Cytoskeleton: #2

Exam 3

Detailed structure of the 9+2 axoneme found in cilia and flagella

* All microtubules of the axoneme have the same polarity: their __plus ends__ are at the __tip of the projection__ and their __minus ends__ are at the __base__.
* __Each peripheral doublet is made of one complete microtubule__, the __A tubule__, and __one incomplete microtubule__, the __B tubule__, the latter containing __10 or 11 subunits rather than the usual 11__.
* ==Basic structure:==
* 9 Doublets and a pair of singlets

Important features of the 9+2 axoneme found in cilia and flagella

* The dynein (motor protein) arms are “fuzzy” projections from the wall of the complete microtubules
* When looking at the axenome of a protist, you can see the structure of the microtubular fibers, the __two types of dynein arms__ (3-headed outer arms and 2-headed inner arms), __the nexin links between the boulets, the central sheath surrounding the central microtubules, and the radial spokes projecting from the outer doublets toward the central sheath.__
* ==Detailed components==
* Nexin (elastic type protein-stretching) links connect doublets around the perimeter and keeps the stable together
* Central sheath surrounds the central microtubules
* Radial spokes from outer doublets to the central sheath
* Incomplete (A tubule) vs complete (B tubule) doublet

Structure of ciliary dynein

* 3 Heavy chains
* Heads with stalks sticking out that bind B tubule/microtubule

Function of ciliary dynein

(motor protein) cilia move by sliding adjacent microtubular doublets relative to one another. The dynein arms act as swinging cross bridges that generate the forces needed for ciliary/flagellar movement


1. __Step 1__: The dynein arms anchored along the A tubule of the lower double attach to the binding sites on the B tubule of the upper doublet.
2. __Step 2__: The dynein molecules undergo a conformation change/power stroke which causes the lower doublet to slide toward the basal end of the upper doublet.
3. __Step 3__: The dynein arms are detached from the B tubule of the upper doublet.

__Step 4__: The arms agave reattached to the upper doublet so that another cycle can begin

Function of ciliary dynein: step 1

The dynein arms anchored along the A tubule of the lower double attach to the binding sites on the B tubule of the upper doublet.

Function of ciliary dynein: step 2

The dynein molecules undergo a conformation change/power stroke which causes the lower doublet to slide toward the basal end of the upper doublet.

Function of ciliary dynein: step 3

The dynein arms are detached from the B tubule of the upper doublet.

Function of ciliary dynein: step 4

The arms agave reattached to the upper doublet so that another cycle can begin

How does the structure change during dynein activity?

* Each of the 3 heavy chains forms a prominent globular head with an extension (stalk) that functions in linking the dynein arms to the neighboring doublet
* ==Dynein motor function==
* The head rotates, so the stalk moves to the left as it rotates 
* Connect, rotate, pull; (as if it walks on the microtubule, but actually pulling the microtubule)- rotary motor
* Cargo microtubule is stable while the track microtubule pulls

Sliding microtubule mechanism of ciliary/flagellar motion

* When the cilium is straight, all the outer doublets end at the same level (center). Cilium bending occurs when the doublets on the inner side of the bend slide beyond those on the outer side (top and bottom).
* The movement of the dynein arms is responsible for the sliding of neighboring microtubules.
* Mechanism of movement
* Connenct to A
* B moves to the left and A moves to the right
* Pulls and disconnect; beat from one direction to another

What are intermediate filaments?

* Strong, flexible, ropelike fibers that give mechanical strength to cells that are subjected to physical stress, including neurons, muscle cells, and the epithelial cells that line the body’s cavities.
* Group of different filaments that are all somewhat similar in size, so they can group together in size
* All of these have a structural role and contribute to the structure

Where do you find the intermediate filaments of keratin?

__Found__ in the epithelial cells (including epidermal cells, liver hepatocytes, and pancreatic acinar cells)

How do intermediate filaments function in keratin?

__Function__: Keratin-containing IFs can form a network that serves as a scaffold for organizing and maintaining the cellular architecture and for absorbing the mechanical stresses applied by the extracellular environment.

Where do you find the intermediate filaments of neurofilaments?

__Found__ in the neurons of central and peripheral nerves

How do intermediate filaments function in neurofilaments?

__Function__: Once the nerve cell has become fully extended, it becomes filled with __neurofilaments__ that give support as the axon increases dramatically in diameter. Otherwise, aggregation of NFs can be seen in neurodegenerative disorders.

Where do you find the intermediate filaments of desmin?

__Found__ in the muscle

How do intermediate filaments function in desmin?

__Function__: Key structural role in maintaining the alignment of the myofibrils of a muscle cell, and the absence of these IFs makes the cells extremely fragile. (ex of absence: an inherited disease called desmin-related myopathy)

Where do you find the intermediate filaments of lamin?

Found in all cell types as well as the inner lining of the nuclear envelope; book does not mention function

Assembly of intermediate filaments

1. __Step 1__: Each monomer has a pair of globular terminal domains separated by a long a-helical region.
2. __Step 2__: Pairs of monomers associate din parallel orientation with their ends aligned to form dimers.
3. __Step 3__: Depending on the type of intermediate filament, the dimers may be composed of identical monomers (homodimers) or nonidentical monomers (heterodimers). Dimers in turn associate in an antiparallel, staggered fashion to form tetramers
4. __Step 4__: Which are thought to be the basic subunit in the assembly of intermediate filaments. (ex: 8 tetramers associate laterally to form a unit length of the intermediate filament)
5. __Step 5__: Highly elongated intermediate filaments are then formed from the end-to-end association of these unit lengths
6. Step 6: Once formed, intermediate filaments undergo a process of dynamic remodeling that is thought to involve the intercalation of unit lengths of filament into the body of an existing filament.
* ==Process?==
* Monomer, dimer, tetramer, unit
* ==How are new units put it?==
* Pre-existing filament disassociates and a new one comes in
* Turnover within this filament
* Tetromers go in and out; a new one will replace it; doesn’t grow out from the ends, but comes out from the middle


* ==Intermediate filament synthesis==
* Add to the middle; grow off of spots in the middle

**Describe the structure of microfilaments including the protein subunits** and how they are put together to form the filament

1. Actin filaments are 8 nm in diameter and __made of globular subunits of the protein actin__ (most abundant protein in most cells). In the __presence of ATP, actin monomers polymerize to form a flexible helical filament__. 
2. As a result of its subunit organization, an actin filament is a __2-stranded structure with 2 helical grooves running along its length__
3. __All of the monomers within an actin filament are pointed in the same direction, resulting in a polar filament with so-called barbed (plus end) and pointed ends (minus end).__
* ==Microfilament structure==
* Protein subunits
* Globular subunits of the protein actin; actin monomers
* F-Actin vs G-Actin (individual globular monomers)
* Structure of filaments
* Two strands with a helical structure
* ATP binding cleft (minus end); pointed end vs polar barbed (plus end)

Describe the structure of microfilaments including the protein subunits and **how they are put together to form the filament**

* A proteolytic fragment of myosin (called __S1) binds tightly and “decorates” the sides of actin filaments__. When __S1 fragments are bound, one end of the actin filament appears pointed__ like an arrowhead, while the __other end is barbed.__
* ==Decorating actin with myosin==
* Actin filament can be decorated with myosin (motor protein) which binds to actin; myosin sticks to actin
* S1 piece stick to myosin and has an orientation (pointed/barbed)

How are microfilaments assembled? Include at which end of the filament assembly occurs and the role of ATP. How or where are they diassembled?

1. Step 1: Start by addition performed actin filaments (seeds) to a solution of actin in the presence of ATP
2. Step 2: As long as the concentration of ATP-actin monomeres remains high, subunits will continue to be added at both ends of the filament
3. Step 3: As the monomers int he reaction mixture are consumed by addition to the ends of the filaments, teh concentration of free ATP-actin continues to drop until a point is reached where net addition of of monomers continues at the barbed end, which has a lower critical concentration of ATP-actin, but stops at the pointed end, which has a higher critical concentration for ATP-actin.
4. Step 4: As filament elongation continues, the free monomer concentration drops further. At this point, monomers continue to be added to the barbed ends of the filaments, but a net loss of subnits occurs at their pointed end.
5. Step 5: As the free monomer concentration falls,a points is reached where the 2 reactions at opposite ends of the filaments are balanced, so that both the lengths of the filaments and the concentration of free monomers remain constant
6. ==What is treadmilling?==

Step 6: Since subunits are being added to the barbed end and removed form the pointed ends of each filament at steady state, the relative position of individual subunits within each filament is continually moving: a process called treadmilling

Microfilament assembly (summary)

* ==Which end grows?==
* Add to plus and lose on the minus end
* ==Role of ATP?==
* Energy to attach
* ==How are they disassembled?==
* ==Treadmilling?==
* Add to plus and lose on the minus end: doesn’t really grow, but stays the same length

What is the function of myosin?

1. One type of ATp-dependent motor protein that interacts with actin filaments. Myosins are divided into two broad groups: conventional (type II) myosins and unconventional myosins.
2. All myosins share a characteristic motor (head) domain. The head has a site that binds an actin filament and a site that binds and hydrolyzes ATP to drive the myosin motor. Meanwhile, the head domains of different myosins are similar, the tail domains are highly divergent.
3. It has 2 pairs of light chains. The 2 filaments that Myosin forms are made of many myosins connected with alternating directions.

Describe in detail the structure of a conventional myosin and discuss its functions

1. Primary motors for muscle contraction
2. Are needed for splitting a cell into 2 during cell division, generating tension at focal adhesions, cell migration, and the turning behavior of growth cones
3. Each myosin II molecule is made of 6 polypeptide chains- one pair of heavy chains and 2 pairs of light chains
4. ==The fibrous tail portion of a myosin II molecule plays a structural role allowing the protein to form filaments==


1. (1) One pair of globular heads that have the catalytic site of the molecule
2. (2) One pair of necks, each consisting of a single, uninterrupted a-helix and 2 associated light chains
3. (3) One, long, rod-shaped tail formed by the interwining of long a-helical sections of the 2 heavy chains
4. Myosin II molecules assemble so that the ends of the tails point toward the center of the filament and the globular heads point away from the center. As a result, the filament is bipolar, indicating a reversal of polarity at the filament’s center.

Function of unconventional myosin

1. Unlike muscle myosin, this smaller unconventional myosin had only a single head and was unable to assemble into filaments in vitro
2. None of the unconventional myosins are capable of filament formation and instead appear to operate primarily as individual protein molecules
3. Involved in organelle transport
4. Ex: myosin I; myosin I often serve as a cross-link between actin filaments of the cytoskeleton and the lipid bilayer of the plasma membrane. Suggestions have been made that myosin I can exert tension of the plasma membrane, which could play a role in processes that need movement/deformation of the membrane

What are actin binding proteins?

1. Proteins that interact with actin filaments which determine the behCapping proteins regulate the length of actin filaments by binding to one or the other end of the filaments, forming a cap that blocks both loss and gain of subunits. Avior within the cell
2. Effect the localized assembly or disassembly of the actin filaments, their physician properties, and their interactions with one another and with cellular organelles.

What are the 8 basic categories of actin-binding proteins?

1. Filament nucleating
2. Monomer-sequestering
3. End-blocking (capping)
4. Monomer polymerizing
5. Depolymerizing
6. Cross-linking and bundling
7. Filament-serving
8. Membrane binding

Filament nucleating

* Slowest step
* Requires 3 axon monomers that come together in proper orientation to start the formation of the polymer. Actin nucleators enhance the rate at which polymers are formed from actin monomers.  Arp ⅔ is activated by binding to the side of an actin filament and to an activating protein (causes 2 Arps to adopt a conformation providing a template in which actin monomers can be added). Formins (nucleating proteins) track with barbed end even as new subunits are inserted at that site. Can promote very rapid elongation of the filaments that they help make.

Monomer sequestering

Thymosins are proteins that bind to actin-ATP monomers that prevent them from polymerizing. They are responsible for high concentration of monomeric actin in non muscle cells. Because of their ability to bind monomeric actin and stabilize the monomer pool, changes in the concentration, or in the activity can SHIFT the monomer-polymer equilibrium in particular regions of a cell.

End-blocking (capping)

Capping proteins regulate the length of actin filaments by binding to one or the other end of the filaments, forming a cap that blocks both loss and gain of subunits. If the barbed end of a filament is capped, depolymerization may proceed at the opposite end which results in the disassembly of the filament. I the pointed end is also capped, depolymerization is blocked. The thin filaments of striated muscle are capped at their barbed end at the Z line by a protein called capZ and at their pointed end by the protein tropomodulin.

Monomer polymerizing

Profilin is a small abundant monomer-binding protein that binds to the same site on an actin monomer as does thymosin. Prolifin promotes the growth of actin filament by attaching to an actin monomer and catalyzing the dissociation of its bound ADP, which is replaced with ATP. Profilin-ATP-actin monomer can then assemble onto the free barbed end of a growing actin filament, which leads to the release of profilin

Depolymerizing

Members of the cofilin family of proteins (including cofilin, actin-depolymerizing factor (ADF), and depactin bind to actin-ADP subunits present within the body and at the pointed end of actin filaments. Has 2 apparent activities: it can fragment actin filaments, and it can promote their depolymerization at the pointed end. Play a role in the rapid turnover of actin filaments at sites of dynamic changes in the cytoskeletal structure. Essential for cell locomotion, phagocytosis, and cytokinesis.

Cross-linking and bundling

Cross-linking proteins are able to alter the 3-D organization of the population of actin filaments. Cross-linking proteins can alter the 3D organization of a population of actin filaments. Promote the formation of loose networks of filaments interconnected at near right angles to one another. These networks allow for an elastic gel that resists local mechanical pressures. Other cross-linking proteins have a more globular shape and promote the bundling of actin filaments into tightly knit, allowing them to act as a supportive internal skeleton for these cytoplasmic projections.

Filament severing

These proteins have the ability to bind to the side of an existing filament and break it into 2. Also, promote the incorporation of actin monomers by making additional free barbed ends, or they may cap the fragments they generate.

Membrane binding

The forces generated by the contractile proteins act on the plasma membrane, causing it to protrude outward or invaginate inward. These activities are generally facilitated by linking the actin filaments to the plasma membrane indirectly, by the attachment to a peripheral membrane-binding protein. Ex: the incorporation of short actin polymers into the skeleton of erythrocytes and the attachment of actin filaments to the membrane at focal adhesions and adherens junctions.

Outline the steps of cell locomotion over a substratum. How is actin assembly/disassembly and myosin pulling on actin involved in this process

* Protrusion extends>attaches to substratum>most of the cell moves over this new attachment>the rest of the cell attaches to the back which disconnects and is pulled forward to the rest>actin filaments attach to myosin heads which are then polymerized
* __Step 1__: Shows the protrusion of the leading edge of the cell in the form of a lamellipodium
* __Step 2__: Shows the adhesion of the lower surface of the lamellipodium to the substratum, an attachment that is mediated by integrins residing in the plasma membrane. The cell uses this attachment to grip the substratum.
* __Step 3__: Shows the movement of the bulk of the cell forward over the site of attachment, which remains relatively stationary. This movement is accomplished by a contractile (traction) force exerted against the substratum.
* Step 4: Shows the cell after the rear attachments with the substratum have been severed and the trailing portion of the cell has been pulled forward

Cell locomotion over a substratum: step 1

Shows the protrusion of the leading edge of the cell in the form of a lamellipodium

Cell locomotion over a substratum: step 2

Shows the adhesion of the lower surface of the lamellipodium to the substratum, an attachment that is mediated by integrins residing in the plasma membrane. The cell uses this attachment to grip the substratum.

Cell locomotion over a substratum: step 3

Shows the movement of the bulk of the cell forward over the site of attachment, which remains relatively stationary. This movement is accomplished by a contractile (traction) force exerted against the substratum.

Cell locomotion over a substratum: step 4

Shows the cell after the rear attachments with the substratum have been severed and the trailing portion of the cell has been pulled forward

Discuss how actin and microtubules are involved in the cell shape changes necessary for the formation of the neural plate and neural tube

1. Microtubules elongated the cells and contraction of the microfilaments caused the cell to shrink at one end. This leads the cells to curve the layers inward.
2. The outer (ectodermal) cells situated along the embryo’s dorsal surface elongate and form a tall epithelial layer called the __neural plate__


1. The cells of the neural plate elongate as microtubules become oriented with their long axes parallel to the cell’s axis
2. After elongation, the cells of the neural epithelium become constricted at one end causing them to be wedge-shaped and the whole layer of cells to curve inward.
3. This change in cell shape is brought about by the contraction of a band of microfilaments that assemble in the cortical region of the cells just beneath the apical cell membrane.
4. Eventually, the curvature of the neural tube causes the outer edges to contact one another, forming a cylindrical, hollow tube