Lecture 18: The Cytoskeleton Flashcards
(48 cards)
1
Q
Cytoskeleton
A
- A system of protein filaments that provides structure and mechanical support for the cell
- Made up of three main types of fibers:
- > Microtubules – 24 nm in diameter, polymers of tubulin (Largest in diameter)
- > Intermediate filaments – average 10 nm in diameter, polymers of helical proteins (large family) (Called intermediate because they are intermediate in size compared to the other components)
- > Microfilaments – 6 nm in diameter, polymers of actin (Smallest in diameter)
2
Q
Cytoskeletal structures
A
- Equilibrium between the small soluble subunits and the large filamentous fibers
- Cytoskeletal structures are constructed by the polymerization of monomeric protein subunits through noncovalent attractions
- Disassembly and reassembly allow for changes in cell shape and/or internal movements of organelles/vesicles relatively rapidly
- > Provides for a lot of flexibility of the cytoskeleton
3
Q
The nature of polymerization of cytoskeletal proteins
A
- The polymerization of cytoskeletal monomers requires nucleoside triphosphates in the form of either GTP (binds tubulin) or ATP (binds actin) – we’ll abbreviate these NTP.
- Cytoskeletal monomers containing NTP have higher affinity for their binding partners than do cytoskeletal monomers containing NDP.
- There is a lot of intrinsic hydrolysis within these cytoskeleton components
- Soluble subunits are mostly bound to the NTP form, while polymers are a mixture of NTP and NDP bind subunits
4
Q
The nature of polymerization of cytoskeletal proteins #2
A
- New filaments are added to the positive end, so subunits on the positive end are more recently added than the ones at the negative end
- The longer the subunit has been attached to the polymer, the more likely it will have hydrolyzed the NTP to NDP
- > Therefore hydrolysis moves from the negative end to the positive end
- If loss of monomers at the negative end is slower than the gain of monomers at the positive end, then the polymer grows and the polymer will shrink if vice versa
5
Q
Actin
A
- Makes up microfilaments
- Composed of a network of flexible filaments dispersed throughout a cell – highly concentrated just beneath the plasma membrane (‘cortex’).
- Form the basis of cell shape and structure
- Form the contractile rings of dividing cells
- Aid in the contraction of muscle cells
- Propel vesicles and other cellular compartments through the cytoplasm
6
Q
Actin #2
A
- Soluble, globular protein
- > Most abundant protein in a typical eukaryotic cell
- > Most highly conserved proteins among eukaryotes
- Approx. 40 kDa M.W.
- ATP-G actin monomers bind more tightly than ADP-G actin monomers
- > ATP-G actin in filaments eventually hydrolyze into ADP-G actin
7
Q
Globular actin (G actin)
A
- Actin monomers
- Polymerize into F actin
8
Q
Filamentous actin (F actin)
A
- Actin polymers
- 2 strands of G-actin monomers wound together into a helical filament
9
Q
“Treadmilling”
A
- When addition at + end of microfilaments is equal to removal at - end
10
Q
The nature of polymerization of cytoskeletal proteins - end vs + end (Actin)
A
- Actin monomers bound to ATP are added to the plus end of the growing filament.
- Actin-ADP monomers are lost from the depolymerizing minus end.
- Actin exhibits dynamic instability called “treadmilling”
- There are many proteins that control the polymerization and depolymerization of the actin cytoskeleton
- > There are proteins that bundle the cytoskeleton in order to make thicker fibers
- > There are also proteins that allow the fibers to interact with membranes
11
Q
Arp2/3 complex
A
- Regulates the initial steps of making a new fiber (nucleation)
- One of the slow steps in generating a new fiber
12
Q
Thymosins
A
- Regulate binding to the monomers to reduce the amount of free monomers in solution
- Increases the rate of depolymerization
13
Q
Tropomodulin
A
- Regulates binding to the ends of the polymers to stabilize them and inhibit growth
14
Q
Profilin
A
- Promote the extension of polymers
- Acts like a nucleotide exchange factor by exchanging ADP- for ATP-bound monomers
15
Q
Cofilin
A
- Interact with the fiber and promote depolymerization
- Can speed up the hydrolysis of ATP
16
Q
Gelsolin
A
- Can cut the fiber to decrease the viscosity
- Also provides more ends for depolymerization and polymerization to happen
17
Q
Fimbrin
A
- Crosslink the cytoskeleton to provide structural stability
18
Q
Rho-GTP
A
- Acts as a molecular switch to control actin polymerization dynamics by regulating the activity of actin-binding accessory proteins
- Participates in actin bundling to create bundled stress fibers
- Stress fibers contain actin/myosin
- Helps cells respond to stretching and compression events (stress)
- There’s regulated control of the activation of this GTPase
19
Q
Rac-GTP
A
- Participates in actin polymerization
- Creates lamellipodia (sheet-like plasma membrane projections) and membrane ruffles
- There’s regulated control of the activation of this GTPase
20
Q
Cdc42-GTP
A
- Regulates similar proteins as Rac-GTP and Rho-GTP
- Creates filopodia (tube-like plasma membrane projections) and short cell protrusions called microspikes
- There’s regulated control of the activation of this GTPase
21
Q
Microtubules Form:
A
- A network of rigid tubules that radiate through the cytoplasm of all eukaryote cells
- Mitotic spindles of dividing cells
- The core of motile appendages: cilia and flagella
- Not distributed everywhere but radiate from the cell
22
Q
Microtubules are formed from:
A
- Heterodimers alpha and beta tubulin
- Diverse family of soluble, globular proteins
- Approx. 50 kDa M.W.
23
Q
Tubulin Structure
A
- Both alpha and beta tubulin bind GTP
- Only beta tubulin hydrolyzes GTP
- Tubulin heterodimers polymerize into protofilaments, which assemble into tubules (microtubules)
- 13 protofilaments are needed to form a full microtubule (forms a hollow structure with a lumen inside)
24
Q
Tubulin Structure #2
A
- Beta tubulin faces the positive end while the alpha tubulin faces the negative end
- To form a microtubule, first a protofilament must be formed, which is single polymerized chain that contains repeating beta and alpha subunits
- Can extend or make the microtubule smaller by adding or removing the alpha and beta subunits
25
The nature of polymerization of cytoskeletal proteins - end vs + end (Microtubules)
- The plus end contains more GTP-bound dimers than the minus end
- The plus end of the microtubules grows by the addition of tubulin dimers bound to GTP.
- With time the GTP is hydrolyzed to GDP.
- The minus end of the microtubules contains more GDP and grows more slowly.
- Tubulin dimers are lost (slowly) from this end.
- Normally the rate of polymerization at the plus end is more rapid than the rate of GTP hydrolysis so the plus end maintains a GTP cap (tubulin-GTP dimer).
- If the rate of GTP hydrolysis exceeds the rate of polymerization, the GTP cap is lost and the plus end undergoes rapid depolymerization (catastrophe)
- When there’s enough GTP in the plus end, more GTP can bind more rapidly (rescue)
26
Microtubule Regulation: Stability
- The growth or shrinkage of microtubules can be regulated by altering the balance between the addition and removal of tubulin dimers
27
Stathmin
- Binds microtubule subunits and prevents their assembly
28
Kinesin 13
- Interacts with tubules and enhances diassembly
29
Katanin
- Severs microtubules
30
MAP (Microtubule associated protein)
- Binds alongside tubules and stabilizes them
31
XMAP
- Stabilizes the positive end of tubules
32
+TIPS
- Associate with positive ends of tubules and links them to other structures (e.g. membranes)
33
Microtubule Regulation: Orientation
- How is it that microtubules assemble in specific locations and orientations in a cell?
- Assembly of microtubules has two phases:
- > Nucleation – small portion of tubule formed at the beginning
- > Elongation - addition of tubulins and the GTP-cap
- Microtubule Organizing Centers (MTOC’s) play a role in nucleation
34
MTOC: Classic Example - Centrosomes
- The pair of centrioles in a centrosome are at right angles to each other.
- Each centriole contains nine fibrils in a pinwheel pattern, each composed of three microtubules A,B,C
- One found in each animal cell.
- Divides before cell division begins.
- A complex structure: Two barrel-shaped centrioles & Surrounded by electron-dense centrosome matrix (CM)
- In addition to alpha and beta tubulins, centriole microtubules contain a special ring of gamma tubulins at their ends that allows nucleation of new microtubules
- Large numbers of microtubules converge onto the centrioles
35
Myosins
- Are a huge family of motor proteins that bind to actin microfilaments.
- Family can be divided into “conventional” type II myosins and “un-conventional” myosins (14 types of the latter)
36
Myosin II structure
- Heteromer with 6 polypeptide chains – one pair of heavy and two pairs of light chains
- Each heavy chain contains:
- > An “S1” head w/ATP-ase activity
- > A “neck” region
- > A coiled-coil tail
37
Myosin II
- Myosin II molecules can associate into filaments
- These filaments are highly stable in muscle cells, they form a basic structural unit of the contractile machinery.
- In non-muscle cells, myosin II filaments are only formed transiently, as needed to move elements of the cytoplasm around
- Myosin II can move actin filaments by attaching to actin filaments, moving (the “power stroke”) and then detaching
38
Kinesins
- The first kinesin was isolated as a motor protein responsible for moving vesicles and organelles along nerve axons from the cell body to the synaptic terminals.
- > Composed of 2 light chains and two heavy chains.
- > Heavy chains are entwined to create a stalk region made of coiled alpha-helices.
- The two globular “heads” of the heavy chains:
- > Have ATP-binding sites.
- > Bind to microtubules initiating ATPase activity and the movement of the kinesin molecule (and cargo) along the microtubule
- > Movement of kinesin only occurs in one direction from the (-) to the (+) end of the microtubules.
- > How is this?
39
Kinesins #2
- The cargo is bound to the other end of the stalk, the tail region that also contains the light chains.
- Cargos may include vesicles, protein complexes and organelles, which are bound to specific members of the kinesin family by adaptor proteins
40
Dyneins
- Another family of microtubule based motor proteins
- Dimer of heavy chains with two ATP-binding “heads” and a stalk.
- Intermediate and light chains surround the stalk. The light chains can be bound to a cargo through a complex of proteins called dynactin
- Dynein molecules move along microtubule protofilaments in a similar manner to kinesin, but from the (+) end to the (-) end of the microtubule
41
Intermediate Filaments
- Composed of a group of related long helical proteins
- Provide mechanical strength to cells
- Not present in every cell type, or even every eukaryotic organism
- Filaments form via coiled-coil interactions of a-helical proteins
- Proteins first form dimers, which assemble in staggered fashion to form ropelike filaments
42
Studying the Cytoskeleton: Manipulation
Various drugs can bind cytoskeletal proteins and affect their assembly/disassembly dynamics
43
Latrunculin
- Binds G-actin molecules and prevents assembly of filaments
44
Phalloidin
- Binds F-actin filaments and prevent disassembly
45
Cytochalasins
- Binds positive ends of filaments and supresses filament dynamics
46
Nocodazole
- Binds tubulin dimers and prevents microtubule assembly
47
Taxol
- Stabilizes microtubules and prevents disassembly
- Can be used to inhibit cell division
- Chemotherapy (cell division of other cells also affected --> hair loss)
48
Colchicine
- Copolymerizes into the microtubule latice, which suppresses microtubule dynamics
