lect 11: cytoskeleton

Question 1

what are vertebrate skeletons composed of? what does the human skeleton do?

Answer 1

vertebrate skeleton: composed of hardened elements (bones)
-human skeleton: supports the structure of our bodies and facilitates bodily movement

Question 2

what are the learning objectives?

Answer 2

-the basic elements and functions of the eukaryotic cell cytoskeleton
-the structure and functions of intermediate filaments (IF)
-the structure and functions of actin filaments (F-actin)
-actin-binding proteins
-cell motility
-muscle contraction in relation to F-actin

Question 3

what is the definition of the cytoskeleton?

Answer 3

cytoskeleton: “skeletal system” of eukaryotic cells is an intricate network of protein filaments that extends throughout the cytoplasm
-composed of a network of three filamentous structures
-together, they form interactive and dynamic network with different structures, properties and functions

Question 4

what are the three filamentous structures that the cytoskeleton is composed of?

Answer 4

-intermediate filaments (IF)
-actin filaments (F-actin or microfilament)
-microtubules (MT)

Question 5

what are the functions of cytoskeletal components?

Answer 5

1. provide structural support that determines cell shape
2. direct the movement of cargo and organelles within the cell
3. generate forces needed for cellular locomotion
4. position various organelles in the cell (spatial organization of cell organelles)
5. make up an essential part of the cell division machinery responsible for DNA separation

Question 6

what are intermediate filaments?

Answer 6

-intermediate filaments are strong, unbranched and ropelike
-IF strengthens cells against mechanical stress (epithelial, neurons, muscle)
-in specific cell types, such as epithelial cells, IF connect cells to other cells or the extracellular matrix via desmosomes or hemidesmosomes
-chemically heterogeneous group of structures (70 different genes)

Question 7

what is common intermediate filament (IF) architecture?

Answer 7

1. each monomer has a pair of globular terminal domains separated by a long fibrous alpha-helical region (polar)
2. pairs of monomers are associated in parallel orientation to form dimers (polar)
3. dimers associate in an anti-parallel, staggered fashion to form tetramers (nonpolar)
4. 8 tetramers associate to form a unit length of the IF (nonpolar)
5. these unit lengths associate with one another in an end-to-end fashion to form the elongated intermediate filament (nonpolar)

Question 8

what is the first part of intermediate filament architecture?

Answer 8

IF monomer is polar

Question 9

what is the second part of intermediate filament architecture?

Answer 9

IF dimer is polar

Question 10

what is the third part of intermediate filament architecture?

Answer 10

IF tetramer is nonpolar

Question 11

what is the fourth and fifth part of intermediate filament architecture?

Answer 11

IF is nonpolar

Question 12

what are the four major classes of intermediate filaments?

Answer 12

these classes can include numerous subtypes. Humans, for example, have more than 50 keratin genes

Question 13

what are keratin containing IFs? what are their functions?

Answer 13

keratin containing IF-structural proteins of epithelial cells
-IF radiate through the cytoplasm, tethered to the nuclear envelope in the center of the cell, and anchored at the outer edge of the cell by desmosomes and hemidesmosomes
-IF are also connected to microtubules and F-actin via plectins
-they organize and maintain cellular architecture and absorb mechanical stress

Question 14

what is epidermolysis bullosa simplex?

Answer 14

a mutant form of keratin makes skin more prone to blistering. The defective protein assembles with the normal keratins and thereby disrupts the keratin filament network in the skin

Question 15

what are neurofilaments (NFs)? what are their functions?

Answer 15

neurofilaments: bundles of IF located in the cytoplasm of mature neurons-oriented parallel to the axon to provide strength and stability
-aggregation of NFs is seen in several human neurodegenerative disorders such as parkinson’s disease
-these NF aggregated may block axonal transport on microtubules leading to neurons death

Question 16

what is the assembly and disassembly of intermediate filaments?

Answer 16

-none of the IF assembly steps require the direct involvement of either ATP or GTP
-instead, assembly and disassembly of IFs are controlled primarily by subunit phosphorylation and dephosphorylation (ex. phosphorylation of vimentin filaments by protein kinase A leads to their disassembly)
-once assembled, the subunits are not incorporated at one end of the filament but into the filament’s interior in a dynamic fashion

Question 17

what is the relationship between intermediate filaments and linker proteins?

Answer 17

-IF are often interconnected to other IF and cytoskeletal filaments by thin, wispy cross-bridges consisting of a dimeric protein called plectin (has various isoforms)
-each plectin dimer has a binding site for an IF at one end and a binding site for another IF, F-actin, or microtubule at the other end

Question 18

what is the structure of actin filaments?

Answer 18

-actin filament (F-actin, microfilament) is a thin and flexible helical filament composed of actin monomers

actin protein is:
-one of the most abundant proteins in almost all cell types
-a major contractile muscle protein

-F-actin is a two-stranded helix with a plus end and a minus end (polar filament)

Question 19

what is actin filament assembly and disassembly?

Answer 19

-actin filaments can grow by the addition of monomers at either end, but their rate of growth is faster at the plus end (10 times) than at the minus end
-before it is incorporated into a filament, an actin monomer binds an ATP molecules, and therefore, Actin is an ATPase
-the initial nucleation event in filament formation occurs slowly in vitro, whereas the subsequent stage of filament elongation occurs much more rapidly
-the ATP associated with actin monomer is hydrolyzed to ADP at some time after it is incorporated into the end of a growing filament

Question 20

what is the first and second step of actin assembly/disassembly in vitro?

Answer 20

1. preformed actin filaments (seed) are added in the presence of ATP-actin
2. if the concentration of ATP-actin monomers is very high, subunits are added to both ends

Question 21

what is the third step of actin assembly/disassembly in vitro?

Answer 21

3. as ATP-actin monomers are consumed by addition to the ends of the filaments the concentration of free ATP-actin drops, until a point is reached where net addition of monomers continues at the plus end by stops at the minus end

Question 22

what is the fourth step of actin assembly/disassembly in vitro?

Answer 22

4. as filament elongation continues, the free monomer concentration drops, such that monomers continue to be added to the plus ends of the filaments, but a net loss of subunits occurs at their minus ends

Question 23

what is the fifth step of actin assembly/disassembly in vitro?

Answer 23

5. a point is reached where the two reactions at opposite ends of the filaments are balanced, such that the rate of monomer addition (at the plus end) equals that of monomer loss (at the minus end), the filament remains the same length (assembly=disassembly)-> this leads to F-actin moving in the direction of assembly, known as ‘treadmilling’

Question 24

what is important in actin filament assembly and disassembly?

Answer 24

by controlling the assembly/disassembly, the cell can rapidly reorganize its actin cytoskeleton when and where is needed for dynamic processes such as:
-cell locomotion
-changes in cell shape
-phagocytosis
-cytokinesis

-assembly/disassembly rate can be influenced by several actin-binding proteins

Question 25

what are actin-binding proteins?

Answer 25

-patterns of F-actin in living cells include various types of bundles as well as crosslinked and branched networks (but not in vitro) -the organization and behavior of actin filaments inside cells are determined by the interaction of actin with a variety of actin-binding proteins

Question 26

what are the categories of actin-binding proteins?

Answer 26

actin-binding proteins can be divided into categories based on their presumed function in the cell: 1. nucleating 2. monomer-sequestering 3. bundling 4. end-blocking (capping) 5. cross-linking 6. filament-severing 7. actin filament depolymerizing

Question 27

what is essential in cell motility (crawling)?

Answer 27

the actin cortex is essential for cell locomotion -three interrelated processes are known to be essential for cell motility

Question 28

what are the three interrelated processes that are known to be essential for cell motility?

Answer 28

1. the cell sends out protrusions at its "front", or leading edge (by actin polymerization); 2. these protrusions adhere to the surface over which the cell is crawling (forming focal adhesions); and 3. the rest of the cell drags itself forward by traction on these points of anchorage

Question 29

what does the graph of polymerization of F-actin in cell motility (crawling) look like?

Answer 29

Question 30

what is the graph of what cell motility (crawling) looks like?

Answer 30

Question 31

what are myosins?

Answer 31

the molecular motors of F-actin -a protein superfamily of many classes generally divided into two broad groups: -myosin II: muscle cells (discovered first) -other myosins (such as myosin I): most other cells

Question 32

what is the function and structure of myosins?

Answer 32

-function as molecular motors that operates in conjunction with actin filaments -move towards the plus end of an actin filament (with one exception) -all myosins share a characteristic motor (head) domain -whereas the head domains of various myosins are similar, the tail domains are highly divergent the head domain contains two sites: -a site that binds an actin filament -a site that binds and hydrolyzes ATP to drive the myosin motor

Question 33

what is myosin I?

Answer 33

-the head domain of myosin I binds to an actin filament and has the ATP-hydrolyzing motor activity that enables it to move along the filament in a repetitive cycle of binding, detachment and rebinding -it moves towards the plus end of F-actin -the tail binds to vesicles or the plasma membrane leading to their translocation

Question 34

what are type II myosins?

Answer 34

-myosins II are primary motors for muscle contraction and also found in a variety of nonmuscle cells -these proteins form dimers, with two globular ATPase heads at one end and a single coiled-coil tail at the other -like most other myosins, myosin II heads bind F-actin and move toward the plus end of the filament

Question 35

what do myosin II consist of?

Answer 35

myosin II consists of: 1. a pair of globular heads that contain the catalytic site of the molecular (binds to ATP) 2. a pair of necks 3. a single, long, rod-shaped tail formed by the intertwining of long alpha-helical sections

Question 36

what does interacting F-actin and myosin II lead to?

Answer 36

muscle contraction -the fibrous tail of myosin II plays a structural role so it can form filaments -clusters of myosin II molecules binds to each other through their coiled-coil tails, forming a bipolar myosin filament -because they are bipolar, the myosin heads at the opposite ends of a myosin filament can interact and pull actin filaments toward one another

Question 37

what is muscle organization and contraction?

Answer 37

-a skeletal muscle fiber contains hundreds of cylindrical strands called myofibrils -each myofibril consists of a chain of contractile units called sarcomeres

Question 38

what is the organization of sarcomeres?

Answer 38

-sarcomeres are highly organized assemblies of two types of filaments: actin filaments (thin filaments) and myosin II filaments (thick filaments) -the myosin II filaments are centrally positioned in each sarcomere -the thin actin filaments extend inward from each end of the sarcomere and are anchored by their plus ends

Question 39

what is the sliding filament model of muscle contraction?

Answer 39

Question 40

what is the attached state of the sliding filament model of muscle contraction?

Answer 40

attached -at the start of the cycle shown in this figure, a myosin head lacking a bound ATP or ADP is attached tightly to an actin filament in a rigor configuration (so named because it is responsible for rigor mortis, the rigidity of death) -in an actively contracting muscle, this state is very short-lived, being rapidly terminated by the binding of a molecule of ATP to the myosin head

Question 41

what is the released state of the sliding filament model of muscle contraction?

Answer 41

a molecule of ATP binds to the large cleft on the "back" of the myosin head (that is, on the side farthest from the actin filament) and immediately causes a slight change in the conformation of the domains that make up the actin-binding site -this reduces the affinity of the head for actin and allows it to let go of the actin filament -the space drawn here between the head and actin emphasizes this change, although in reality the head probably remains very close to the actin

Question 42

what is the cocked state of the sliding filament model of muscle contraction?

Answer 42

the cleft closes like a clam shell around the ATP molecule, triggering a large shape change that causes the head to be displaced along the actin filament by a distance of about 5nm -hydrolysis of ATP occurs, but the ADP and inorganic phosphate (P) produced remain tightly bound to the myosin head -dashed lines show the position of the myosin head prior to ATP hydrolysis

Question 43

what is the rebinding and power stroke state of the sliding filament model of muscle contraction?

Answer 43

weak binding of the myosin head to a new site on the actin filament causes release of the inorganic phosphate produced by ATP hydrolysis -this release allows myosin to bind more tightly and triggers the power stroke-the force-generating change in shape during which the head regains its original conformation -in the course of the power stroke, the head loses its bound ADP, thereby returning to the start of a new cycle

Question 44

what is the attached state of the sliding filament model of muscle contraction?

Answer 44

at the end of the cycle, the myosin head is again bound tightly to the actin filament in a rigor configuration -note that the head has moved to a new position on the actin filament, which has slid to the left along the myosin filament