dystrophin and glycoprotein complex and muscle staining

1. DYSTROPHIN GLYCOPROTEIN COMPLEX
AND STAINING OF MUSCLE TISSUE
Dr. Nandimalla vinay kumar
DM Neurology

2. INTRODUCTION
• Striated muscle contraction requires precise and coordinated activity of both the nervous
and somatic systems, from excitation of individual myofibers at the neuromuscular
junction to the ATP-regulated power stroke of myosin.
• Muscle contraction in both heart and skeletal muscle results in cellular deformation and
shortening.
• Throughout this process, the contractile machinery inside the myofibers must remain
intimately connected with the membrane and extracellular matrix.
• One function of the dystrophin glycoprotein complex (DGC) is to provide a strong
mechanical link from the intracellular cytoskeleton to the extracellular matrix.

3. Proteins of myofibril
1. More than 20 proteins where 6 constitute app 90% of total
myofibrillar proteins (MP)
2. Myosin, actin, titin, tropomyosin, troponin and nebulin
• On basis of function-
 Contractile-actin, myosin
 Regulatory-tropomyosin, troponin
 Cytoskeletal-titin, nebulin( integral to structure of Z disk)

4. Contractile proteins
• 1. Actin-
 20% of MP
 Globular shaped app 5.5 nm in diameter
 G shaped actin-monomeric form
 G actin monomers polymerize to form F actin
 2 strands of F actin are spirally coiled around one another to form “super helix”

5. • 2. Myosin-Fibrous protein , 45% of MP
 Elongated rod shaped with a thickened portion at one end (head)
 Head region is double headed and projects laterally from the long axis of the filament
 Portion between head and tail is known as neck
 Myosin filaments are arranged in opposite directions on either side of M line.
 Mysoin heads-active site which forms cross bridges with actin filaments during contraction

6. Regulatory proteins
Tropomyosin-
 5 % of MP, Lies in close contact with actin filament
 Each strand lies alongside, within each groove of actin super helix
 Single molecule extends length of 7 G-actin mol .
Troponin-
 5% of MP
 Present at well defined intervals in grooves of actin filament
 Lies along the tropomyosin strands
 1 mol of troponin for every 7-8 G actin molecules

7. Cytoskeletal proteins
1. Titin-most abundant, 10% of MP.
 3rd filament.
 Largest polypeptide known (25000 aa).
 Extend longitudinally in each half sarcomere from M line to Z disk.
 Portion of titin in A band is inelastic and that in I band is elastic.
 Binds to the outside shaft of the thick filament and C protein that encircles and stabilizes the thick filament
Nebulin–4% of MP
 Located close and parallel to actin filament
 Extends along the length of the thin filament from A band to Z disk
 In Developing muscle-organization of thin filaments
 In Mature muscle-serves as scaffold for stability of thin filaments, anchors thin filaments to Z disk

8. DYSTROPHIN GLYCOPROTEIN COMPLEX
• It is a multiprotein complex.
• Functions as a structural link between sarcolemma, cytoskeleton and extra cellular matrix.
• It aids in blood flow regulation and muscle fatigue recovery.
• Decrease in function cause fibers to become weak and degeneration.
• It regulates – recruitment of Nnos, signaling molecule important for relaxation, catalyzes the
production of NO.
• When muscle relaxation occurs, NO diffuses through the muscle cells causing muscle to relax.
• The DGC is composed of transmembrane, cytoplasmic, and extracellular proteins.
• Components of the DGC include dystrophin, sarcoglycans, dystroglycan, dystrobrevins,
syntrophins, sarcospan, caveolin-3, and NO synthase

9. DYSTROPHIN GLYCOPROTEIN COMPLEX

10. Extrajunctional muscle membrane: Associated proteins 2

12. DYSTROPHIN
• Dystrophin is a 427kDa cytoskeletal protein that localizes to the cytoplasmic face of the
sarcolemma and is enriched at costameres in muscle fibers.
• It has four main functional domains;
1. actin-binding amino-terminal domain (ABD1)
2. central rod domain,
3. cysteine-rich domain
4. carboxyl-terminus.

13. ACTIN BINDING DOMAIN
• ABD is located at amino terminal of dystrophin.
• Alpha actinin is normal component of actin filament in skeletal and smooth muscle.
• ABD is involved in crosslinking F actin and there by connecting filamentous elements of
cytoskeleton of cell membrane.

14. CENTRAL ROD
• It contains 24 spectrin like repeats and each repeat is 110 a.a in size forms triple alpha helical
bundles.
• A & B forms – long helix
• C form – short helix
• Alpha helical coil repeats are interrupted by 4 hinge portions.
• At the end of 24th repeat is 4th hinge region and immediately followed by WW domain.

15. CYSTEINE RICH DOMAIN
• It contains two EF hand motifs and ZZ domain.
• EF hand motifs consist of two alpha helices implicated in calcium binding.
• WW domain along with two neighbouring EF hands binds the carboxy terminus of beta
dystroglycan anchoring dystrophin at sarcolemma.

16. CARBOXY TERMINAL
• It contains two polypeptides that fold in to alpha helical coils similar to spectrin repeats in the rod
domain.
• These coils are involved in protein-protein interaction.
• CT domain provide binding sites for dystrobrevin and syntorphins.

17. Functions of dystrophin
• Provides structural integrity link between sarcolemma and
cytoskeleton.
• Acts as molecular shoch absorber during contraction and relaxation.
• Aids in signaling pathway.

18. Dystroglycan
• Two components- alpha and beta.
• It acts as receptor for extracellular ligands such as laminin.
• Alpha is tightly attached to beta component of dystroglycan which
interacts with dystrophin.

19. SARCOGLYCAN COMPLEX
• This complex is tightly associated with beta dystroglycan.
• It contains 4 transmembrane proteins
 alpha sarcoglycan
 beta sarcoglycan
 gamma sarcoglycan
 delta sarcoglycan
• It provides phosphorylation sites for CAMP dependent protein kinases , protein
kinase C , casein kinase II.

20. •Sarcoglycan proteins: Types
α-Sarcoglycan (Adhalin) : LGMD 2D
β-Sarcoglycan : LGMD 2E
γ-Sarcoglycan : LGMD 2C
δ-Sarcoglycan : LGMD 2F
ε-Sarcoglycan : Myoclonus-Dystonia syndrome
o General features Family: Homologous transmembrane proteins with single membrane spanning
domain
o All Sarcoglycans (SG) have
o Glycosylation
o Extracellular region
o Contains cysteine cluster
o Location
o N-terminal (Type I): α- & ε-SG
o C-terminal (Type II): β-, γ- & δ-SG
•

21. o Organization: Heterotetramers
o β & δ subunits
o Core of complex
o Sarcoglycan pair most tightly bound to each other
o α, γ (& e) subunits
o Bind to complex independently
o Added to complex in Golgi apparatus
o Tissue composition of sarcoglycan oligomers
o Skeletal Muscle: α, β, γ, δ sarcoglycans
o Smooth Muscle: e, β, z, δ Sarcoglycans
o Myelinating Schwann cell: β, δ, e
o molecules interacting with Sarcoglycan complex
•β-Dystroglycan: May bind to γ-Sarcoglycan
•α-Dystrobrevin, N-terminal: Binds to Sarcoglycan complex
•Sarcospan: Stabilized at the membrane by Sarcoglycan complex, especially δ-Sarcoglycan
•Filamin-2: Binds to γ & δ Sarcoglycans
•Biglycan : Extracellular leucine-rich repeat (LRR) protein
•No direct association with dystrophin

22. • Sarcoglycan complex: Possible functions
• Stabilization of Dystrophin-Glycoprotein Complex
• Especially Dystrophin–Dystroglycan interaction
• Regulation of adhesion of Dystrophin-Glycoprotein
Complex to Laminin-2 in extracellular matrix
• ? Role in vascular function associated with blood
flow: Especially γ-Sarcoglycan
o Mechanical: Bridging between basal lamina &
intracellular actin network
o Signaling: Cysteine cluster & ATPase activity in α-
sarcoglycan

23. SYNTROPHIN
• Syntrophin, first identified as a 58-KDa protein in the electric tissue of Torpedo.
• It interacts directly with the carboxylterminus of both full-length and truncated forms of dystrophin.
• Three syntrophin forms exist, and each isoform contains two pleckstrin homology domains.
• It functions as modular adaptors that localises signaling molecules such as Nnos ,AQP4 channels ,ion channels etc in
association with DGC.

24. SARCOSPAN
• Sarcospan is a member of the tetraspanin family that associates tightly with the DGC.
• It is highly hydrophobic protein whose amino and carboxyl-termini each face the cytoplasm.
Isoforms: Generated by alternative splicing
oIsoform 1: Specific for skeletal & cardiac muscle
oIsoform 2: Widely expressed
• sarcoglycan complex is required for the stability of sarcospan at the plasma membrane.

25. DYSTROBREVIN
• It shares significant homology with thecysteine-rich and
carboxyl-terminal domains of dystrophin.
• Three isoforms of alpha -dystrobrevin, derived from
alternative splicing, are components of skeletal muscle
DGC.
• Important motifs present in the longest isoform, alpha -
dystrobrevin-1, include the Ca2-binding EF hand, zinc
finger ZZ-domain, coiled-coil domain, and a tyrosine
kinase substrate domain.
• Alpha dystrobrevin and syntorphin triplets are
associated with dystrophin and anchors n NOS to
sarcolemma
•β-Dystrobrevin
•Coded by different gene than α-Dystrobrevin
•Location: Neurons of cortex & hippocampus; Not in brain
microvasculature or muscle

26. Filamin C (FLN2; FLNC)
•Tissue expression
oSkeletal muscle: Z-disk
oHeart
•Structure
oDomains
•Actin binding domain: 2 Calponin homology (CH) domains
•Immunoglobulin (Ig) domains: 24; Divided into ROD1 & ROD2 subdomains
•C-terminal: Dimerization domain
•2 transcripts: ± Exon 31
oLong isoform: More during cardiac stress
oShort isoform: Normal expression; 3.5x higher in skeletal muscle
o Dimer: 2 identical filamin proteins
• Interacts with
• Actin: via amino terminal CH domains,γ- & δ-Sarcoglycans, Myotilin, Calpain-3 ,Furin ,Inositol
polyphosphate phosphatase like 1.
• Functions
• Involved in actin reorganization and signal transduction
• Maintains membrane integrity during force application
• Structural protein: Z-disc; Myotendinous junction; Intercalated discs

28. CAVEOLIN
• Caveolin-3 is expressed only in striated muscle.
• caveolin forms, caveolin-3 can oligomerize to form caveolae,small membrane invaginations that participate in
membrane organization and uptake of small solutes.
• Caveolin binds directly to nNOS.
• The absence of caveolin-3 increases myofiber apoptosis.
NITRIC OXIDE SYNTHASE 1 (NOS1; NNOS)
•Association of nNOS with the Dystrophin-Glycoprotein complex: Binding to Syntrophins
•Nitric oxide (NO) is synthesized in skeletal muscle by nNOS
•Production of NO by nNOS may be cell-protective: Several possible mechanisms
oActions as a cytoprotective free radical
oInduction of cGMP-dependent cell survival pathways
oLocal vasodilatory effect by effects on vascular smooth muscle
•nNOS deficiency
oDisorders: Muscle denervation; Myosin-loss myopathy

30. Other membrane proteins
•Caveolin : LGMD 1C
•Dysferlin: LGMD 2B; Myoshi distal myopathy
•Integrins
•Integral membrane proteins
•Receptors for: Laminins 1, 2, 4
•Interactions are independent of the dystrophin-dystroglycan complex
•Integrin α7β1
oSpecificity for skeletal and cardiac muscle
oConcentrated at myotendinous junctions
oAlso detected at neuromuscular junctions and along the sarcolemmal membrane
oIntegrin-α7 mutations: Congenital muscular dystrophy
• β1 integrin
• Required in muscle for innervation by motor axons
• Role in muscle fiber development of normal adhesive & signaling interactions with motor neurons
• Integrin α5β1
• Binds to fibronectin

32. CONNECTIVE TISSUE COMPONENTS: SKELETAL MUSCLE
Synaptic Extra-synaptic Synaptic & Extrasynaptic
Agrin (Nerve-derived)
Neuregulins (ARIA)
Acetylcholinesterase (A12)
Laminins: 4, 9, 11
S-laminin
Heparan sulfate
proteoglycans
βGalNAc-terminal
conjugates
Collagens
α3 (IV); α4 (IV); α5 (IV)
NCAM
S-nidogen
Collagens
α1 (IV); α2 (IV)
Laminin-2
Tenascin-XB
α-Dystroglycan
Nidogen
Fibronectin
Integrin-α7
Perlecan
Agrin (Muscle-derived)
Decorin
Collagens
α1 (VI); α2 (VI); α3 (VI)
Biglycan

33. COSTAMERIC ARRANGEMENT OF THE DGC
• In skeletal and cardiac muscle membranes, the DGC is
concentrated over costameres.
• Costameres are transverse, rib-like structures that overlie the Z
lines of the sarcomere.
• Focal adhesion proteins such as vinculin, alpha-actinin, beta 1
integrin, and beta-spectrin are also costameric.
• Costameres are thought to transmit mechanical force from the
sarcomere to the sarcolemma, the extracellular matrix, and
even surrounding fibers and require both outside-in and inside-
out signaling.
• The disruption of the costameric arrangement may be the
major initiating factor in the loss of membrane permeability
that is a feature of both cardiac and skeletal muscle lacking
sarcoglycan or dystrophin.

39. Dystrophin associated glycoprotein complex. Dystrophin associated glycoprotein complex and related
proteins that help the anchoring of the sarcolemma to the basal lamina. Within brackets under the different
proteins are the different diseases that result from deficiency of the respective proteins. (Limb girdle
muscle dystrophies (LGDMD); Duchenne muscular dystrophy DMD; Becker muscular dystrophy (BMD);
Congenital muscular dystrophy type 1A (MDC1A); Emery–Dreifuss muscular dystrophy (EMD)) (Adapted
from Diseases of Muscle and the Neuromuscular Junction Part 1).

45. STAINING OF MUSCLE

48. • HEMATOXYLIN AND EOSIN STAIN
cross section shows several fascicles
surrounded by and separated by
sarcoplasm and thin layer of perimysium
the muscle fibers are of relatively uniform
sizeand shapewith nuclei locatedat the
periphery of the cell

50. NADHstain
• Low power view demonstrates 2
populations of muscle fibers.
• Type 1 myofibers stain more
darkly than type 2 myofibers
because of the greater use of
aerobic metabolism by type 1
fibers.
• The sarcoplasm stains fairly
uniformlyacrossthecell.

52. Myosin (ATPase) stain
• Myosinadenosine
triphosphatase(ATPase) at
pH10.5stainstype 2
myofibers brown.
• Type1fibers arestained withaneosin
counterstain sothatthey are visible.

53. Myosin heavy chain histochemical stain
• IHC fiber typing stain for myosin heavy
chain ,slow type in which type 1 fibers are
brown.
• The eosin counter stain makes the type 2
myofibers visible with pink colour.

54. Myosin heavy chain histochemical stain
• IHC fiber typing stain for myosin heavy chain
,fast type.
• Type 2 myofiber sare brown and type 1
myofibers are pink due to eosin counterstain.

55. Periodic acid Schiff staining
• It stains sugar moiety so that glycogen, MPS and
glycoproteins are highlighted.
• This method is most useful in evaluating glycogen
storage disorders.
• It also provides information about vasculature.
• It can highlight fibers that are degenerating or
necrotic and demonstrate inclusion bodies

56. Modified gomori trichrome stain
• The modified Gomori trichromestainis
valuable in evaluatingmitochondrial
myopathies, inclusion body myositis, and
some other disorderswithintracellular
inclusions.
• The nuclei and mitochondria are stained red,
cytoplasm is mostlyblue-green, connective
tissue is green.

57. Sudan black stain
• Sudan Black stain for lipid demonstrates
slightly more staining of type 1 myofibers
because of their higher lipid content
due to their greater dependence on
aerobic metabolism compared with type 2
myofibers.
• Hereditary and acquired disorders of lipid
metabolism show excessive staining of
fibers.
• Some of the mitochondrial myopathies are
associated with increased intracellular lipid
content.

58. Paraffin sections
• It provides more cytological details than a frozen
material, so it improves identification of cells involved in
inflammatory disorders.
• Detailed structure of vascular walls can be seen in
paraffin sections.
• This section is usually larger than frozen and offers more
material for examination.
• LPV shows fibers aligned linearly in longitudinal section.
• HPV shows the strations clearly.

59. MUSCLE PATHOLOGY
Neurogenic changes in muscle pathology-
Neurogenic disorders have thefollowing characteristicsonmusclebiopsy:
1. Angulated atrophic fibers
2. Group atrophy
3. Target fibers
4. Nuclear clumps

60. Myopathy can have thefollowing characteristicsonmuscle biopsy:
1. Myofiber necrosis
2. Myophagocytosis
3. Regeneration
4. Round atrophic fibers
5. Myofiber atrophy and splitting
6. Increase in internal nuclei
7. Fibrosis
8. Nuclear chains
9. Moth eaten fibers
10. Ring fibers
11. Whorled fibers

62. Fiveimportant groupsofdisordersthatcanbediagnosedbymusclebiopsy
include the following:
• Myositis
• Muscular dystrophies
• Glycogen storage diseases (metabolic myopathy)
• Mitochondrial myopathies (metabolic myopathies)
• Congenital myopathies

63. REFERANCES
• The Dystrophin Complex: structure, function and implications for therapy Quan Gao1 and
Elizabeth M McNal doi:10.1002/cphy.c140048.
• Treating pediatric neuromuscular disorders: The future is now September 2017 American
Journal of Medical Genetics Part A 176(25) DOI:10.1002/ajmg.a.38418
• MEMBRANE-ASSOCIATED PROTEIN COMPLEXES Skeletal Muscle Fibers & Connective
Tissuehttps://neuromuscular.wustl.edu/musdist/dag2.htm#sg
• Bradley and daroffs neurology textbook

64. Thank you