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Lecture 16 Ch 9 Cytoskeleton II Intermediate Filaments and Microfilaments
Terms in this set (22)
Compare and contrast the physical characteristics of microtubules, microfilaments and intermediate filaments (Table 9.1)
Describe the general structure of intermediate filament proteins (including the basic filament unit)
Intermediate filaments are fibrous proteins that terminate in globular domains.
+ There are 5 different families:
- Acidic keratin
- Basic keratin
- Vimentin and others
They are different because they have diverse amino acid sequences (difference sin primary structure).
- Central, rod shaped, alpha helical domain of similar length flanked on each side by globular domains of variable size.
- Thus, they have similar tertiary structure, that is fibrous in nature. Dimers and tetramers of fibrous proteins (long and extended)
- They have the same polar shape, being an extended fibrous protein, there is an N and C terminus with globular domains (not a globular protein) - Specificity- of what bind to the intermediated filaments is based on N and C globular domains sticking outwards from the long fiber. Which allows intermediate filaments to hook to something (Other IF's, microtubules, actin microfilaments). A molecule that can do that connection role is Plectin.
- Intermediate filament resist the force of stretching. They don't tare when they are pulled on because they are strong, fibrous proteins. (VERY STRONG)
- There are more than 70 genes (divided into 5 classes)
When two monomers wrap around one another to form a dimer, they do so in a polar way so if your asked, "Is the intermediate polar?", well the monomer is polar, and the dimer is polar, but the polarity disappears when a tetramer (two dimers aligned) is made. When two dimers align together, they align in an antiparallel fashion, thus the two ends look identical and it is no longer a polar molecule. These tetramers will align to make a basic unit of intermediate filaments, but they are non-polar so full length of IF is nonpolar. And one-unit link of filament will join with other links of filaments to form full intermediate filament. When they do, IFs are generally 10 nm in diameter (smaller than microtubules but larger than microfilaments).
Basic unit of filament (Intermediated filament)
8 tetramers align and create it
Relate the subcellular distribution and the molecular structure of intermediate filaments to their functions. Identify the role of plectin in formation of the cytoskeleton.
Intermediate have flexile strength a, especially in cells that experience mechanical stress. Multiple binding domains connect and support the cytoskeleton scaffold.
Plectin is responsible to crosslink IFs to:
- Other Ifs
- Actin microfilaments
If it extends from the nuclear envelope, it is most likely for 3D cell support.
If it is a neurofilament, then it is for neural support.
Lamins support the underneath of the nuclear envelope
Compare and contrast the assembly of intermediate filaments with that of microtubules and microfilaments (directionality, order, energy requirements, etc)
Directionality and energy requirement
IF are non-polar so it cannot serve as a good molecular highway since they don't have polarity/ no directionality. Microtubules as well as microfilaments are polar.
To create these filaments doesn't require that the monomers be bound to GTP or ATP. To create microtubules or microfilaments, you need GTP or ATP.
Size:Microtubules are the largest (25), intermediate filaments are the second largest ( 10-12nm) and microfilaments are the smallest (8nm)
New monomers are added to the growing polymer of microtubules and microfilaments at one end or the other. But in IF, new filaments can be added internally.
Polymerization and depolymerization in microtubules happen in the side while for IF it can happen in the middle. Microfilaments also polymerize and depolymerize.
All eukaryotic cells have microtubules and microfilaments
IF are specific to animals
All units are found in cytoplasm, but IF are the proteins that stabilize structure of nucleus. (cytoplasm and nucleus)
From their point of origination, MTOC, microtubules tend to go out in particular direction (directionality) which serves as a molecular highway. The microtubules are large and strong.
IF's run from space that is connected to ECM (Hemidesmosome and desmosomes) to anchor point for pulling two cells together (one cell connected to ECM and another cell) while also connecting the plasma membrane ECM or other cell to the nucleus. (Everything connected)
Identify the relevance of the phosphorylation status (phosphorylated/dephosphorylated) of intermediate filaments to their structure/function
Phosphorylation changes shape of a variety of proteins, and often an outcome of such change. Thus, what would be the result if incubating cells with an agent that leads to phosphorylation of keratin? They might fall apart; the keratin will be falling apart.
Intermediate filament assembly and structure is dependent upon the phosphorylation status of the filament unit. PHOSPHORYATION CAN CHANGE THE STRUCTURES THAT ARE MADE OF INTERMEDIATE FILAMENTS.
What if you incubated the cells with an agent that blocked GDP/GTP exchange on cytoskeletal elements?
Nothing, if it is only intermediate filaments or microfilaments However, if we were talking about microtubules, their incorporation/stability is depended on GTP/GDP exchange. Microfilaments on the other hand, are dependent on ATP/ADP exchange.
what are the three different types of IF that are most important?
Associate the intermediate filament families with their physiological roles:
Keratin- helps bind to hemidesmosomes and desmosomes from the nuclear envelope for 3D support.
Lamins- support the underneath of the nuclear envelope.
Neurofilaments- support microtubules of the axon
Extends from nuclear envelop to desmosomes and hemidesmosomesfor 3-D cell support. (This will be found in epithelial tissue - skin, lining of GI tract, lungs - which will be very strong. We need cells to be held very tightly accomplished, plasma membrane and the nucleus to other parts) - Epithelial cell strength and they also support cell and epithelial tissue integrity.
support microtubules in axonal extensions, because they can be too long, and we need stabilization. Thus, disturbance of neurofilaments is thought to contribute to neurodegenerative disease.
- When there are tangle of neurofilaments, it interferes with messages traveling through the axon: results in ALS (Amyotrophic Lateral Sclerosis).
- Thus, it may hinder axonal cargo trafficking
If we consider ropes from hemidesmosome, desmosomes to nucleus it will pull really hard, so we need to undergird the nuclear envelop with lamin. Support nuclear envelope.
Describe the fluorescence microscopy experiment that showed internal incorporation of filament proteins into existing intermediate filaments
OVERALL: Keratin was produced in a test tube, and labeled with a biotin. And compared the location of artificial keratin to the distribution to the endogenous naturally made keratin.
1. The gene for keratin was cloned
2. Keratin was then expressed and labeled in vitro
3. Labeled keratin (exogenous) was injected into cells
4. Cells injected with biotinylated keratin where incubated with anTi-bioton antibody to reveal location of newly incorporated keratin. Distribution of the labeled keratin in the cell was compared to distribution of endogenous keratin.
5. Anti-keratin antibodies revealed distribution of intermediate filaments: new filament units are internally incorporated
Make predictions about other experiments utilizing fluorescence microscopy to analyze effects on cytoskeletal proteins (in this, be able to interpret the meaning of yellow fluorescence when only red/green stains or tags are utilized)
Picture show images of epithelia cells that have been stained with fluorescent labeled antibodies to keratin 18 and keratin 8, both intermediate filaments.
A pattern is distinct for each keratin, but also similar where there is a linkage to the nuclear membrane. And then out from there keratins linking to various points of the plasma membrane to keep everything together.
In the picture where keratin 8 was depicted, keratins were well attached at the plasma membrane. As oppose to Keratin 18 who appeared to have more connection to the nuclear membrane
In both pictures, the plasma membrane has the color red. However, . given that keratin 8 was green and had multiple point of connection at plasma membrane, the overall depiction of the color at the plasma membrane was yellow.
What if you incubate the cells with an agent that leads to phosphorylation of keratins?
- They might fall apart. Keratin is falling apart due to phosphorylation
- Intermediate filament assembly structure is dependent on phosphorylation
- Phosphorylation can change structure that are made of IF
IF ASSEMBLY AND STRUCTURE IS DEPENDEN ON THE PHOSPHORYLATION STATUS OF THE FILAMENT UNIT
Relate microfilaments and intermediate filaments to adherens junction, desmosome, hemidesmosome and focal adhesion structures (Fig. 7.19, 7.16, 7.24, 7.25)
- INTERMEDIATE FILAMENTS: BIG CABELS HOLDING EVERYTING TOGETHER
- MICROTUBUES: PATHWAYS
- MICROFILAMENTS: FOR SMALL CHANGES THAT ARE OCURRING CLOSE TO THE PLASMA MEMBRANE OR FOR SHAPES NEAR THE PLASMA MEMBRANE
Focal Adhesions and adheres junctions. They connect ECM to integrin, and then integrin attached to actin (actin microfilament). Intermediate filaments connect the actin microfilament to nucleus (cytoplasm + nucleus).
Intermediate filaments (keratin) extend to hemidesmosomes and desmosomes, for cell support.
OVERALL:Intermediate filaments bind to desmosomes and hemidesmosomes from the nuclear envelope to help provide structure. At focal adhesions microfilaments are where the cell attached to the other surface
Offer examples of the roles of actin microfilaments in the cell and how the molecular structure supports these functions—use the terms helical, plus, minus, barbed, pointed, ADP, ATP, ATPase, S1 myosin
1) They can help cell break in cytokinesis, help with hearing, encourage muscle contractions, and help with organelle migration. Its structure is that of a globular helix protein with a growing + barbed end and a shrinking - pointed end. In order for the barbed end to grow, the proteins serve as ATPase and have ATP bind to them and hydrolyze ADP. This structure allows proteins to walk along them for cell transport, for them to grow out to help with hearing, to grow out and then pull together for cytokinesis, and also pull together for muscle contraction.
- Ex: linen of small of intestine, because it increases surface are due to the need of absorptions. Villi that make linen intestine is near cell surface made out of microfilaments.
- Cell cortex: just under the plasma membrane providing support for the plasma membrane.
- Focal Adhesions: connect ECM àintegrin àactin. ANCHORING
-Axonal outgrowth: Leading edge is made of microfilaments
-Vesicle transport path
-Stereocilia of cochlear hair cells that convert sound waves to signal
-Facilitates neural crest cell migration during embryonic development as well as immunocyte motility in inflammation - filopodia
Microfilaments are the main driver in non-flagellar, non-ciliary cell locomotion. HOWEVER, THE COOPERATION OF CYTOSKELETAL ELEMENTS OVERALL HELP!!!
Actin microfilament have an inextensible helicalform that is considered to be flexible. Suh helical filament has a plus(barbed) end, and a negative - minus(pointed)end. New actin monomers are added to the barbed (+) end 10x faster than the (-) end. However, Before the actin monomer is even incorporated into a filament, the actin monomer must be bound to a molecule of ATP. (ATP hydrolyzed to ADP at some time point after it is incorporated into the end of a growing. After ADP-actin is released from the polymer, ADP/ATP exchange must occur before reincorporation possible.) actin protein is considered to be an ATPase because hydrolysis ATP to ADP. S1 Myosinis what surrounds such actin filament.
Describe the dynamic and reversible process of actin microfilaments assembly (and disassembly) and relate to the function of actin microfilaments within the cell; relate the stability of microfilament segments to their binding to ATP versus ADP
While actin monomers can be gained or lost at either end (minus, pointed) or (plus, barbed). The plus end required a lower minimum concentration of ATP-actin than the minus end for polymerization.
o Conformationally different (different structure)
o Sometime after incorporation, monomer bound ATP is hydrolyzed to ADP
o Bulk of polymer is ADP-actin, expect the plus end
o Minus end become largely ADP-actin
§ Less stable as polymer
§ Lower affinity for ATP actin.
In addition, proteins can determine if the actin is:
· Stable (treadmilling) - one end is shrinking at the same rate it is growing
With adequate description, be able to identify which ends of microfilaments are capable of adding new actin monomers and compare the relative rates
Both ends are capable of adding new acting proteins, however the + or barbed end can add it 10x faster than the pointed or - end can.
This is because the plus end requires a lower minimum concentration of ATP-actin than the minus end for polymerization.
Relate the cell's energy status/availability of actin-ATP to the relative rate of actin polymerization at the plus (versus minus) ends of the microfilaments
Your microfilament at either end, can be growing, shrinking or stable (tread- milling).
- The end is shrinking as fast the other end is growing
The more ATP-actin, the more binding on both ends, but the + end would still be 10x faster than the - end. This is because the + end has a lower ATP concentration required to join.
After pressure is applied, keratin filaments provide a ring and spoke pattern that make the cell be connected.
Epithelium: responsible for protecting inside of the body
- Outside: Basal layer is attached to ECM where you find collagen (integrin--> laminin --> Collagen)
- Inside: integrin --> Plectin --> IF like keratin
Basal layer contains the more stem like cells and can divide and replace one another. Or if they divide and form daughter cells that are more differentiated they populate the upper layer of the skin (as you go up you are more differentiated) and as you go up you have a layer of no longer living cells, only sacs of IF. But they are still very connected to one another that even H2O cannot pass.
Without skin you don't have barrier to keep water in.
Wild-Type Mouse epidermis (To see expression of intermediate filaments in epidermal tissue)
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