Eukaryotic Cell Structure, cont.

CYTOSKELETON

An intricate network of protein filaments that extends throughout the cytoplasm of all eukaryotic cells. Network of filaments helps to support the large volume of cytoplasm in a eukaryotic cell--especially important in animal cells with no cell wall. ABSENT in prokaryotic cells.

 Highly dynamic, continuously reorganizing.

 Provides machinery for intracellular movements as well as large scale movements such as amoeboid motion, contraction of muscle cells, beating of cilia and flagella and changes in cell shape.

Built on a framework of three types of protein filaments:

intermediate filaments - assembled from a family of different fibrous proteins

microtubules- assembled from tubulin subunits

actin filaments- assembled from actin subunits

 

Features:

• ~10nm in diameter [Intermediate in diameter between actin filaments (7nm) and microtubules( 25nm)]
• great tensile strength
• enable cells to withstand mechanical stress
• play basically a structural role by providing mechanical strength
• toughest and most durable of the three types
• Intermediate filament proteins - include a variety of proteins expressed in different types of cells.
• Intermediate filaments are like ropes with many long strands twisted together.

 

 Cytoplasmic intermediate filaments can be grouped:

• Keratin filaments - in epithelial cells
• Vimentin and Vimentin-related filaments - in connective tissue cells, muscle cells, white blood cells, and supporting cells of the nervous system
• Desmin filaments - specifically expressed in muscle cells
• Neurofilaments - major intermediate filaments of mature neurons - critical role in support of sometimes very long axons.
• Lamin filaments - Whereas cytoplasmic intermediate filaments are like ropes, the intermediate filaments lining and strengthening the inside surface of the inner nuclear membrane are organized as a 2 Dimensional mesh. NUCLEAR LAMINA

Features:

• Long, relatively rigid, hollow rods ~25nm in diameter [thickest cytoskeletal elements]
• composed of a single type of globular protein known as tubulin
• dynamic
• continually undergoing assembly and disassembly

Functions:

•  Microtubules determine cell shape and play a role in a variety of cell movements:
• -cell locomotion by cilia and flagella
• -the intracellular transport of organelles
• -separation of chromosomes during mitosis

Structure:

• Microtubules are built from subunits- molecules of tubulin.
• Each subunit is a dimer composed of two similar tubulins (alpha tubulin and beta tubulin) bound tightly together by noncovalent bonding.
• The tubulin subunits are then stacked - to form the wall of a hollow cylindrical microtubule.
• The microtubule appears as a cylinder with 13 protofilaments [each a linear chain of tubulin subunits made with alpha and beta tubulin alternating along its length.

Polarity:

• Alpha tubulin exposed at one end of microtubule and beta tubulin exposed at the other. Same for all protofilaments so microtubule itself has polarity.
• One end (beta tubulin end) plus end
• Opposite end (alpha tubulin end) minus end
• This polarity is important in determining the direction of movement of motors such as Kinesin and Dynein along microtubules.
• Microtubules in most cells extend outward from a microtubule-organizing center in which the minus ends of the microtubules are anchored. In animal cells, the major MTOC is the centrosome.
• Centrosomes contain gamma tubulin. Gamma tubulin appears to play a key role in microtubule nucleation. Gamma tubulin rings serve as a start site or nucleation site for the growth of one microtubule.
• The centrioles, also found within the centrosome have no role in the nucleation of microtubules and their function remains somewhat of a mystery. [plant cells lack centrioles]

 Microtubule assembly and disassembly

• In a living cell there is a mixture of microtubules and free tubulin subunits.
• The mitotic spindle itself is maintained by a continuous balanced addition and loss of tubulin subunits.
• Microtubule will grow if subunits are added more quickly to plus end than they are removed from the minus end (treadmilling)

 Dynamic Instability

Individual microtubules alternate between cycles of growth and shrinkage. This dynamic behavior is termed dynamic instability. This behavior stems from the intrinsic capacity of tubulin subunits to hydrolyze GTP.

Each free tubulin dimer contains one tightly bound GTP molecule that is hydrolyzed to GDP shortly after the subunit is added to the microtubule.

GTP associated subunits pack tightly together while GDP associated subunits bind less strongly to one another.

When polymerization is proceeding rapidly, tubulin molecules add to the plus end faster than the GTP that they carry is hydrolyzed. A cap (the GTP cap) prevents depolymerization.

If the rate of polymerization slows, the GTP at plus end will be hydrolyzed to GDP. If this occurs, the GDP-bound tubulin will dissociate resulting in rapid depolymerization.  Now, the microtubule can depolymerize by loosing subunits from the plus end.

 Function of this behavior: the centrosome is continually shooting out new microtubules in an exploratory fashion in different directions and retracting them through depolymerization.

A microtubule can be prevented from disassembling if its plus end is stabilized by attachment to another molecule or cell structure. Analogy to fisherman "waiting for a bite".

 Mitosis

Microtubules must completely reorganize during mitosis resulting in the formation of a mitotic spindle. Duplicated centrosomes at beginning of mitosis move to opposite poles.

Microtubule motors and movements

• Two large families of motor proteins are the:
• kinesins
• dyneins
• These motor proteins power the variety of movements in which microtubules participate. [energy derived from repeated cycles of ATP hydrolysis]. They bind, release, and then re-bind to the microtubule. Kinesin moves along microtubules toward the plus end and dynein moves toward the minus end.
•  The movement of kinesin transports vesicles and organelles away from the cell body. Dynein moves organelles back in the opposite direction.
•  The transport of membrane vesicles and organelles through the cytoplasm is a major role played by microtubules.

 

Cilia and Flagella

Cilia and flagella are actually microtubule-based projections of the plasma membrane. They are responsible for the movement of a variety of eukaryotic cell.

 Cilia beat in a coordinated manner in a back and forth motion which functions to either move fluid over the surface of the cell or move the cell itself through the fluid.

 Flagella are much longer but very similar in structure. They have a more wavelike pattern of beating.

Structure:

•  The fundamental structure of both cilia and flagella is the axoneme.
• The axoneme is composed of microtubules and associated proteins.
• Microtubules are in a characteristic 9 + 2 arrangement with a central pair of microtubules surrounded by 9 outer doublet microtubules. Outer doublets are connected to central pair of microtubules by radial spokes.
•  Two arms of dynein are attached to each A tubule and the motor activity of these dyneins drives the beating of cilia and flagella.
•  The minus ends of the microtubules are anchored in a basal body which is similar in structure to centriole. It contains 9 triplet microtubules and is known to play a clear role in organization of the axoneme microtubules. Each of the outer microtubule doublets of an axoneme is formed by extension of the two microtubules present in triplet in the basal body. Thus, the basal body serves to initiate the growth of axonemal microtubules as well as anchoring cilia and flagella to the surface of the cell.
•  Movements of cilia and flagella result from the sliding of the outer microtubule doublets relative to each other, powered by motor activity of dynein. It is not known how dynein action is coordinated.

Structure:

• Thin, flexible filaments approximately 7nm in diameter, several micrometers in length
• particularly abundant beneath the plasma membrane
• many more actin filaments in a cell than microtubules
• Composed of subunits of actin.
• Actin represents 20% of total cell protein in muscle cells.
• Actin monomer [globular G actin]
•  Individual monomers polymerize to form F actin (filamentous actin).
• Filaments have a distinct polarity with a an end designated as the plus end and an end designated as the minus end.

Assembly and Disassembly:

Polymerization begins with the formation of small aggregates consisting of 3 actin monomers. ------NUCLEATION

 Filaments then grow by adding monomers to both ends. The plus end typically grows at a rate 5-10X faster than the minus end. The filament is inherently unstable and can also disassemble from both ends.

 The monomers bind ATP. ATP is then hydrolyzed to ADP shortly following assembly. The ATP is not required for the polymerization. As with the GTP of tubulin, hydrolysis of bound ATP to ADP reduces the strength of the binding between monomers and decreases the stability of the polymer.

 Polymerization is reversible [depolymerization occurs when necessary].

 The equilibrium between monomers and filaments is dependent upon the concentration of free monomers in the cell.

 Assembly and disassembly is regulated in part by actin binding proteins

Actin binding proteins
thymosin - is the most abundant actin binding protein

• Its role is to sequester actin monomers preventing them from being assembled into filaments.
• profilin - has the same activity. However, it can promote monomer incorporation into filaments by stimulating the exchange of bound ADP for ATP.
• capping proteins - bind to the ends of actin filaments and prevent the loss or addition of actin monomers.

-Organization of Actin Filaments

• actin filaments rarely occur in isolation in the cell, generally found in:
• actin bundles
• filaments are cross-linked into closely packed parallel arrays
• actin networks
• filaments are loosely cross-linked in orthogonal arrays that form a 3D meshwork with the properties of semisolid gels.
• Actin Bundling Proteins
• fimbrin- has 2 adjacent actin-binding domains allowing it to hold 2 parallel actin filaments closely together.
• a-actinin serves as a cross-linking protein and binds to actin as a dimer. Because it is a dimer, the filaments are `40nm apart as opposed to 14nm apart. The increased space allows the motor protein myosin to interact with the actin filaments- enabling them to contract.
• ACTIN NETWORKS Actin networks are held together by large actin binding proteins such as filamin
• Filamin binds as a dimer of 2 (280kda) subunits.
• It is a flexible V-shaped molecule with actin-binding domains at the ends of both arms. When it is bound it forms cross-links between orthogonal actin filaments. This network underlies the plasma membrane and supports the surface of cells.

 Functions of Actin

 Cell crawling depends on actin

The formation of filopodia and lamellipodia both depend upon actin polymerization.

 lamellipodia- thin, sheet-like extensions of the plasma membrane

filopodia-thin, stiff protrusions of the plasma membrane

Both are exploratory, motile structures that form and retract with great speed. Both are generated by rapid local growth of actin filaments, which are nucleated at the plasma membrane and push out the membrane without tearing.

 In addition,  most cells have specialized regions of the plasma membrane that form contacts with adjacent cells, tissue components, or other substrates.

 These regions also serve as attachment sites for bundles of actin filaments that anchor the cytoskeleton to areas of cell contact.

 The sites of attachment - focal adhesions - also serve as attachment sites for large bundles of actin filaments that are termed stress fibers.

 Stress fibers - are contractile bundles of actin filaments cross-linked by alpha-actinin. These bundles anchor the cell and exert tension against the subsratum. They are attached to the plasma membrane at focal adhesions via interactions with integrin.

 The actin cytoskeleton is similarly anchored to regions of cell-cell contact called adherins junctions.

 This attachment is mediated by a family of proteins known as adherins.

ACTIN ALSO PLAYS A MAJOR ROLE IN MUSCLE CONTRACTION