Lecture 11 Flashcards
What is the function of intermediate filaments?
They are strong, but flexible cytoskeleton components that provides mechanical support for metazoan (multicellular) animals
What would happen if the intermediate filaments were expanded 1 million times?
They would resemble a 10mm braided plastic rope
Are intermediate filaments found in plants?
They are not found in plants, but are in prokaryotes (organisms without a defined nucleus)
Why did they name the filaments “intermediate”?
They were first identified as “intermediate” sized based on their thickness in skeletal muscle (between the thick [myosin] and thin filaments [actin])
Why are they called intermediate in todays world?
Now, they are most commonly referred to as intermediate in diameter (~10nm) as compared to actin filaments (~6nm) and microtubules (~25nm)
Are intermediate filaments found in vertebrate cells?
Yes, although they are in most vertebrate cells, their function was not know until 1991!
What are intermediate filaments involved in?
Mechanical support and it is also found in the nucleus (at interphase), lining the inside of the nuclear envelope.
How do intermediate cells provide mechanical support?
They do this by attaching to 2 different types of cellular junctions:
1) Desmosomes- transmits forces between cells (adjacent cells)
2) Hemidesmosomes- transmits forces between cell and the extracellular matrix
3) The Junction- intermediate filament network provides tissues their mechanical integrity
How many intermediate filament proteins are there?
There are over 70 different genes in humans, and they are grouped into 6 classes. There is expressed selectivity in different cell types (depends on where you are and what types of cells you have).
What is the structure of intermediate filaments?
1) The monomers of IFs have an alpha helical coiled coil “rod-like” core and variable N and C terminal domains .
2) The N and C domains as well as their amino acid sequences give the IFs their unique features.
3) 2 monomers form POLAR coiled-coli dimers.
4) Anti-parallel staggered tetramers from 2 (adjacent) coiled-coil dimers form APOLAR protofilaments.
5) 2 protofilaments form a protofibril.
6) 4 protofibrils make a 10nm IF.
Is the entire filament polar or apolar?
The whole filament is non-polar, but the dimer is polar! The protofilaments are non-polar.
Is it known how IFs assemble in vivo?
No, there are no known nucleating, capping sequestering or severing proteins.
Are intermediate filaments stable?
Intermediate filaments are very stable because their subinits exchange very slowly (eg. in minutes to hours during cell division).
What can intermediate filaments resist?
They can resist high temperatures, high salt concentrations and detergents.
Can intermediate filaments act as tracks for moecular motors?
Intermediate filaments do not act as tracks for molecular motors, they can be transported as cargo by molecular motors on other tracks (Actin filaments of microtubules)
Where do post-translational modifications occur?
Post-translational modifications (mainly phosphorylation) occur at many sites on intermediate filament proteins.
What are some variable effects of post-translational modifications?
1) Oten destabalization of the intermediate filaments and block their assembly.
2) Usually occurs during mitosis.
3) eg. A specific protein kinase that control parts of the cell cycle (Cdk1-CyclinB) phosphorylates 2 sites flanking the rod domain of one of the nuclear intermediate filaments (called laminin)
4) This disrupts the head to tail overlap needed for elongation and lateral associaion of the filaments.
What do neurofilaments do?
Neurofilaments (intermediate filaments in neurons) work in the opposite manner. Phosphorylation stabalizes the filaments by binding to their lage C-terminal ends.
How many nuclear lamins do all animal cells have?
All animal cells have at least 1 of the 3 types of nuclear lamins.
Do the cytoplasmic intermediate filaments vary amongst cell type?
Yes. Most have 1 or 2 different classes of intermediate filaments proteins.
Examples:
1) Epithelial cells: Class 1 and Class 2 keratins
2) Muscle cells: Desmins
3) Mesechymal cells (stem cells): Vimentin
Do the cytoplasmic intermediate filaments vary amongst cell type?
Yes. Most have 1 or 2 different classes of intermediate filaments proteins.
Examples:
1) Epithelial cells: Class 1 and Class 2 keratins
2) Muscle cells: Desmins
3) Mesechymal cells (stem cells): Vimentin
What is so unqiue about tumor cells?
Tumor cells often express alot of intermediate filament protein based off of the differentiated cells that they arose from (useful in diagnosing metastatic tumors).
What is hair made from?
Hair is made of keratin intermediate filaments. They are chemically cross-linked to each other.
What is hair associated with?
Hair is associated with matrix proteins. Through disulfide bonds and amide bonds between lysines and acidic residues, lots of cysteines (~14% of hair is cysteine).
What do you smell when you burn hair?
Sulfur. It creates a tough material that is able to be modified (that’s what hair stylists do)
How can the crosslinks be modified?
The crosslinks can be modified during “permanents” (Perms). First reduce the disulfide bonds then reform them into a new shape.
What are the 2 main classifications of keratins?
1) alpha-keratin
2) beta-keratin
What is alpha-keratin?
Structure= primary alpha-helices.
Includes hair, nails, horn, claws, hoovers, mammalian skin, hagfish slime.
What is beta-keratin?
Structure= beta sheets.
Includes nails, shell, feathers, beaks, reptile skin.
How does classification between the two keratin types occur?
1) The structure of the keratins being used
2) Disulfide bridges (and other bonds)
3) The amount of water absorbed
4) Other matric proteins change the characteristics of the final products resulting in stronger or more flexible structures
What is the acidity/basicity of kertain based off of?
Their amino acids
How was the function of intermediate filaments shown?
The generation of a mutant mouse that has a truncated (shortened) form of keratin-14 in the basal layers of its skin epidermic by Elaine Fuchs.
What were the results of this mutant mouse?
The mouse had extensive skin blistering following mild trauma. Many mice died as neonates. The phenotype resembled epidermolysis bullosa simplex (EBS). Then, sequencing of keratin 14 in EBS patients showed a mutation in the conserved arginine 125 residue.
Are intermediate filaments elastic?
Yes, to a point!
Can intermediate filaments stretch?
Unlike actin filaments and microtubules that break when bent or pulled more than 1% of their size, individual intermediate filaments can stretch ~33% of their size and return to their native states…like rubber bands.
Can intermediate filaments stretch to 80%?
They can stretch more (over 80%), but at tht point, alpha-helices turn into beta-sheets and do not recoil.
How much can intermediate filaments to stretched until they break?
A single intermediate filament can be stretched/deformed 250% before breaking.
How much can intermediate filaments stretch in whole cells?
In whole cells, stretched ~133% of their size, the intermediate filaments return to their native states. Beyond that, they buckle.
What are the largest family for intermediate filament binding proteins?
Plakins, including a large protein family called Plectin. All have plakin domains or plakin repeats (or both) in the proteins.
What is Plectin?
It is in most tissues, but not neurons. It binds intermediate filaments, actin and microtubules.
What do vimentin-based intermediate filaments do?
In embryonic tissues, vimentin-based intermediate filaments stabalize the positions of organelles and enhance the elastic behaviour of cells.
What organelles do cytoplasmic intermediate filaments associate with?
Nucleus
Mitochondria
Golgi apparatus
What does binding organelles to cystoplasmic intermediate filaments do?
Binding to organelles can control the organelle’s shape and position.
What do cytoplasmic intermediate filaments directly bind to?
Cytoplasmic intermediate filaments directly bind to the nuclear envelope through envelope proteins that form the “Linker of the nuceloskeleton to the cytoskeleton” (LINK) complex
Can intermediate filaments bind indirectly?
Yes, intermediate filaments can also bind indirectly to the nucleus through microtubule or actin-associated proteins that bind to the LINK complex.
What is Nesprin-3 and what can it do?
Other proteins like Nesprin-3 (an outer nuclear membrane protein) can bind to intermediate filaments through plectin
What binds to mitochondria?
Keratin, veminetin, desmin and neurofilaments. These influence mitochondrial distribution (they act of anchoring structures) and their metabolic function. An example of this is that Vimentin binds to mitochondria through plectin 1b (found on the outer membrane of the mitochondria)
What do mitochondria in Vimentin KO cells do?
The cells are more motile and mitochondrial shapre, memrbane potential and ATP production are all altered.
What kind of function do Plectin 1b KO cells have?
They have oddly shaped mitochondria, but have normal mitochondrial function.
What happens when actin-based motility intermediate filaments are altered at the wound edge (skin)?
Keratins 6,16, and 17 are all increased, but Keratins 1 and 10 are decreased.
Do tumour increase when actin-based motility intermediate filaments are altered at the wound edge (skin)?
Invasive tumors often have increased levels of intermediate filament proteins.
When an epithelial transforms from a cell in a tissue to one that will migrate during cancer that is an epithelial to mesenchymal transformation (EMT). Vimentin in a marker of EMT.
What does intermediate filament depletion cause?
Intermediate filament depletion usually causes a decrease in the speed of cell movement.
What increases the migration of breast-cancer cells?
Keratin 14. The effects of other keratins on cell migration is cell-type specific.
What does keratin 19 do?
Keratin 19 increases invasion of hepatocellular cancers, but decreases invasion by breast cancer cells.
How can intermediate filaments influence cell migration?
Yes, lamellipodia are actin-rich structures, but keratin and vementin-based intermediate filaments are also within them as well as at the trailing regions.
What can mutations to intermediate filaments (eg. Keratin) or the junctions that they dock into cause?
It can cause blistering diseases
What is Epidermolysis Bullosa Simplex?
Results in extensive blistering and sensitivity to mechanical stress. Skin rips off during birth
Can most animal surive without Keratin-8 and -18?
Most animal without Keratin-8 or -18 die embryologically, but if they survive they have few problems (only modest problems with their colons and liver)
Do Desmin KO mice survive?
Desmin KO mice survive, but will die with vigorous exercise.
What happens do humans with Desmin mutations?
Humans with Desmin mutations usually have general muscle failure.
What do actin and microtubules use for their polymerization?
Nucleotides (ATP and GTP), but they evolved independently
How did the actin gene come about?
The common ancestor of life must have had an actin gene ~3 billion years ago, however that ancestor and that gene are gone
What 2 homologous domains does the actin monomer have?
The actin monoer has 2 homologous domains that form the cleft for Mg-ATP binding
What is the size comparison of the actin precursor to the current actin?
It is thought that the actin precursor was likely 1/2 the size of our current actin. There was likely gene duplication of that gene that made the actin of today.
Do genes for actin exist in prokaryotes (bacteria and archaea)?
Yes
What is actin like in prokaryotes?
Their sequences have diverged, but structurally they are similar.
What is the evolution of eukaryotic actin?
Eukaryotic actin is formed by 2 strands aligned in the same direction and form a helix, some prokaryote actin is single stranded… others are double stranded, but with strands in opposite directions. Eukaryotic actin came from an organism related to the archaean called Lokiarchaeota. That mystery organism is also thought to have given rise to Eukaryotes as a whole. Further duplication and divergence of actin genes have rise to many of the actin -related proteins (eg. Arp 1,2,3)
What do Bacteria and Archaea have in common?
Bacteria and Archaea both have a protein that is similar in function to tubulin, but has essentially no sequence similarities. The protein is called FtsZ.
What is FtsZ?
It has the same fold as tubulin.
What do monomers of GTP-FtsZ form?
Monomers of GTP-FtsZ do not form tubules, but do form protofilaments.
What does GTP hydrolysis by FtsZ cause?
It causes the protofilaments to dissassemble.
Do Eurkaryotes have the FtsZ gene?
Yes, eukaryotes have the FtsZ gene, which came from the symbiotic relationship with bacteria that formed chloropplats and mitochondria in eukaryotic cells
What is FtsZ involved in, and what is it not involved in?
It is involved in the division of chloroplasts, but not mitochondria.
Is there much known about intermediate filaments evolution?
There is much less known about intermediate filament evolution.
What is known about the intermediate filaments evolution?
What is known is that the genes are scattered throughout the phylogenetic tree and have been acquired and lost over the pas billion years
Do bacteria have intermediate filament?
Some bacteria have intermediate filament (eg. Caulobacter crescentus has cresentin).
