Lecture 3 - Intermediate filaments and microtubules Flashcards
Describe intermediate filaments
Large diversity in sequence and size (contrast to MFs & MTs)
5 major classes of intermediate filament proteins (Types I-V) based on sequence similarities
Lamins make up nuclear intermediate filaments (important for maintaining nuclear structure)
Keratins make up cytosolic intermediate filaments that extend to the cell membrane in skin epithelial cells (keratinocytes) (give these cells and hair, nails etc derived from them, tensile strength)
Describe keratins
(Type I, acidic keratins)
(Type II, basic keratins)
In outer epithelia
Prominent in skin, hair and nails
Heterodimers of basic and acidic subunits
Mutations involved in skin disease.
Usually form heterodimers to form intermediate filaments.
Fibrous proteins found in skin, hair nails and other epidermal keratincoytes (such as outer epithelial cells stained in colon section on the left of this slide) - staining of keratin often used as a way to identify outer epithelial cells in pathology allowing pathologist to orientate what they’re looking at
Describe vimentin
(Type III)
Widely distributed (stromal tissues, lymphocytes, endothelial cells, fibroblasts)
Supports cell membranes & keeps nucleus & organelles in position
Intermediate filaments containing vimentin play an important structural role in maintaining cell morphology and as such are highly expressed in stromal cells of supporting tissues such as endothelial cells surrounding blood vessels
Describe neuronal IF proteins
(Type IV)
Neurofilaments
Structural role in axons
Determine axon diameter & hence their speed of conduction
Type IV intermediate filaments proteins play an important structural role in axons – in particular determining and maintaining axon diameter which in turn controls the speed of nervous impulses along those structures. Left hand image shows parallel bundle arrangement of these filaments within axons.
Describe lamins
(Type V)
Fibrous network supporting inner nuclear membrane
Type V intermediate filaments are made up of lamins. These nuclear intermediate filaments form a network to provide support for the inner nuclear membrane – also appears to have a role in organising different types of chromatin and contribute to regulation of gene expression.
How are intermediate filaments assembled?
Intermediate filament proteins consist of a globular head and a globular tail (N- and C-termini respectively), separated by an extended alpha helical region.
Intermediate filaments wrap around each other to form parallel dimers, two of which then go head-to-tail to form an antiparallel tetramer. Antiparallel tetramers stack end-on-end to form protofilaments, which double up to form proto-fibrils. 4 proto-fibrils wrap around each other to form a ~10nm diameter intermediate filament.
Assembly of intermediate filaments does not require ATP or GTP but is rather a spontaneous process that assembles individual proteins into either homo or hetero polymers consisting of a central filament with the globular head and tail domains projecting away.
What are microtubules?
- largest diameter of the three types of cytoskeletal filaments.
- Polymers assembled from dimers of globular tubulins, arranged into a tube like structure.
- Like microfilaments, microtubules are also used to move components around the cell – e.g. separation of chromatids during mitosis, transport of organelles; MTs act as tracks for motor proteins (kinesins and dyneins which act in a manner akin to how myosins walk along microfilament tracks discussed in lecture 2).
Describe microtubule structure
The subunits that assemble into microtubules are heterodimers of tubulin with each heterodimer consisting of one alpha and one beta subunit.
Both alpha- and beta- tubulin bind GTP, but do so in different ways.
- alpha tubulin binds GTP in an irreversible manner - this GTP is not hydrolysed and is referred to as non-exchangeable GTP.
- beta tubulin binds GTP in a reversible manner and can hydrolyse its bound GTP to GDP, and then exchange that GDP for GTP.
GTP hydrolysis and GDP exchange by beta-tubulin occurs as the microtubule extends - note similarity with microfilament assembly where actin hydrolyses bound ATP when part of F-actin. Another similarity that microtubules have with actin microfilaments is that they have polarity-– arising from the way in which the tubulin dimers polymerise.
How are microtubules assembled?
An assembled microtubule: alpha tubulin with GTP bound, and the beta subunit with GDP bound. Note how the dimers are organised end-on-end. This assembly occurs in a stepwise process, firstly of dimers into protofilaments which then form sheets that curled into tubules.
Individual tubulin dimers assemble to form short protofilaments (similar nucleation of microfilament assembly). Several protofilaments then associate laterally into sheets which can also simultaneously extend longitudinally (through addition of more dimers to the assembled sheet).
The sheets then roll into hollow tubules (which again continue to extend longitudinally through the addition of more dimers).
Dimers can be added to both ends, but as with microfilaments the growth is usually faster at the plus end (dimers in the tubule are oriented the same way with beta tubulin at plus end). The GTP on the beta-tubulin is hydrolysed when the subunit is part of the tubule, so the closer a dimer is to the plus end of a tube the more likely it is to have hydrolysed its GTP and therefore have GDP bound.
How are microtubules organised in the cell?
Most MTs in a cell are anchored at their (-) end
Organising them into a microtubule organisation centre (MTOC).
Located near the nucleus, this directs assembly and orientation of MTs, direction of vesicle traffic and orientation of organelles.
In animal cells, the MTOC is the centrosome - made up of a pair of centrioles which themselves are built from short microtubules associated with other components.
Centrioles are associated with the pericentriolar matrix that contains proteins (e.g. gamma tubulin, pericentrin) that serve to anchor the minus end of microtubules allowing growth and shrinkage at the plus end (where the GTP caps form).
Not all microtubules are connected to an MTOC e.g. nerve cells with very long dendrites that receive nerve impulses from other neurons have stable microtubules.
How do microtubules function in mitosis?
Centrioles replicate during mitosis and are moved to opposite poles of the dividing cell. Microtubule dynamic instability plays a major role in separating out daughter chromatids (microtubule disassembly increases ~10 fold during this stage of the cell cycle).
What are the other types of MTOC other than the centrosome?
In addition to a centrosome cells with flagella have a MTOC (the basal body) to organize the microtubules found in that structure.
Describe how microtubules are used for transport
Microtubules are the cellular tracks for trafficking vesicles, organelles and chromosomes inside a cell.
They are used by microtubule motor proteins (kinesin and dynein) that play a role in transport/movement in the cytoplasm.
Work in opposite directions to each other.
Microtubules are used as transport tracks within the cell –in a similar way to microfilaments - in conjunction with motor proteins –kinesins and dyneins (rather than myosins that attach to actin microfilaments).
Kinesins and dyneins carry various structures along microtubules, but in different directions.
Kinesins carry cargo away from the microtubule organizing centre (anterograde transport)
Dyneins carry cargo towards the microtubule organizing centre (retrograde transport)
