Cell Biology: Cytoskeleton

Front 1

MF and MT are dynamic filament networks.

Back 1

Unpolymerized pool and polymerized pool of protein subunits

Subunits exchanged between pools

Various drugs can alter steady state between two pools.

Front 2

MF and MT are polar.

Back 2

Fast growing "plus" and slow growing "minus" ends

Specific motor complexes that recognize this polarity

Organized in cytoplasm to appropriately exploit this polarity.

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Microfilaments: General facts

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Actin- form double helical conformations. Diameter: 5-7nm

Is not hollow

Ubiquitous and well conserved.

Actin binding proteins may promote polymerization or depolymerizations, may link different MFs together.

Front 4

Myosin
Filamin
Gelsolin
a-actin
spectrin, ezrin and moesin.

Back 4

Myosin: MF based motor molecule. Generates force during muscle contraction.

Filamin: promote formation (meshwork x-linkers)

Gelsolin: promote disassembly

a-actin: bundling (cabling) proteins.

Spectrin: membrane attachment proteins. Ezrin and moesin are ERM family proteins.

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Microfilaments: Assembly

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Nucleation: Three subunits interact in proper orientation to bind together and form a seed. RATE LIMITING step.

Elongation: More subunits add on head to tail. Rapid.

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Cytochalasins

Back 6

Break down MFs

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Phalloidin

Back 7

Mushroom toxin. Stabilizes MFs. Can be used to stain MFs.

Front 8

Microfilaments: Structures and functions

Back 8

Cell cortex: Cytoplasmic region near plasma membrane, contains a bunch of MFs and binding proteins (spectrin). Organelles generally absent in this area, except secretory vesicles.

Muscle sarcomeres/myofibrils: Actin are major components of contractile apparatus in muscle. Motor protein myosin II

Stress fibers: Bundles of MFs + proteins in muscles. Found along plasma membrane of non-muscle type cells, esp endothelial cells of large arteries. Contain actin-binding proteins a-actin and myosin II.

Front 9

Microfilaments: Structures and functions II

Back 9

Microvilli: Specialized area of cortex found in intestinal epithelial cells. Core bundle of MFs (20-30) + myosin, fimbrin, villin.

Contractile ring: Circumferential ring along with myosin cross bridges and a-actin anchors, found adjacent to cytoplasmic surface of plasma membrane of dividing cells

Focal contact: accessory structure that hooks up microfillaments

Front 10

Duchenne/Becker muscular dystrophy

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Defective dystrophin (actin binding protein that binds to both actin and plasma membrane of muscle and neuronal cells).

Has to do with strength and flexibility of muscle cell plasma membranes during cycles of contraction and relaxation.

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Neurofibromatosis type II gene

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Encodes merlin, actin binding protein. Binds actin and plasma membrane in non-muscle cells. Part of ERM family of actin binding proteins.

Front 12

Plant profilin

Back 12

Causes immune response, pollen allergies.

Front 13

Troponin I and T

Back 13

Cardiac muscle actin binding protein. Shed into blood stream after mild heart attack, good way to predict a much larger heart attack.

Front 14

Microtubules: General facts

Back 14

Also abundant and ubiquitous. ~1-3% of total protein, up to 15% of soluble protein in brain tissues

Alpha, beta, and gamma share ~35% identity.

Part of G-protein superfamily, have GTPase activity and tubulin diamers have been strongly implicated in some signal transduction pathways.

Front 15

Microtubules: Structure

Back 15

25nm diameters.

Hollow with 14nm lumen

13 longitudinally oriented protofilaments. Each protofilaments in cross section corresponds to one subunit.

Subunit protein is dimer of alpha and beta tubulin

Most MTs are initially formed at centrosome and remain anchored there after polymerization.

Front 16

Centrosome

Back 16

2 centrioles and pericentriolar material (PCM-protein aggregates that associate specifically with centrioles)

Centrioles not directly involved in nucleating growth of cytoplasmic MTs but are used as templates for the growth of cilia and flagella.

Centrioles posess 9 triplets of short ~0.4um MTs held together by protein linkers. 2 of 3 MTs are incomplete MTs (10 protofilaments)

Centrioles w/in pair are arranged orthogonally (perpendicularly)

PCM contains gamma-tubulin, directly responsible for MT nucleation and elongation.

2 centrioles + PCM = centrosome.

Front 17

Microtubules: Assembly

Back 17

Tubulin heterodimers assemble into MTs. Diameter = 25nm

Grow from centrosome. Must have GTP bound to polymerize. Will hydrolyze into GDP when polymerized.

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Microtubules: Associated proteins (MAPs)

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Brain MAPs 1, 2, tau, non-neural MAP4: Structural MAPs, aid in MT assembly, cross link MTs.

Kinesin: Moves cargo towards plus ends (out to the plasma membrane)

Dynein moves towards the minus end (back towards the centrosome)

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Cytoplasmic MTs

Back 19

From centrosome, distal ends are plus.

Intracellular transport of vesicles, mRNAs, and signaling complexes.

Spatial distribution of major organelles, including the ER, mitochondria, lysosomes, and golgi.

Controls whole cell migration

Changes cell shape

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Mitotic apparatus

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Structure upon which chromosomes are separated to daughter cells

Formation of mitotic apparatus, or spindle

Attachment to kinetochores of chromosomes

Separation of chromosomes in anaphase

Induction and placement of the cytokinetic furrow

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Axonemes of flagella and cillia

Back 21

Cilliated cells usually have a lot of cillia (200-300, 2-10um long, 0.3-0.5um diameter) at apical surfaces. Used for clearance of mucous by respiratory epithelium, passing of an oocyte along oviduct.

Flagella: Usually only 1 (100-200um, 0.3-0.5 diameter). Sperm- motility.

Axoneme generates motion by sliding of filaments against eachother, uses axonemal dynein and ATP.

Front 22

Cillary or Flagellar Axoneme

Back 22

Interaction of MTs with other associated proteins (nexin, radial spoke protein, dynein)

Dynein has outer arm and inner arm. Attached to the A subfiber. B subfiber is attached to A subfiber on the other side. Between the circle is nexin link, and radial spoke is between the central pair and outer circle.

Front 23

Basal body

Back 23

Subplasmalemmal centriole with specialized function. 9+2 arrangement.

Front 24

Primary cilia

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Axonemal arrangement of MTs may vary. Plamsma membranes are enriched with receptors specific for signal transduction pathways.

Sensory antenna (serotonin, PDGF, somatostatin, etc.)

Olfactory cells, hair cells of inner ear, light sensing cells of retina.

Front 25

Primary cillary dyskinesia (PCD or Kartagener's syndrome)

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Immotile cilia and flagella. AR- immotility of axoneme caused by defect in inner or outer dynein arms, or in radial spokes.

Frequent respiratory tract infections (rhinitis, sinusitis, otitis media, bronchiectasis). Immotile sperm, infertility, ectopic pregnancies.

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Abberant mitoses in neoplasia

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Indicator for the development of cancer, often forming too many centrosomes.

Front 27

Cancer chemotherapy

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Vincristine and vinblastine kill dividing cells by polymerizing mitotic spindle MTs.

Paclitaxel halts cell division by stabilizing mitotic spindle and not allowing MTs to depolymerize. Ovarian or breast cancers.

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Proteins that can be involved in cancer drug treatment

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Agains mitotis-specific kinesins

Kinases like aurora and polo-like kinases that act during mitosis

Other proteins that regulate spindle assembly checkpoint.

Front 29

Colchicine

Back 29

Gout

Supression of the activation of neutrophiles.

Also used for chromosome isolation for karyotype analysis

Front 30

Alzheimer's disease

Back 30

MAP- Tau is abnormally phosphorylated. Makes the paired helical filaments found within neurofibrillary tangles.

Front 31

PKD

Back 31

Primary cilia- can not sense flow of urine, divid too much to form large cysts

Front 32

Retinal degeneration

Back 32

Inner and outer segments of rods and cones are connected by primary cillum that is required for proper transport of material.

Front 33

Bardet-Biedl syndrome

Back 33

Retinal degeneration, early onset obesity. Defect in cillium/basal body construction.

Front 34

Intermediate filaments: History and evolution

Back 34

First found in muscle, intermediate size.

Primordial gene may have been nuclear lamin-like. Nuclear localization signal must have been deleted.

Cytoplasmic IFs not in all eukaryotes.

More than 50 different IF proteins

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Intermediate filaments: Structure

Back 35

10nm diameter, long alpha-helical coiled coils in center (allow tight binding, highly conserved), 2 globular ends (may be exposed on surface of IF, may impart tissue specific functions).

Hard to disassemble

Front 36

Subunits:

Type I Keritins
Type II Keritins

Back 36

Type I- form obligate heteropolymers with Type II proteins.

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Subunits:

Type III

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1. Vimentin- mesenchymal tissue (fibroblasts

2. Desmin- Muscle tissues

3. GFAP- Astrocytes

4. Peripherin

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Subunits:

Type IV: Neurofilaments

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NF-L
NF-M
NF-H
alpha-internexin

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Subunits:

Type V

Back 39

Nuclear lamins

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Plectin

Back 40

IF associated proteins (IFAPs)

Link IFs--> cytoskeletal networks

Found in hemidesosomes

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Desmoplakin

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IFAP
Found in cell-cell junctions (desmosomes)

Front 42

BPAG1

Back 42

IFAP

IF-->hemidesosomes in epithelial cells. When mutated, blistering disease.

Front 43

Filaggrin

Back 43

IFAP

Found in skin, bundles of keratin (epithelial cell-specific intermediate filaments) together.

Front 44

Intermediate filaments: Assembly

Back 44

Multiple stages

Monomers bind to form parallel, coiled coil dimers

Dimers bind to eachother in anti-parallel fashion to form staggered tetramers. Tetramers equiilant to actin monomers in solution, but not structurally polar.

Tetramers form 8 parallel protofilaments.

Rapid, phosphorylation may induce breakdown. Lamin hyperphosphorylation induces nuclear envelope breakdown at end of prophase. N-terminal domain and coiled-coil domain are essential.

Front 45

Intermediate filaments: Functions

Back 45

Mechanical strength and continuity to cell layers and muscle tissues

Nuclear shape

Lamins have been linked to DNA replication machinery in S-phase

Maintaining diamer of large-caliber axons

Front 46

Cell type of origin

Back 46

Can be stained by pathologists to figure out where the cancer came from

Front 47

Epidermolysis bullosis

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Mutant cytokeratin proteins- faulty assembly abilities. Hemidesmosomes lack structural integrity. Skin tissue blisters and does not hold together well

Front 48

Amyotrophic lateral sclerosis (Lou Gehrig's disease)

Back 48

Mutagenized neurofilament proteins.