelsolin
- severs actin filaments and binds to plus end
- Capping protein
- prevents assembly and disassembly at plus end
- Tropomyosin
- stabilize filament
- Cofilin
- Filament bundling, cross linking and attachment to membranes
- Fimbrin, alpha-actinin, filamin, spectrin (RBC cytoskeleton and HS), ERM family (Ezrin, Radixin, Moesin)
Accessory Proteins that Control Assembly and Position of Cytoskeletal Filaments: Microtubules
- Tubulin dimers
- Staminin
- binds subunits, prevents assembly
- gamma-TuRC
- (gamma-tubulin ring complex) nucleates assembly and remains associated with minus end
- TIPS
- (plus end tracking proteins) remain associated with growing plus ends, and can link them to toher structures (e.g. membrane)
- Staminin
- Microtubules
- Kinesin 13:
- enhances catastrophic disassembly at plus end
- Katanin:
- (Japanese work for “sword” severs microtubules
- MAPS: (Microtubule associated protein)
- stabilizes tubules by binding along sides
- XMAP215
- A microtubule associated protein that stabilizes plus ends and accelerates assembly
- Kinesin 13:
- Filament cross linking
- Tau (a MAP protein), MAP2:
- both cause bundling of microtubules
- Plectin
- cross-linking protein
- links microtubules to intermediate filaments
- Tau (a MAP protein), MAP2:
Microtubules are nucleated from a specific location, called
microtubule-organizing center (MTOC)
_____ is responsible for the nucleation of microtubule growth
gamma-tubulin ring complex (gamma-TuRC)
a centrosome consists of a fibrous centrosome matrix to which the gamma-TuRCs are attached- greater than ___ copies of gamma TuRCs
50
embedded in the centrosome is a pair of cylindrical structures, called _____, arranged at right angle to each other
centrioles
Microtubules are nucleated at the centrosome at their _____ end, with ____ pointing outward and grow toard the cell periphery
minus end, plus end
nucleation of actin filaments occurs at or near the
plasma membrane, thus actin filaments mostly accumulate at cell periphery
Lamellipodia
flat protrusive veils of actin
Filopodia or microvilli
spiky bundles of actin
actin polymerization is regulated by
ARP complex and Formins
Actin-Related Protein or ARP (Arp 2/3) Complex
- Two ARP proteins, Arp2 and Arp3, are 45% identical to actin
- Function similar to gamma-TuRC, the ARP complex nucleates actin filament growth from the minus end, allowing elongation at the plus end
- Require activating factor
- in abscence of activating factor, Arp2 and Arp3 are masked by their accessory proteins to prevent them from nucleating a new actin filament
- Binding to the activating factor induces conformation change that resembles the plus end of actin filament, allowing actin monomers to bind, bypassing the rate-limiting step of filament nucleation
what is ActA
- it is a surface protein of Listeria
- It activates Arp 2/3 complex causes local nucleation of actin filaments which are cross linked
- Growing filaments are the driving force to push cell through cytoplasm
ARP comlex works most efficiently when it is
- bound to the side of preexisting actin filament-filaments cross linked
- filament branch grows at 70 degress angle relative to the original filament
_____ nucleate the growth of straight and un-branched actin filaments
Formins
Formins
- Nucleate the growth of straight and un-branched actin filaments
- A large family of dimeric proteins, with each formin subunit having a binding site for an actin monomer
- Formin dimers nucleate actin filament polymerization by capturing two monomers at the plus end or the growing end of an actin filament
- Formin proteins form a dimeric complex that can nucleate the formation of a new actin filament and remain associated with the rapidly growing plus end as it elongates
- different from gamma-TuRCs, nucleating tubulin polymers, because they bind to minus end of filament
How is there a large pool of actin polymers always kept available with all the nucleation of actin filaments occurring in the cell
- Tymosin
- keeps actin monomers soluble so they are readily available for generating filaments
- Actin monomers bound to thymosin are in locked state, where they cannot associate with actin filaments, this causes high concentration of soluble actin monomers in cells
- keeps actin monomers soluble so they are readily available for generating filaments
______ recruits actin monomers to the actin filament for polymerization
- Profilin
- Profilin binds to the actin monomer to expose the site of actin that binds to the plus end of the actin filament
- The addition of an actin monomer to the filament induces conformation change in the actin that reduces its affinity for profilin
- so the profilin falls off, leaving actin filament one subunit longer
- profilin competes with thymosin in binding to individual actin monomers
Proteins that regulate the availability of actin monomers for actin polymerization
- Thymosin
- Profilin
_______ are proteins that bind along the sides of microtubules, that alter the filament’s stability and mechanical properties
- Microtubule-associated proteins (MAPs)
- stabilize microtubules against disassembly, contain at least two domains, one domain for binding to microtubule and another that project outward
MAP2 vs. Tau
- MAP2 has a long projecting domain with a second microtubule-binding domain at the other end, thus forming bundles of stable microtubules that are kept widely spaced
- Tau binds to the microtubule at both its N- and C-termini, with a short projecting loop, forming bundles of more closely packed microtubules
Regulation of Stability proteins of Actin Filament
- Tropomyosin
- Actin filaments are stabilized by the binding of tropomyosin (an elongated protein)
- binding of tropomyosin prevents the actin filament interacting with other proteins (tropomyosin is a key protein in erythrocyte membrane skeleton)
- Cofilin
- actin depolymerizing factor
- binds to both actin filament and free actin subunits
- binding of cofilin forces the flament to twist a little more tightly, weakens the contacts between actin subunits, making the filament brittle and more easily severed
- Cofilin prefers to bind to ADP-containing actin filaments, thereby efficiently disassembling the older filaments
- Actin filaments can be protected from cofilin by tropomyosin
Kinesin-13 proein family
- Catastrophe factor
- increase the rate at which a microtubule switches from a growing to a shrinking state
- They bind to microtubule ends and pry protofilaments apart by lowering the activation energy barrier that prevents a microtubule from sprining apart into the curved protofilament
Actin filaments are organized into
two types of arrays, Bundle (Formin makes bundles) and web-like(gel-like) network (formed by ARP complex)
Actin filament cross-linking proteins help to stabilize and maintain these structures and are divided into 2 classes:
- Bundling protein
- cross-links actin filaments into a parallel array
- alpha-actinin, fimbrin, and villin
- gel-forming protein
- holds two actin filaments together at a large angle to each other to create a looser meshwork
Alpha-actinin vs. fimbrin
- alpha actinin
- cross-links actin filaments into loose bundles, allowing myosin II to enter to make actin filaments contractile
- Fimbrin
- cross-links actin filaments into tight bundles, excluding myosin II
- They tend to exclude on another because of different functions
villin is associated with
microvilli
_____ connect the sides of actin filament bundle to the plasma membrane in microvilli
Lateral sidearms (myosin-I, calmodulin)
filamin promotes theformation of losse and highly viscous gel-like network by clamping together
2 actin filaments roughly at right angles
cells require the actin gel formed by filamin in order to
- extend membrane projections which help cells ot crawl across a solid surface
- cells without filamin cannot crawl properly, and they produce disorganized membrane blebs
Cell migration can be conceptualized as a cyclic process, involving three distinctive activities
- Polarization and Protrusion
- The molecular processes at the front and the back of a moving cell are different. Actin-rich structures are pushed out at the front of the cell
- Adhesion (Attachment) and Traction:
- adhere to extracellular matrix (ECM) or adjacent cells via transmembrane receptors linked to the actin cytoskeleton. Adhesions serve as traction sites for migration as the cell moves forward over them.
- Re-traction
- adhesions are disassembled at the cell rear, allowing the bulk of the trailing cytoplasm to be drawn forward
Different types of protrusive structures, all have a dense core of actin filaments and no membrane-enclosed organelles
- Filopodia (microspikes)
- formed by migrating fibroblasts
- one-dimensional
- contain a core of long, bundled actin filaments
- Lamellipodia
- formed by epithelial cells, fibroblasts and neurons
- two-dimensional, sheet-like structure
- contains cross-linked mesh of actin filaments, most lie in a plane parallel to the solid substratum
- Pseudopodia
- Formed by amoebae and neutrophils
- Three-dimensional projections filled with an actin-filament gel
