Intermediate Filaments

Cell Biology

How many classes of intermediate filaments are there?

5 classes

Light Description: List the IF classes, their proteins, and their locations

• Class I: Acidic Keratins, epithelial cells

• Class II: Basic keratins, epithelial cells

• Class III:

  • Vimentin, Mesenchymal cells

  • Desmin, muscle cells

  • Glial Fibrillary Acidic Protein (GFAP), glia

  • Peripherin, PNS neurons

• Class IV: Neurofilament proteins (NF-L, NF-M, NFM, NF-H, alpha-internexin), neurons

• Class V: Nuclear lamins (A, B, C), nuclear lamina

Describe the structure of intermediate filaments: monomer to Dimer to TETRAMER to Unit Length Filament

Intermediate filaments: made of long alpha coiled-coil loops with a middle intermediate, not globular

They intertwine and stack on each other longitudinally (unlike actin and mts)

Dimer: Organizes C-Terminuses together

Tetramer: Two dimers joint at opposite ends

ULF (intermediate): 32 monomers (8 tetramers) bind together antiparallel to ensure the entire filament is nonpolar

Full IF: These bind together to form chemically identical ends.

How are IF polymers different from microtubules and actin filaments?

• There’s no NTP binding/hydrolysis

• Non-polar/chemically identical ends

  • No motor proteins travel along IFs

• Initial nucleation is slower

• Subunits exchange along the length instead of along the edge

What is vimentin?

Another type of intermediate filament that fits in between intermediate filaments

What is the elementary piece of a Intermediate filament? What is it’s analogue in actin and mts?

IF: the tetramer

Actin: one globular protein

MT: Alpha-Beta tubulin dimer

How do you depolymerize intermediate filaments

Phosphorylation: by introducing phosphates anywhere along the length of the filament causes disassembly

Why is it necessary that IFs depolymerize depolymerize during mitosis?

• they’re too interconnected to everything and too stretchy and adaptable to pulling stress

Order the flexibility of cytoskeletal strflexibleMostucturers from most felxible to least

Most: IFs: will not break easily under stress, rather it will extend

Middest: Actin: wiggly but not super strong

Lowest: is microtubule: girl will snap like a pretzel the moment you pull

Who is the Plakin family

These are cross-linkers. The region next to the n-terminus is full of alternatively spliced regions that can create different structures for binding. They allow IFs to integrate into the entire cytoplasm

• Full of famous girls like plectin (a ubiquitous protein), BPAG & desmoplakin (both are epithelial cell localized)

Who is plectin? Describe her structure

Plectin is a major IF that links longer IFs to actin

• Isoform-specific domain=N-terminus: localized plectin to a particular organelle or cell

• Actin binding domain: links IF to actin

• Variable alternative splicing creates different binding structures

• Coiled-coil domain: stretchy tail of plectin

• IFBD: Intermediate filament binding domain.

What is the MAIN FUNCTION of IFs?

Increasing the mechanical strength of cells and tissues

Describe the structure and composition of Class I & II IFs

Acidic Keratins and Basic Keratins:

They dimerize with each other in epithelial cells, and then form acid-base tertamers

What do keratin intermediate filaments attach to

To the nuclear envelope and to desmosomes (cell-cell adhesion structures between epithelial cells)

How is keratin anchored at intermediate filaments?

Intermediate filament←c-terminus of desmoplakin←plakoglobin & plakophilin (adaptor proteins)← bound to specialized cadherins that hold cells together

What is a hemidesmosome?

The region binding a cell and extracellular filaments. It’s not mediated by adherins, but by integrins.

What happens if BPAG or plectin mutate in their location?

If they mutate in the epithelia, the epidermis will detach from the lower layers of skin, creating terrible blisters. Because they bind to laminins (laminal proteins), they are bound by intergrin.

List the Class III IFs and their localizations

• Vimentin: Mesenchymal and undifferentiated cells (but can also be expressed in differentiated cells… WHEN THEY’RE CANCEROUS!)

• Desmin: muscle cells, localized by alpha actinin, so in z-disks

• Glial fibrillary acidic protein (GFAP) in glia

• Peripherin in PNS neurons

What does desmin do?

They’re IFs that wrap around myofibrils, in order to organize the sacromere. They can wrap z-disks and mitochondria, for example, to keep them away from damaging contractile filaments and keep an ATP source close

What are costamers?

Linkers of IFs to the extracellular matrix. They link to DGCs: dystrophin-glycoprotein complexes. They serve as cytoplasmic attachement sites

How do mutations in desmin affect muscle cells?

A loss can lead to muscle cells over-contracting in myofibrils, leading to tears in filaments and damage

Name and localize Class IV IFs

Neurofilament proteins (NF-Light, NF-Medium, and NF-Heavy), localized in neuronal tissues

HOW do neurofilament proteins polymerize

The long tails form coil-coil dimers. Every dimer must be at least 50% NFL. NFLs can dimerize with themselves, but the other NFs must join

Describe the structure of NFs

• All three have n-terminus heads and coil rod sections of the same time.

• C-terminus heads have tails of light, medium, and heavy length that all very.

What do NFs do thanks to their structure?

• Their coil tails dimerize and then form anti-parallel tetramers to create lateral projections in neurofilaments.

• They’re surrounded by protofibrils, and the tails that stick out interact with other filaments. They can thus form bridges with other NFs

What are the functions of NFs

1. Their stretch and recoil provide structural integrity to the non-stagnant axons that are deformed by muscle acfivity

2. Increased IF density strengthens and improves the conduction of electrical signaling

   1. They thus define the diameter of axons

3. They strengthen long and thin neuronal processes

4. They integrate neurons to skin and extracellular surfaces.

Describe and localize the Class V IFs

Lamins: localized around the nucleus

Describe the connectivity between the ER and the nuclear membrane.

The outer nuclear membrane is continuous with the ER membrane like some sort of wrap, but the inner nuclear membrane is separate and lined with lamina to bind that to the cytoskeleton

How do lamins get localized into the nucleus?

Via nuclear localization signal on the very long C-terminus. This signals delivery through the treacherous nuclear pore.

How are lamin tetramers regulated?

They’re disassembled by phosphorylation into their dimers to allow the nuclear envelope to dissolve during mitosis.

Describe the structures of lamins?

They dimerize as long coil-coils with intermediate space, and have long streamy c-terminal tails that house nuclear localization signals. Their dimers form tetramers that for netlike webs in the inner nuclear membrane

Who are the binding partners of lamina?

Lamina bind to SUN-domain intermembrane proteins, who are bound to KASH-domain intermembrane proteins in the perinuclear space.

Who are the binding proteins of KASH-domain proteins?

SUN-domain proteins (who are bound to nuclear lamina or chromosomes

In the cytoplasm, KASH-domain proteins can specialize and bind to

• motor proteins cargo domains, who’s motor heads are BOUND to microtubules or centrosomes

• plectin (for stabilization)

• actin

BRIEF functions of lamina

• nucleus stability

• nucleo-cytoplasmic coupling

• distribution of nuclear pores

Yk we needed a label question…

A - Chromatin

B - ER membrane

C - ER Lumen

D - nuclear envelope

E - Inner nuclear membrane

F - Outer nuclear membrane

G - Perinuclear space

H -  nuclear lamina

I - Nuclear pore complexes

J - Links from lamina to cytoskeleton

Describe the VAGUE structure of the nuclear pore

• They have 8-fold pyramidal symmetry to form the basket

• actual complexes that build into the nucleus

All nuclear pores are built the same! SO HOWWW are they built

• The nuclear basket extends into the inner nucleus

• Cytosolic fibrils extend out into the cytosol

• The disordered region of channel nucleoporins 

• scaffolding nucleoporins hold them to the side

• The nuclear envelope holds the width of the pore

The nucleoporin is DENSE: let’s talk about light descriptions of objects rolling in: how big are they

It’s a maze molecules must travel and fit through. No molecules over 40nm in length can pass through freely

What is the nucleus import signal?

• A short, positively charged 

  • Lys, Lys, Lys, Arg, Lys (KKKRK)

What is the export nuclear code?

• Shortish, hydrophobic sequences 2, 3, and 3 random amino acids apart.

  • Met, X,X, Leu, X,X,X, Leu, X, X, X, Phe (MXXLXXXLXXXF) 

How do mutations in localization signals affect movement?

Even just one mutation can prevent nuclear localization.

Describe nuclear localizations in terms of T-Antigens

T-Antigens stop DNA transcription and start viral transcription. They’re from viruses, and they’re the mechanism by which they hijack the cell’s DNA replication. For those proteins to even enter the nucleus, however, they require the nuclear import signal of KKKRK (or several positive amino acids in a row) to enter through the nuclear pores.

What are kariopherins?

Nuclear transport receptors of the Importin Beta family: they bind to nuclear localization signals on cargo proteins and then carry them through nuclear pores. If a protein does not have her own kariopherin, an adaptor protein with a localization signal is then carried into the pore via karioprotein while being attached to cargo.

How do cargo proteins know to release cargo in the nucleus if they’re not pH mediated?

They’re Ran-GTPase gradient mediated

• inactive Rans exists in the cytoplasm, while active Rans exist in the nucleus

  • GEF for RanGTPase is always bound to chromotin, and can only change Ran’s comformation when it gets in

  • GAP for RanGTPase is always bound to the nucleofibrils of the cytoplasmic side of the nucleoporin. 

Why can’t the nucleus be pH mediated

Hydrogen protons are so motherfreaking small. As such, the protons would just equilibrate through the nucleopores.

Who are exportin and importin?

Kariopherins (nuclear transport receptors) that IMPORT INto the cell cargo proteins and export old proteins out

Describe (in detail) Ran-Dependent directionality of nuclear transport into the nucleus

1. Importin binds to EITHER cargo or Ran-GTP

2. In the nuclear space, Ran-GTP bound outcompetes the cargo (its at high concentration) and is then attached to importin to leave via the pore

3. There, Ran encounters its GAP on the nucleoporin fibrils and exchanges it with GDP and stays in the cytoplasm

Ran-GDP bound cannot cannot bind to importins.

There is No Ran-GTP-Bound GTPase in the cytosol

Describe (in detail) Ran-Dependent directionality of nuclear transport into the nucleus

1. Exportin must bind to BOTH Ran-GTP-Bound GTPase and cargo, or NOTHING, before carrying them out to the cytoplasm

2. When the complex then goes outside of the nucleus, it will interact with the GAP on the nucleoporin fibril.

3. Cargo gets delivered to the cytosol and the Ran, which cannot bind without being bound to GP

What are FG repeats?

Phenylalanine-Glycine repeats on the nucleoporincomplex fibrils. They bind to FG-binding sites on importin and exportin to help them get through the nucleoporin maze

What cation signals an immune response from the nucleus.

Calcium

Who does calcium activate in beginning an immune response?

calcineurin, a protein phosphatase

Who is NF-AT

Nuclear factor of activated T-cells: a transcription factor who intitiates gene transcription as an immune response.

What does NF-AT, (nuclear factor of activated T-cells) look like in it’s inactive form?

A protein that is bound to 3 phosphates with it’s nuclear export signal exposed while it’s in the cytosol

Who does calcineurin dephosphorylate?

NF-AT (nuclear factor of activated T-cells). This dephosphorylation exposes its nuclear import signal while the calcineurin then binds to the nuclear export signal.

Describe the full process for shuttling proteins in an immune response:

1. High Calcium ion concentration activates calcineurin

2. Calcineurin dephosphorylates NF-AT and binds to the nuclear export signal, blocking it 

3. The nuclear import signal is also then exposed, allowing NF-AT to bind to importin and enter through the nuclear pore

4. It will then activate transcription

5. Calcineurin will then dissociate in the low calcium concentrations, exposing the export signal and phosphorylating NF-AT again for transport

6. Exportin can then bind and take it out

Cyclosporine A inhibits calcineurin. How does this inhibit the shuttling protein pathway.

Inhibition prevents the dephosphorylation of the NF-AT, which will therefore prevent its entrance back into the nucleus. This will prevent gene transcription and whatever cell growth we wanted to create.

What are the FUNCTIONS of neurofilaments?

1. shock absorber (for damage when they move)

2. providing elasticity to the neuron and strengthening the long length

3. Appropriately distributing neuronal organelles along the axons

4. conducting electrical signals

5. integrating the neuron to extracellular surface ie skin