Microtubules, Microfilaments And Intermediate Filaments

Front 1

Name the two proteins that form Microtubules.

Back 1

-Alpha tubulin
-Beta tubulin

Front 2

How are the proteins in microtubules arranged?

Back 2

-They form long protofilament strips in which alpha-subunits are bound to Beta-subunits in a polar fashion.
-13 protofilament strips form a hollow cylinder that is the microtubule assembly

Front 3

What is a catastrophe in microtubule assembly?

Back 3

-When the GTP at the "capped" (+) end of the microtubule is hydrolyzed to GDP and the microtubule begins to shrink
-This occurs normally in low concentrations of free tubulin
-Causes the microtubule to split like banana

Front 4

What property makes microtubules associate and dissassociate rapidly?

Back 4

-Dynamic instability

Front 5

What is required for Micro tubule assembly? How is this process revered?

Back 5

-Guanine triphosphate (GTP) is needed in order for tubulin dimers to bind
-It exists in the exposed E-site on the beta subunit which can be hydrolyzed and the alpha unit has a GTP nucleotide bound at a non-exchangable site

Front 6

Name some features of tubulin

Back 6

-Acidic (pI 5.1.-5.4)
-COntains many Asp and glutamine residues
-Has calcium ion binding sites
-can bind with MAPs

Front 7

How is Tubulin regulated?

Back 7

-Tyrosination of C terminus of alpha-tubulin is associated with growing MT
-Tyronsine addition is catalysed by tubulin-tyrosine ligase (increased in axon outgrowth or cell division)
-Tyrosine removal is catalysed by tubulin-tyrosine carbocypeptidase

Front 8

What is the function of the TAU protein?

Back 8

-TAU is a microtubule associated protein that maintains the microtubule stability by phosporylation of the tubulin
-It is only present in axons and not dendrites
-Anti sense RNA (opposite to mRNA) of TAU blocks polar growth

Front 9

Name a protein that destabilisizes the microtubule and how is it regulated?

Back 9

-Stathmin
-Regulated by phosphorylation of Ser and Thr amino acid residues on tubulin.

Front 10

Name some motor proteins associated with microtubules?

Back 10

-Kinesin is a plus end directed motor that walks towards axon synapse, it is an ATPase that moves mitochondria and membrane bound vesicles along the axon length
-Dyesin is similar except moves towards the minus end (cell body)

Front 11

Define the Blood brain barrier

Back 11

specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream while supplying the brain with the required nutrients for proper function

Front 12

What cells is the blood brain barrier comprised of?

Back 12

-Endothelial cells
-Pericytes
-Assoctiated glial cells

Front 13

Name 5 pathways in Blood-brain barrier transport

Back 13

-Paracellular aqueous pathway that transports water soluble agents through tight junctions between cells
-The transcellular lipophillic pathway transports lipophillic substances through cells
-Transport proteins carry glucose, amino acids and alike through cell
-Receptor mediated transcytosis carriers insulin and transferrin in vesicles through cell
-Adsorrptive transcytosis carries albumin and other plasma proteins through a cell

Front 14

What is the blood brain barrier defined as?

Back 14

-A specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream while supplying the brain with the required nutrients for proper function

Front 15

What is the BBB comprised of?

Back 15

-Endothelial cells
-Pericytes
-Tight junctions
-Basal lamina
-Astrocytic end feet

Front 16

What is the basal lamina?

Back 16

-The extracellular matrix that is in contact with both the endothelial cells and the astrocytic feet

Front 17

What are the pericytes ? What is the function of them in the BBB?

Back 17

- They are flat contractile cells which wrap around the endothelial cells of the venules and capillaries
-Lack alpha-actin isoform typical of contractile pericytes so debated on there role ion regulating blood flow to brain
-Have recently been found to produe ~84% of blood flow increase when sensory input is increased
-Stuck in constricted state during ischaemia (target for stoke damage)
-Are possibly of microglial origin.

Front 18

What limits the transport of substances into the brain?

Back 18

-Tight physical junctions and metabolic enzymes created by the endothelial cells , this forms the rate limiting factor for entry of many therpeutic drugs into the CNS

Front 19

List some of the features of the endothelial cells of the blood brain barrier?

Back 19

-Flattened cells which are anchored to the lamina densa (basal lamina) they attach to eeach other in a number of ways such as VE-cadherins, Occludins and claudins
-They express a large number of transport proteins when found in the BBB

Front 20

What are some fautures unique to the ccerebral capillary endothelial cells ?

Back 20

-They form tight junctions creating blood vessels that are seemingly impermeable
-Have protein bridges of occuldins and claudins for cell to cell junctions
-They interact with cytoplasmic proteins (such as the zona occludens )
-Is uniformly thick with few pinocytic vesicles or openings
-High number of mitochondria in to support active transport
-High levels of metabolic proteins which break down blood borne drugs and molecules before entering CNS, specifically neuroactive agent enzymes
-Have a polarity between the lumen and ablumenal surfaces

Front 21

Name five ways in which molecules can cross the BBB?

Back 21

-Through water in the paracellular aqueous pathway
-straight through cell in the transcellular lipophillic pathway
-Through transport proteins
-Through receptor mediated transcytosis
-Through adsorptive transcytosis

Front 22

What do molecules have to cross in order to get into CNS?

Back 22

-First pass through cappillary endothelium, then the cytoplasm of the endothelial cells then the albumenal membrane to the paricytes of lamina densa

Front 23

What diameter are the Microfilaments (MF) ? Where are they found?

Back 23

-5nM in diameter
-Found mainly in the growth cones

Front 24

How can microfilaments be inhibited?

Back 24

-By actin binding drugs such as phalloidin and cytochalasin

Front 25

What are microfilaments composed of ?

Back 25

-Actin core and actin binding proteins

Front 26

What are some key features of actin?

Back 26

-Highly conserved structure meaning it is highly optimised
-Is an enzyme that can bind ATP and hydrolyze it
-can be found in the isoform of alpha, beta and gamma
-Is a helical arrangment of subunits with a groove running along them
0Is a bilobal globular protein of 42 kDa

Front 27

How are the microfilaments regulated?

Back 27

-they are regulated via ATP hydrolysis,-Interactions with actin binding proteins that stabilise, destabalise, cross link filaments or cross link them with other cyctoskeletal elements or membranes e.g. cantitins

Front 28

What roles does microfilaments play in neurons?

Back 28

- They form the contractile ring, regulate growth cone advance and cell migration
-Is dependent on ATP hydrolysis and interactions with actin binding proteins

Front 29

Name an actin binding protein involved in enriching growth cones and increasing microfilament dynamics?

Back 29

-Actin destabilising factors of the cofilin family of proteins
-Regulated by ser phosphorylation

Front 30

How many types of intermediate filament is there?

Back 30

-there are 6 types
-1+2 are acidic and basic cytokeratins
-Type 3 contains glial fibrillary acidic protein (GFAP)
-Type 4 contains neurofilaments
-5 are the nuclear lamins
-Type 6 are the nestins (undifferentiated neural stem cells)

Front 31

List some common features of the intermediate filaments?

Back 31

-10nm in diameter with a alpha helical rod domain
-No nucleotide needed for assembly (unlike MF and MT)
-Very stable

Front 32

How many neuronal specific types of neurofilaments are there?

Back 32

- Heavy chains of around ~200 kDA (NFH
-Medium chain (~120 kDa) NFM
-Light chain (~68kDa) NFL

Front 33

How are neurofilaments isolated?

Back 33

-Taken from the spinal cord or sciatic nerve they are given an osmotic shock and homogenised
-This is the ultra-centrifugated (with a high sal conc.)
-It is then assayed using diagnostic reconstitution which requires it to be put in 8M urea where it is soluble, non-soluble proteins are centifuged and discarded.
-Dialysis is performed to then remove urea

Front 34

How are normal NFs structured?

Back 34

-with pure NFL forming the normal 10nm filaments
-Abnormal filaments are made up of purely of NFM or NFH
-It is a normal assembly when all three are present and NFM and NFH bind to the pure filament core of NFL

Front 35

List some of the properties and functions of NFs

Back 35

-Expression is increased during axon development
-Its phospphyraltion by kinase like map Kinase ERK 1/2 is increased along axons and during development
-Found mainly in the axon as seen with immunoflourscent staining
-Controls axon girth and stability
-NFH projects out from the filament core
-Highly expressed in mature neuron axons

Front 36

What is the structure and function of glial cell intermediate filaments ?

Back 36

- a 52 kDa protein that forms homopolymeric filaments of 10nm diameter .
-Forms a cytoplasmic network in astroglial cells and is regulated by phosphorylation of its N-terminal
-Exact role in astrocyte development is uunclear

Front 37

Why are the intermediate filaments given there name? What is there overall role in the neuron?

Back 37

-They are intermediate in diameter between MFs and MTs
-Play a structural role in neurons and are highly stable members of the cytoskeleton