Cytoskeleton and Muscles Flashcards
What are the functions of Microtubules?
Vesicular movement and Organelle attachment/movement
Chromosome movement during cell division.
Cell motility
Maintain cell structure (“support beams”)
What are Intermediate filaments for?
Stretch structure.
What are the Actin filaments for?
They are the support for the structure of the cell (located just beneath membrane).
What are the primary Motor Proteins associated with the cytoskeleton and what direction do they move?
Dynein - Microtubules
- Move objects towards center of the cell (Retrograde)
Kinesin - Microtubules
- Move objects towards periphery of the cell (Anterograde)
Myosin - Actin
- Important for contraction of actin filaments (think muscle!)
Describe the structure of a typical Microtubule.
Hollow tube whose wall consists of 13 columns of tubulin subunits.
Diameter: 25nm with 15nm lumen.
What are the subunits used to build a Microtubule? Which points in which direction?
alpha and Beta subunits. They form a dimer with +/- polarity. Beta is + and alpha is -.
Describe the Tubulin GTP cycle and how it puts the Microtubule subunits together.
Beta subunit can bind GTP. This makes the alpha/Beta subunit bind with high affinity to other subunits.
Beta subunit will eventually hydrolyze GTP to GDP, which will end the high-affinity state.
What happens if the region of GTP-hydrolysis in Microtubules ‘catches up’ to the dimer addition region?
“Catastrophe”
Microtubule will shrink and protofilaments will ‘peel away’ from microtubule wall.
What are the different configurations of Microtubules and what are their functions?
Singlets (Carry/move vesicles, organelles, chromosomes)
Doublets (Make up cilia/flagella; aka motility)
Triplets (Make up centrioles and basal bodies)
Describe the directionality of Microtubules.
+ points to periphery of cell
- points to/is anchored in MTOC
Name the 3rd subunit located in the MTOC (Microtubule Organizing Center).
Gamma tubulin. It acts as the scaffold for growth for Microtubules.
What is a Centrosome?
Two centrioles that are 90 degree angles to each other. Microtubules will grow from gamma tubulin ring complexes in a sphere surrounding the centrioles (pericentriolar material).
What do Microtubule Motor Proteins (Dynein, Kynesin) require to work? What do the proteins have that allows them to use this required substrate?
ATP.
Each has 2 ATPase heads.
What are Microtubule associated Proteins?
MAPs (i.e tau proteins) protect from Microtubule disassembly (catastrophe from GTP hydrolysis) by inhibiting tubulin dissociation.
Describe the structure of a MAP.
2 Domains:
- 2 tubulin binding domains (stabilizes the MT)
- 1 tubulin binding domain and 1 binding domain to other cellular structures (for anchoring MT in specific location).
What is the pathology of Alzheimer’s Disease?
Defect: tau proteins (MAPs) are hyperphosphorylated.
Symptoms:
Intraneuronal: Formation of Neurofibrillary Tangles (NFTs), which are composed of tangled clumps of Tau proteins.
- Reduces functional MTs, which disrupts axon transport)
Extracellular: Beta amyloid/senlie plaques, causes progressive neuronal damage.
What types of Alzheimer’s are there?
Autosomal Dominant: Late Onset AD
Multifactorial
- Apo E4 = earlier than Late Onset AD
- Apo E2 = reduced risk
(CHECK SLIDES 33 TO 37 AGAIN)
What is the pathology of Chediak-Higashi syndrome? (RECALL)
Defect: Mutation of CHS1/LYST (lysosomal trafficking regulatory protein involved in vesicle fusion). Results in delayed formation of phagolysosome in leukocytes, albinism, and granular defects in NK cells/platelets.
Symptoms: Hypopigmentation/Albinism, mild coagulation defects, recurrent lifethreatening infections.
Note that USMLE says a defect with microtubule polymerisation is what causes problems with cytoplasmic granules.
What are the 3 types of Microtubules involved in formation of the Mitotic Spindle?
Astral (unattached) MTs
Kinetochore MTs
- attach to Kinetochores on chromosomes to pull the chromatids apart
- Dynein escorts chromatid while MT shortens (tubulin dimer breakdown)
Polar MTs
- MT motors push Polar MTs apart to elongate spindle.
What is an axoneme?
It is the cytoskeletal structure in cilia and flagella. 9x2 (outer MT doublets) + 2 (central MTs) arrangement.
What is a Basal body?
The MTOC of the axoneme.
Has a 9-triplet structure (resembles centriole)
How do Cilia/Flagella generate movement?
They create power strokes (waving motions).
Each outer MT doublet is associated with inner and outer Dynein arms. The Dynein arms will slide these doublets relative to one another to generate power strokes. (Recall the direction Dynein moves as a motor protein: towards cell center)
Linking proteins prevent MTs from sliding.
Describe the Pathology of Primary Ciliary Dyskinesia (PCD).
Defect: Immotile cilia and sperm
Symptoms: Retain secretions and recurrent infection, infertility
50% = Kartagener Syndrome
- Situs Inversus = cell motility during embryogenesis can affect location of organs/structures during development
What are some drugs that disrupt MT activity and how?
Bind Tubulin subunits and prevent Polymerisation.
- Colchicine (gout treatment)
- Vincristine, Vinblastine (treat cancers with high mitotic index)
Bind and stabilize MTs, inhibiting Depolymerisation.
- Paclitaxel/Taxol (Can’t break down mitotic spindle = block mitosis, helps treat cancers)
Describe the structure of a typical Intermediate Filament.
Fibrous proteins that are supercoiled into thicker ‘cables.’
Diameter: 8-12 nm
What are the main functions of Intermediate Filaments?
Maintains cell shape (tension-bearing)
Anchors nucleus and certain organelles
Forms the Nuclear Lamina
Connect to IF of adjacent cells and to ECM components to form anchoring junctions, desmosomes, hemidesmosomes.
How are the building blocks of an Intermediate Filament (IF) arranged?
2 Monomers coil together to form a Dimer
2 Coiled-coil dimers stagger to form staggered Tetramer (Staggering forms ‘stick ends’)
8 Tetramers twist into the rope-like filament of an Intermediate Fiber. (Lateral contacts > longitudinal contacts)
Is there polarity to the IFs?
NOPE
Is ATP required to put the subunits of an IF together?
No.
How are IFs organized?
They extend from the outer Nuclear Memebrane to the cell membrane and across the cytoplasm.
What are the two classes of IFs? What are examples of each?
Cytoplasmic
- Keratins (in epithelia)
- Vimentin/-related (CT, Muscle cells, neuroglial cells)
- Neurofilaments
Nuclear
- Nuclear lamins
How can IFs help determine cancer origin?
Tumor cells typically lose their normal appearance, meaning it’s difficult to determine cell type based on morphology.
IF expression is still retained, which can help determine tissue of origin.
What does Keratin do in epithelial cells?
It anchors epithelial cells to the basal lamina and holds them together under stretch conditions.
What is the pathology of Epidermolysis Bullosa Simplex?
Defect: Mutated Keratin 5 or 14
Symptoms: fragile skin; blisters can very easily form.
What is the pathology of Epidermolytic Hyperkeratosis?
Defect: Mutated Keratin 1 or 10
Symptoms: Weaken structural stability of keratinocytes. Easy blistering, thickened scaly skin (from chronic wounding)
What is the pathology of Epidermolytic Plantopalmar Keratoderma?
Defect: Mutated Keratin 9 (palms and soles only)
Symptoms: Prominent blistering in palms and soles only.
What is Desmin?
IF of skeletal muscle cells. Forms a protective network for mechanical stress around myofibrils around Z disk.
What is GFAP?
Astrocyte IFs that contribute to mechanical strength.
What are Lamins?
…Dude.
Nuclear lamins. Go back and look it up if you don’t remember.
What are Neurofilaments?
IFs that cross-link to strengthen longer axons. They contribute to tensile strength, while MTs are responsible for transport.
Describe the structure of a typical Actin filament.
Two intertwined strands of actin, each of which is a polymer of actin subunits.
Diameter: 7 nm
Subunits: G-actin –polymerizes–> F-Actin
HAS POLARITY.
What are the main functions of Actin filaments?
Maintain Cell shape (also bear tension, like IFs)
Muscle Contraction
Cytoplasmic streaming/Cell Motility (Pseudopodia)
Cell division (Cleavage furrowing)
Describe F-Actin and polarity.
F-Actin is polymerized subunits of G-Actin.
- Thin, Flexible, Double-helix structure
- Dynamic
Polarity:
+ end is Fast Growing
- end is Slow growing
Is Actin polymerization ATP-dependent?
Yes.
How does Actin polymerization “treadmill”?
Actin-ATP cap stabilizes microfilament and allows to grow. Once polymerized, Actin-ATP will become Actin-ADP, which will dissociate once it reaches the ‘end’ of the microfilament.
Note, Actin-ATP will add faster to + end than - end
What does it mean when Actin ‘treadmills?’
It is constructed at one end and dissociate at other end. The length itself never changes.
What is Actin Nucleation? Where does it occur?
The formation of an actin filament by pulling G-Actins into a dimer/trimer nucleus around which new G-Actins are added. This is stimulated by Arp2/3
Monomers need bound ATP to add to the nucleus/growing filament. Profilin will exchange ADP for ATP to meet this need.
It occurs at the PM.
What does the ARP complex (Arp2/3) bind more efficiently to? What does that result in?
It binds/nucleates filaments more efficiently wh en bound to the side of a pre-existing actin filament.
This results in a branched actin network/web.
Where would the ARP complex typically be found?
At the leading edge of the cell.
What do Formins do?
They nucleate growth of STRAIGHT, UNBRANCHED actin filaments.
Name the types of Actin-Binding/Related Proteins and their examples.
Cross-linking proteins (filamin, fimbrin, villin)
Severing Proteins (gelsolin, cofilin)
How do which Actin-binding proteins modify Filament elongation?
Thymosin
- Binds G-Actin to prevent the assembly of F-actin
- Is a competitor of Profilin which binds to G0-Actin in order to promote assembly (ADP -> ATP)
Severing Proteins: Cofilin - Promotes disassembly at - end Gelsolin - Caps + end to prevent further growth.
What are the non-muscle functions of actin?
Form microvilli
Act as Stress Fibers
Cell movement (lamellipodia/filipodia)
Cytokinesis
What are Myosins, and what are the 3 we mentioned and their functions?
They are motor proteins.
Myosin I: microvilli
Myosin II: muscle/non-muscle contractile activity
Myosin V: Vesicle/Organelle Transport
What are the 3 types of Actin organization in the cell?
Contractile bundle
Gel-like
Tight Parellel bundle
What is the function of Filipodia if it doesn’t actually move the cell?
Sense where the cell is going.
Which Cross-linking Proteins (recall) form what Actin organizations?
Fimbrin and a-actinin (and villin) = parallel cables and bundles
Filamin (and Spectrin) = filament webs/gels.
Differentiate between Fimbrin and a-actinin and the way they link actin..
A-actinin = loose packed bundles, CONTRACTILE
- stress fibers
Fimbrin (and villin) = tight packed bundles, NON-CONTRACTILE
- microvilli
Where do Stress Fibers bind for movement?
Focal adhesions on the ECM via INTEGRIN.
Allows cell to push actin network forward for movement.
What is the function of Dystrophin?
Anchors actin cytoskeleton to the sarcolemma (muscle cell membrane) in skeletal muscles.
It can bind actin, dystroglycans, synaptrophins, and dystrobrevin.
What is the function of Vinculin and Talin?
Attaches actin cytoskeleton to membranes in focal adhesions.
What actin interaction allows cell-to-cell contact?
Cadherins (attachments of actin filaments)
They are extensions of actin outside the cell that bind to each other between cells. Adhesion belts?
What are Rho GTPases?
They are proteins that activate the Nucleating proteins (Arp2/3 complex and Formins)
Lamellipodia (rac)
Filipodia (cdc42)
Stress Fibers (rho)
What are the GTPases/Small G Proteins and what are their functions?
Small G Proteins/GTPases:
- Arf, Rab, Ran, Sar = switches for intracellular trafficking
- Ras = Transduction of growth factor signals
- Rho GTPases = Regulation of the actin cytoskeleton (rho, rac, cdc42)
What happens in order to facilitate Cell Movement?
Rho GTPases activate Arp2/3 complex: actin polymerization at leading edge of cell pushes cell forward
Rho GTPases also activate Cofilin: actin depolymerization at trailing edge and reorganization of leading edge
New focal contacts are made at the front, and old ones at rear are disassembled as cell moves forward.
What is the pathology of Wiskott-Aldrich Syndrome?
Defect: Mutated WASP, which is expressed in haematopoietic cells. WASP leads to Arp2/3 activation.
Symptoms: Failure of actin nucleation, disruption of signalling pathways.
- Death before adulthood
What are the contractile behaviors of certain actin structures in non-muscle cells?
Stress fibers = tension across cell allows cell movement via pulling on ECM
Adhesion belts = alter shape of epithelial sheets, formation of tubes.
Cytokinesis = actin filaments and Myosin II form contractile ring just under PM that pinches dividing cell in 2
What are the three drugs that inhibit Actin-based function (block cell movement)?
Cytochalasins (Fungi) - block depolymerisation Phalloidin (toadstool mushroom) - block depolymerisation Latrunculin (sea sponge) - bind free monomers (prevent polymerisation
How does Myosin II interact with other Myosin II units?
Tail-to-tail bipolar thick filaments.
What interacts with the Actin thin filament to prevent it from being moved before intended?
Tropomyosin/Troponin complex covers binding sites on Myosin II until contraction occurs.
What are the boundaries of a single Sarcomere?
One Z Disc to another Z Disc.
Where are the thin filaments capped? zby what?
Cap Z caps the + ends. The actin filaments will attach to the Z disc.
Tropomodulin caps the - ends.
What comprises the Z-disc?
Cap Z and a-actinin.
What anchors the Myosin (thick filament) to the Z disc?
Titin.
How is Muscle Contraction triggered?
Nerve signal triggers AP in muscle cell membrane, which spreads into T-tubules, then across gap junctions to the SR, which will release Ca2+
Ca2+ binds to troponin C, which uncovers the Myosin binding sites, meaning the Myosin heads can now bind.
Describe the mechanism of a Myosin II power stroke (and walking along thin filament).
- Ca2+ influx frees up myosin binding site on thin filament.
- Myosin head binds to thin filament.
- ATP introduced to Myosin head, causing it to release the thin filament
- ATP is hydrolyzed to ADP + Pi, which causes the Myosin head to shift in the direction of contraction.
- Release of Pi leaves ADP on Myosin head, which causes the Myosin head to rebind to the thin filament
- ADP is removed, generating the Power Stroke. Myosin head will return its normal ‘position,’ sliding the thin filaments towards the - end.
What component of the muscle unit is most commonly mutated?
Titin.
Describe the pathology of Dilated Cardiomyopathy.
Defect: Mutation in genes encoding components of sarcomere, cytoskeleton, nuclear lamina.
(Think Actin and Titin)
Symptoms: weakness, fatigue, risk of sudden death
What is the pathology of Familial Hypertrophic Cardiomyopathy?
1 cause of sudden cardiac death in athletes.
Defect: cardiac isoforms = myosin II (70%), tropomyosin, troponin
Symptoms: (Asymptomatic/Mildly Symptomatic)
- Dyspnoea
- Angina
- Palpitation
- Syncope
- Fatigue
- Fatal Cardiac Arrest
What are the Dystroglycan Complexes and their overall function?
They confer structural stability during contraction.
- alpha-dystroglycan: binds ECM glycoprotein (laminin!) and proteoglycan (agrin)
- Beta-dystroglycan: binds dystrophin and other adapter proteins, and intracellular signalling proteins (GRB2)
What happens if Dystrophin is not present in the muscle cell?
The sarcolemma becomes fragile due to the absence of a physical link, and muscle degeneration becomes a risk during repeated cycles of contraction and relaxation.
What is involved in Muscle repair and growth?
Satellite cells and Stem cells (side population cells).
Describe the process of Muscle repair.
Satellite cells chill in the basal lamina until it’s time to work.
- Stress/trauma activates them, prompting them to differentiate into myoblasts.
- These myoblasts will attach to the surface of the myotubes.
- Myoblasts fuse to the myofibers and form mature muscle.
Note; Asymmetric division prevents satellite cell pool from ‘drying out.’
What happens to Muscle repair in Muscular Dystrophies?
The repair response is not fast enough to keep up with repeated damage, which results in connective tissue and fat accumulation.
Describe the simple pathway of Muscle destruction.
- Lack of dystrophin –> Increased susceptibility to contraction-induced injury.
- Repeated degeneration/regeneration and ongoing inflammation and necrosis (Ca2+ influx causes apoptosis/necrosis)
- Eventual Muscle destruction
What is a general symptom of Muscular dystrophies (MD)?
Increased serum creatine kinase. It is typically only found inside muscle cells, and damaged muscle cells would lead to creatine kinase release.
Describe the pathology of Duchenne MD (DMD).
Defect: NO dystrophin in muscle whatsoever. X-linked recessive (lethal in males; 2/3 from carrier mother and 1/3 from germline mosaicism)
Symptom: Total loss of (muscle?) function, as opposed to Becker’s MD, which allows partial function.
- Gower’s sign (standing method for children)
- Lordosis, Scoliosis
- Contractures
- Wheelchair by 10-12 years
Note: Manifesting heterozygotes for females exist. (8% of carriers)
Describe the pathology of Becker MD (BMD).
Defect: Mutated (but present) dystrophin; allows partial function. (X-linked)
Symptom: Slower progression/onset but significant variability
- Think of it as a watered down version of DMD
Describe the pathology of Emery-Dreifuss MD (EDMD).
Defect: Mutated Emerin or Lamin A/C leads to fragile nuclear envelope; affects cells frequently subjected to physical stress. (X-linked)
Symptoms: Early onset
- Early contractures
- Cardiomyopathy, conduction defects/arrhythmias in adulthood: need pacemaker by 30
Describe the pathology of Myotonic Dystrophy.
Defect: Trinucleotide repeat disorder? CTG
Autosomal dominant
Symptoms: CATARACTS
- Onset 20-40
- Weakness in hands and legs, sternomastoids
- Facial muscles weakend (ptosis and haggard appearance)
Describe the pathology of Facioscapulohumeral MD (FMD).
Defect: deletion of subtelomeric tandem repeat??? Autosomal Dominant
Symptoms: Winged Scapula
- onset 10-40
- unable to puff cheeks, facial weakness
- weakness in upper arms and shoulders.
Describe the pathology of Limb Gerdle MD (LGMD). Type 1 and 2.
DEFECT:
- LGMD 1: laminopathies; Autosomal Dominant
- LGMD 2: sarcoglycanopathies (links to dystrophin with dystroglycan complex); Autosomal recessive
Symptoms: “Limb Girdle” = weakness of proximal musculature of Hip and Shoulder.
- heart unaffected
- Slow and variable rate of progression
- earlier onset = faster progression
Describe the muscle groups affected by the Various MDs.
Duchenne/Becker = Shoulders, upper torso, hips/thighs and lower legs.
Limb Girdle = Shoulders (and proximal muscles on upper torso), hips/thighs
Fascioscapulohumeral = Facial, Shoulders (and proximal muscles on upper torso), lower legs
Emery-Dreifuss = Shoulders (and proximal muscles on upper torso), lower legs
