Lecture 12 Flashcards
What is spectrin?
Spectrin is another major component found in the cytoskeleton.
It is found on the inner surface of the plasma membrane.
It can attach to many integral membrane proteins (membrane transporters, channels, receptors).
It was first identified lining the inner memrbane surface of mammalian red blood cells.
Found in all animal tissues (so probably in all metazoan cells)
What is spectrin responsible for?
Thought to be responsible for maintaining plasma membrane integrity and contributes to its elasticity and limits extensibility.
Where is spectrin found?
Spectrin is found in the Golgi, vesicle, ER and the nucleus.
What is spectrin made of?
Made of anti-parallel alpha-(290kDa) and beta-(240kDa) heterodimers
How many dimers form the spectrin tetramer?
2 dimers
How many major spectrin alpha-subunits are there?
There are 2 major spectrin alpha-subunits.
2 alpha-spectrin genes
Alternative splicing gives rise to 8 alpha-spectrin isoforms
How many beta genes are there?
5 beta genes.
Some can be alternatively spliced.
This give large isoform variability.
All of regulated in a time and tissue-specific manner
What is at the end of the spectrin molecule?
Has actin-binding domains (ABDs) at the end of the spectrin molecule.
What are actin-binding domains (ABDs)?
These ABDs are really 2 calponon-homology (CH) binding domains arranged in tandem
What is calponin?
Calponin in smooth muscle has 1 calponin binding domain and cannot bind actin, but that’s where the domain was first described.
What is the binding affinity of CH1 and CH2?
CH1 can bind actin alone, CH2 cannot. Both together bind actin even better.
What does spectrin have that is used for signaling?
Spectrin has a pleckstrin homology domain that is used by many proteins for signaling.
What are some general spectrin problems?
If the spectrin-actin network is partially eliminated, red blood cells fragment into 50nm inverted vesicles.
Mutations to spectrin cytoskeletal components lead to hemolytic anaemias.
(eg. spherocytic [round red blood cells] anaemia [less red blood cells])
What happens when you mutate a spectrin component?
Not every gene has been mutated, but some have, with serious consequences.
Where is Beta4 Spectrin found?
It is mostly found in neurons. Localizes to the nodes of Ranvier.
What does Beta2 Spectrin localize to?
Axons
What does Beta3 Spectrin localize to?
Cell body and dendrites of neurons
What happens to Beta4 Spectrin null mice?
Mice have tremors.
Contraction of their hindlimbs.
Ankyrin-3 (AKA Ankyrin-G) and Voltage gated sodium channels are not correctly localized in the neurons of the mutant mice.
What happens in Akyrin-3 null neurons?
Beta4 Spectrin is not localized propoerly and actin potentials do not fire normally.
Who was the “quivering mouse”?
There was naother mouse that already existed (in 1957) called the “quivering mouse”. It has a naturally occuring Beta4 Spectrin mutation. They have tremors and hearing defects, due to the voltage gated channel mis-localizations.
What are spectrin repeats?
Helical repeating units within spectrin and many other proteins
How many repeats are in alpha-spectrin?
20 repeats
How many repeats are in beta-spectrin?
16 repeats
How many repeats do other proteins have?
Between 2 and 74 repeats
What do the repeats range from in amino acids?
99-122 amino acids
What do spectrin repeats cause?
Spectrin repeats cause a structural fold that is found in ALL spectrin repeats.
What is this structural fold caused by?
It is caused by 3 of the repeats interacting with each other.
What do sequence homologies top out at?
Sequence homologies top-out at 30% similarity
What is the function of repeats?
Repeats function as flexible spaces between the membrane and the cytoskeleton (primarily actin)
Most spectrin superfamily members have more than how many Calponin-Homology (CH) domains?
1
What proteins are included in the Spectrin superfamily?
Alpha-actinin
Spectrin
Dystrophin (muttions cause muscular dystrophy)
Utrophin (upregulated in muscular dystrophy patients)
What is alpha-actinin?
Alpha-actinin is thought to be the ancestor of this superfamily as the rest appear to be slight modifications of it.
What are additional spectrin superfamily proteins called?
Spectraplakins: cytoskeletal/junctional binding proteins
Nesprins 1-4: nuclear envelope spectrin repeat protein
What are some Spectraplakins?
MACF (Microtubule-actin cross-linking factor 1, it does what its name implies)
BPAG (Bullous pemphigoid antigen 1)
Plectin
Desmoplakin
What does Nesprins 1-4 do?
Binds the cytoskeleton in the nucleus
Nesprin 1 and 2 are massive proteins (800kDa-1000kDa)
These 2 genes give rise to ~20 isoforms
These don’t end with spectrin repeats
What is Ankyrin?
Ankyrin mediates the interaction between spectrin and other transmembrane proteins
It binds to the spectrin repeats
How many Ankyrin genes are in vertebrates?
3
How many Ankyrin repeats are there?
24 repeats. Those repeats are also found in many different proteins. These repeats bind to proteins. Hence, Ankyrin can bind to and cross-link more than 1 membrane protein (eg. Na/Ca exchanger, IP3 receptor and Na/K- ATPase)
What does Ankyrin bind to at the plasma membrane?
At the plasma membrane Ankyrin binds spectrin to a protein called Band 3.
What does Band 3 do?
Band 3 (AKA Anion Exchanger-1 [AE1]). It exchanges Cl- ions for HCO3- ions.
What do Ankyrin-2 (AKA. Ankyrin-B mutations) do?
Causes fatal heart arrhythmias.
This defect in Anyrin-2 is one of the causes of the arrhythmias that are fatal in a human disease called inhereted long QT syndrome (results in irregular heartbeats).
What is “QT”?
“QT” refers to the heartbeat interval” that is part of the PQRST chart of a normal heartbeat.
What does the Ankyrin-2 mutation cause?
It causes the mis-localization and decreased expression of proteins known to interact with Ankyrin-2 including:
Na2+ pump
Na2+/Ca2+ exchanger
There is also altered Ca2+ signaling, this likely causes the arrhythmias
How many genes of Adducin are there in vertebrates and invertebrates?
There are 3 genes (ADD 1-3) in vertebrates (all 3 can be alternatively spliced)
Invertebrates only have 1 gene
What is Adducin?
Needs to dimerize or oligomerize to be functional
It promotes binding of spectrin to actin
It is the crucial link for actin-spectrin interactions
It is also an F-actin plus-end capping protein
Binds to the plus-ends and sides of actin filaments.
It can do this alone or with spectrin.
What are considered negative regulators in Adducin?
Calmodulin and protein kinase C (PKC) are negative regulators, blocking the interaction of spectrin with actin
How does Adducin bind actin and spectrin?
1) Spectrin binds to actin with very low affinity
2) Adducin binds to the spectrin-actin complex
3) This recruits additional spectrin to the adducin-spectrin-actin lattice
4) Adducin bundles and caps the barbed end of thea ctin filaments (this blocks additional actin from polymerizing or dissociating)
What is Adducin-1 mutation in mouse?
Also known as alpha-adducin, it is ubiquitously expressed (found in: brain, heart, lung, spleen, etc..)
What is Adducin-3 mutation in mouse?
In the blood it is found in red blood cells and platelets (FYI. Adducin-3 [AKA. y-Adducin] is in platelets too)
Where do Adducin-1 and Adducun-2 interact?
In red blood cells, adducin-1 and adducin-2 interact
Where do Adducin-1 and Adducun-3 interact?
In platelets Adducin-1 and Adducun-3 interact. There is no adducin-2 in platelets).
What does Adducin-1 cause in null mice?
Causes Adducin-2 and -3 proteins to also be gone from RBCs, despite Adducine-2 and -3 mRNA remaining unchanged. Thus, there is no adducin associated with the RBC spectrin in these mice
What Adducin-? is absent from platelets?
Adducin-3 is absent from platelets, but the platelets function normally and are their normal shape.
What happens to the RBCs in Adducin-1 mutations in null mice?
Spherical RBCs (spherocytes), RBC dehydration and osmotic fragility.
Are the mice smaller or bigger in this mutation?
Smaller
Were there are observable changes in organs?
No observable changes in any other organ apart from the brain as 50% get hydrocephalus (leakage of cerebral spinal fluid in their brains) and die.
What is Adducin-2 mutation in mouse?
It is highly expressed in the brain, bone marrow (in humans) and spleen (in mice).
NOTE: Adducin-1 and -3 are found every where (ubiquitously expressed)
What does Adducin-2 cause in null mice red blood cells?
Have red blood cells that are:
Osmotically fragile, spherical and dehyrated as comapred to wild-type conrtols
There is a 30% decrase in adducin-1 at the membrne of the RBCs
There is 5X more adducin-3 at the memrbane of the RBCs
What does Adducin-2 cause in null mice?
More irons in their spleens (there are more blood cells in the spleen (phagocytes and erythrocytes)
Spleens are larger than normal
Also more iron in thier livers and kidneys (this is likeky due to compensation in the generation of more blood cells in these mice)
What is Protein 4.1 (AKA. Band 4.1)?
There are 4 different proteins encoded by 1 gene.
What does Protein 4.1 bind to in the plasma membrane?
Binds to Glycophorin-c in the plasma membrane (and to Band 3) at the plasma membrane.
Shown using proteins 4.1 null red blood cells that had an altered conformation of Band 3 present.
Remember: Ankyrin also binds to Band 3
What else does Protein 4.1 also bind to?
Protein 4.1 also binds spectrin-actin through adducin to the plasma membrane
