19. Key functional proteins in skeletal muscle

Question 1

What is the structure of a muscle fibre?

Answer 1

Skeletal muscle fibres are composed of bundles of myofibrils which are segmented into functional units called “sarcomeres”.

Question 2

What is a sarcomere?

Answer 2

basic unit of measure within muscle, defined as the portion between two adjacent Z lines

It is the functional unit of a myofibril.

• contains:
• Nebulin to help align actin
• Titin to provide elasticity and stabilises myosin
• M line in the middle
• Actin & myosin

Question 3

What is a costamere?

Answer 3

Rib like structures (=”Costal”) on the surface of the sarcolemma

They are specialised sites of transmembrane complexes, occurring over the entire sarcolemma, where the transmission of force is concentrated

Found specifically at the Z disk

Question 4

The 3 states of transmembrane linkage - force transmission through costameres

Answer 4

A. relaxed
B. Contracted, forces transmitted
C. Contracted, forces not transmitted - TM complexes not linked to ECM

Question 5

Where is the Vinculin-Talin-Integrin complex usually localised?

Answer 5

usually localised in adherence junctions, involved in cell adhesion, cell migration, also sometimes cell proliferation

integrin acts as a receptor for laminin at the basal lamina, link to proteins at Z line with interaction with vinculin-talin

Question 6

α7β1 integrin - what are integrins composed of? And which isoforms are the most common?

Answer 6

Integrins are heterodimers composed of an α and β subunit.

22 heterodimers have been identified, composed of 16 α chains and 8 β chains

The α7B and β1D isoforms are the most common integrins found in adult skeletal muscle.

Question 7

What is the function of the α7β1 integrin?

Answer 7

1. Mediate the processes of cell adhesion & migration
2. Regulate the intracellular organisation of the actin cytoskeleton
3. Important role in many signalling processes

Question 8

Talin 1 vs talin 2.

Answer 8

Talin 2 is expressed at higher levels in skeletal muscle than talin 1

Both have a talin head and talin rod.

Question 9

Describe the role of talin 1.

Answer 9

• interacts with the cytoplasmic domain of β1 integrin and with focal adhesion kinase (FAK) and vinculin
• also binds to F-actin to establish a link between β1 integrins and the cytoskeleton (more mechanical function)

Question 10

What happens when the talin 1 (TIn1) gene is lost in SM?

Answer 10

Leads to progressive myopathy caused by mechanical failure of myotendinous junctions (MTJs).

Question 11

What happens when the talin 2 gene is lost?

Answer 11

Mice lacking talin 2 are viable and fertile but develop a myopathy with centrally located nuclei that is associated with defects in the maintenance of MTJs.

Question 12

What happens when you lose both talin 1 and 2?

Answer 12

SEVERE FUNCTIONAL OUTCOME
Severe defects in myoblast fusion and sarcomere assembly.

*talin 1 and 2 are somewhat mutually exclusive but can perform same function

Question 13

Describe the structure of vinculin.

Answer 13

1066 aa

Has 4 Vh subdomains:
Vh 1 - talin, α-actinin, α-catenin, IpaA

Linker region: signalling intermediate
Arp 2/3, ponsin, vinexin, VASP

Vt region: how it interacts with actin
F-actin, PIP2, paxillin

Question 14

What is vinculin involved in? And what is the result of mutations in its splice variant?

Answer 14

Vinculin is a membrane-cytoskeletal protein involved in linkage of integrin to the actin cytoskeleton.

Muscle specific splice variant = Metavinculin
*Mutations in metavinculin are assoc. with Idiopathic dilated cardiomyopathy (DCM) in humans

Question 15

What does the Dystrophin-Glycoprotein Complex do & where is it found?

Answer 15

It is specific to striated muscle fibres and is aka dystrophin associated complex. DGC is localised at costameres.

It is a multimeric protein complex that acts to link the basal lamina to the actin cytoskeleton in SM fibres.

Question 16

What are the physiological roles of the DGC?

Answer 16

• stabilise the sarcolemma during contraction especially during forced lengthening
• DGC forms rib-like lattices on the cytoplasmic face of the sarcolemma - “costameres”
• mechanical links between the sarcomeres within muscle fibre and laminin in the ECM.

Question 17

Describe Dystrophin & its brief structure.

Answer 17

Gene = 427kDa
Large rod-like protein enriched at SM sarcomeres.

Key intracellular component of the DGC.

Contains:

• 2 actin-binding domains (ABD)
• 24 Spectrin-like repeats
• 4 Hinge regions (H)
• 1 Cysteine-rich domain (CR)
• 1 C-Terminal domain (CT)

Question 18

What is the function of dystrophin?

Answer 18

Acts to link the sarcolemma to the actin cytoskeleton by direct interactions with F-actin & βDystroglycan

May also act to modulate signalling pathways through interactions with Syntrophin & Dystrobrevin.

Question 19

Describe in more depth the structure of the Dystrophin protein.

Answer 19

2 ACTIN BINDING DOMAINS:

• One N terminal, composed of calponin homology domains (CH)
• One centrally located, composed of spectrin-like repeats 11, 12, 13, 15, 17

24 SPECTRIN-LIKE REPEATS:
- Create the rod-like structure of the Dystrophin protein

4 HINGE REGIONS:
- regions of flexibility

1 CYSTEINE-RICH DOMAIN:

• composed of WW domain, two EF hand domains and a ZZ domain
• site of protein-protein interactions

1 C-TERMINAL DOMAIN:
- site of interaction with Syntrophin at SBD & Dystrobrevin at DBBD

Question 20

What happens when Dystrophin is defective or absent?

Answer 20

Dissipation of forces through the myofiber become disrupted.

• membranes become permeable
• high rate of muscle fiber breakdown & regeneration

Duchenne’s Muscular Dystrophy: severe complete dysfunction of protein

Becker’s MD: allelic - partially functional protein

In the absence of Dystrophin, other DGC members fail to accumulate on the sarcolemma indicating a role for Dystrophin in stabilising these proteins.
*β-dg, β-sg, α-sg, α-db2, α1-syn, γ-sg

Absence results in:

• muscles containing varying muscle fibre size
• inflammatory cell infiltrate
• accumulating fibrous tissue
• membrane permeability.

Question 21

Dystrophin can also regulate multiple signalling pathways via interactions via…?

Answer 21

Syntrophin/Dystrobrevin - coordinate the assembly of multiple signalling proteins to the DGC

• Grb2
• calmodulin
• SAPK
• nNOS

nNOS - produces NO during muscle contraction to facilitate vasodilation

Question 22

What is the Dystroglycan complex composed of?

Answer 22

Laminin
αDystroglycan
βDystroglycan

αDystroglycan & βDystroglycan come from same protein, cleavage produces alpha

Question 23

What is the function of the Dystroglycan complex?

Answer 23

Acts as a TM linker between the ECM & the plasma membrane/cytoskeleton

Links to the actin cytoskeleton of the muscle fibre by interaction with Dystrophin.

Question 24

What are laminins and what are they composed of?

Answer 24

Laminins are a family of large, extracellular trimeric proteins.

Composed of α chain, γ chain & β chain
*α chain necessary for interaction w receptors

Question 25

What is the function of laminin?

Answer 25

Forms direct link b/w ECM & Dystroglycan to maintain skeletal muscle structure

Question 26

What happens when laminin is defective?

Answer 26

Congenital Muscular Dystrophy (CMD) - genetic deficiency of laminin α2 chain (Merosin) - variable muscle fibre size - presence of immature fibres (central nucleation) - increased connective tissue (fibrosis)

Question 27

αDystroglycan

Answer 27

extracellular protein containing multiple sites for glycosylation requires post-translational modifications for proper function Function: receptor for laminin in SM

Question 28

βDystroglycan

Answer 28

TM protein that binds αDystroglycan & Dystrophin Function: provides link between subsarcolemmal cytoskeleton & ECM

Question 29

Dystroglycan dysfunction

Answer 29

No known association b/w mutations in dystroglycan & human disease. H/e, mutations in some glycosyltransferases result in Dystroglycan dysfunction as a result of lack of glycosylation. Lack of glycosylation doesn't affect Dystroglycan expression at the sarcolemma but it required for proper ligand binding (laminin). P'lation of βdystroglycan targets βdystroglycan protein for destruction if not bound to dystrophin.

Question 30

What is the sarcoglycan complex composed of?

Answer 30

αSarcoglycan, γSarcoglycan, δSarcoglycan, βSarcoglycan and sarcospan

Question 31

Describe the function of the sarcoglycan complex?

Answer 31

Not fully understood, appears to have both mechanical and non mechanical roles that mediate interactions among ECM, sarcolemma & cytoskeleton. Forms lateral association with dystroglycan complex. May function to stabilise the DGC at sarcolemma.

Question 32

What are the consequences of defective sarcoglycans?

Answer 32

Absence of sarcoglycans lead to alterations in membrane permeability, ultimately resulting in cell death. Genetic deletion of sarcoglycan subunits results in a subset of Limb-girdle Muscular Dystrophy (LGMD), referred to as "sarcoglycanopathies".

Question 33

Desmin: what is it & what is its function?

Answer 33

Muscle specific type III intermediate filament protein expressed in skeletal, cardiac & smooth muscle Function: links adjacent myofibers at level of the Z disc and binds myofibrils to the sarcolemma at level of costameres. Desmin is not required for normal muscle development and is essential for the maintenance of myofibril integrity following stress

Question 34

Desminopathy

Answer 34

- very rare diseases (only 60 patients reported to date) - desmin-related myofibrillar myopathy - mutation in desmin gene prevents formation of desmin filaments - results in misalignment of sarcomeres, disorganisation of muscle fibres, and muscle cell death

Question 35

What is Titin?

Answer 35

- Largest known protein in nature - Anchored at Z & M lines of sarcomere - Maintains rigid contact with the thick filament along the A band - flexible along the I band - "molecular bungee cord" of skeletal muscle - functions to protect muscle fibres from damage due to over stretching

Question 36

What are Titin's 4 main roles in skeletal muscle?

Answer 36

1. Provide a structural framework through association with sarcomeric proteins 2. Centering the thick filament within the sarcomere during contraction 3. Act as a molecular spring 4. Signalling mediator (has kinase domain)

Question 37

Titinopathy

Answer 37

aka Tibial Muscular Dystrophy (Limb Girdle muscular dystrophy 2J) - Detected in the Finnish population (1/10000 occurrence) - Alter the structure & function of Titin - May disrupt Titin's interactions with other sarcomeric proteins - Disrupts normal muscle contraction, resulting in muscle weakness and wasting over time - Not known why disease is confined to lower leg muscles.