Cytoskeleton

1
Cytoskeleton
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments

• Overview
• Experimental Methods
• Microtubules
• Microfilaments

(Updated 4/9/08)
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A. Overview
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments

• Definition
• Types of Cytoskeleton Fibers
• Dynamic Polymerization/Depolymerization
• Molecular Motors
• Alberts Fig. 16 1, Panel 16 1, Panel 16 2,
  Fig. 16 11, Fig 16 12, 16 8, 16 7, 16
  10, 16 13, 16 14, 16 15, 16 16, 16 17,
  16 19, 16 56, Table 16 1

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B. Experimental methods
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments

• Visualization Approaches
• Light Microscopy
• Fluorescence Microscopy
• http//www.itg.uiuc.edu/exhibits/gallery/fluoresce
  ncegallery.htm
• Digital/video Microscopy
• Electron Microscopy
• Genetic Approaches
• Biochemical Approaches

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C. Microtubules
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

1. Structure
2. Microtubule-associated proteins
3. Functions
4. Microtubule motors
5. Microtubule organizing centers
6. Dynamic properties of microtubules
7. Flagella and cilia

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C.1. Microtubules Structure
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Structure
• Alberts Fig 16 11
• Structure and composition - hollow, tubular
  found in most eukaryotic cells (cilia, spindle,
  flagella)
• Outer diameter - 24 nm
• Wall thickness - 5 nm
• May extend across cell length/breadth
• Wall composed of globular proteins arranged in
  longitudinal rows (protofilaments)
• Protofilaments are aligned parallel to tubule
  long axis
• In cross section, consist of 13 protofilaments
  arrayed in circular pattern within wall

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C.1. Microtubules Structure
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Each protofilament is assembled of dimeric
  building blocks (one a-tubulin one b-tubulin A
  heterodimer) organized in linear array along
  length of protofilament
• Two types of tubulin subunits have similar 3D
  structure fit tightly together
• Protofilaments asymmetric (a-tubulin at one end,
  b-tubulin at other) All in single MT have same
  polarity Each assembly unit has 2 nonidentical
  components (heterodimer)
• All protofilaments of microtubule have same
  polarity Thus so does full tubule (plus-
  minus-end)
• Plus end - fast-growing (b-tubulins on tip)
  Minus end - slow-growing (a-tubulins on tip)

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C.2. Microtubules MAPs
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Microtubule-associated proteins
• Alberts Fig 16-40, 16-41
• MTs can assemble in vitro from purified tubulin,
  but MAPs are found with MTs isolated from cells
  most found only in brain tissue MAP4 has wider
  distribution
• Have globular head domain that attaches to MT
  side filamentous tail protruding from MT
  surface
• May interconnect MTs to help form bundles
  (cross-bridges), increase MT stability, alter MT
  rigidity, influence MT assembly rate

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C. 2. Microtubules MAPs
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• MAP activity controlled by addition removal of
  phosphate groups from particular amino acid
  residues by protein kinases phosphatases,
  respectively example - Alzheimers disease (AD)
• Abnormally high MAP (tau) phosphorylation
  implicated in fatal neurodegenerative diseases
  like AD neurofibrillary tangles in brains made
  of hyperphosphorylated tau may help kill nerve
  cells
• Excessively phosphorylated tau molecules are
  unable to bind to MTs people with one of these
  diseases, a type of dementia called FTDP-17,
  carry mutations in tau gene, implicating it as
  cause

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C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Functions
• Alberts Table 16-2 Fig 16-23, 66
• Internal skeleton (scaffold) providing structural
  support maintaining organelle position
• Resist compression or bending forces on fiber
  provide mechanical support like girders in tall
  building prevent distortion of cell by
  cytoplasmic contractions
• MT distribution conforms to helps determine
  cell shape flattened, round cells - radiate from
  nuclear area columnar epithelium - parallel to
  cell long axis like aluminum rods support tent

10
C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Elongated cell process (axon, axopods of
  heliozoan protists) - MTs oriented parallel to
  each other axon or axopod long axis help move
  things
• In developing embryo, extend growing central NS
  axons to peripheral NS inhibit (colchicine CO,
  nocodazole NO) outgrowth stops, regresses
  (collapses back to rounded cell body)
• Found as core of axopodial processes of heliozoan
  protozoa many MTs arranged in spiral with
  individual MTs traversing entire length of process

11
C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Plants play similar role in plants affect shape
  indirectly by influencing cell wall formation
  found in cortex just below membrane during
  interphase forming a distinct cortical zone

12
C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Also have role in maintenance of cell internal
  organization (organelle placement) - disrupt MTs
  (CO, NO) gt Golgi disperses to cell periphery
  goes back to cell center when inhibitors removed
• Move macromolecules organelles around cell in
  directed manner (intracellular motility)
• Halt vesicle transport between compartments if
  disrupt MTs so transport dependent on them
• Proteins made in neuron cell body move down axon
  (neurotransmitters, etc.) in vesicles

13
C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Different materials move at different rates
  fastest rate is 5 µm/sec (400 mm/day) vesicles
  seen attached to MTs
• Structures materials moving toward neuron
  terminals are said to move anterograde
• Other structures, like endocytic vesicles that
  are formed at neuron terminals carry regulatory
  factors from target cells, move from synapse to
  cell body in a retrograde direction
• Ex. axons filled with MTs, MFs IFs evidence
  suggests that both anterograde retrograde
  movement are mediated mostly by MTs video
  microscopy shows vesicles moving along MTs
• Confirmed by EM of axons molecular motors move
  vesicles along the MTs that serve as tracks

14
C. 3. Microtubules Functions
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Motile elements of cilia flagella (more later)
• Active components of mitotic/meiotic machinery
  move chromosomes

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C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Microtubule motors
• Alberts Fig 16-58, 59, 60, 62, 63, 64, 67
• Motor proteins convert chemical energy stored in
  ATP into mechanical energy that is used to move
  cellular cargo attached to motor
• Types of cellular cargo transported by these
  molecular motors include vesicles, organelles
  (mitochondria, lysosomes, chloroplasts),
  chromosomes, other cytoskeletal filaments
• A single cell may contain dozens of different
  motor proteins, each specialized for moving a
  particular type of cargo in particular cell region

16
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Collectively, motor proteins are grouped into 3
  broad families myosins, kinesins, dyneins
• Kinesins dyneins move along MTs myosins move
  along MFs None known for ifs
• Motor proteins move unidirectionally along their
  cytoskeletal track in a stepwise manner from one
  binding site to the next
• As they move along, they undergo a series of
  conformational changes (a mechanical cycle)
• Steps of mechanical cycle are coupled to chemical
  cycle, which provides energy fueling movement
• Includes motor binding ATP, ATP hydrolysis,
  product (ADP Pi) release binding of new ATP
• Binding hydrolysis of 1 ATP moves motor a few
  nm along track Cycles repeated many times

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C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Kinesins
• Kinesins move vesicles/organelles from cell body
  to synaptic knobs isolated in 1985 from squid
  giant axons tetramer made of 2 identical heavy
  chains 2 identical light chains smallest
  best understood
• Large protein - pair of globular heads generate
  force by hydrolyzing ATP bind MT each head
  connected to a neck, a rodlike stalk fan-shaped
  tail that binds cargo to be hauled
• Diverse superfamily of kinesins - heads similar
  since roles similar tails vary since they haul
  different cargoes

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C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• In vitro mobility assay - kinesin-coated beads
  move to MT "" end (axon tip) it is a ""
  end-directed MT motor, therefore, kinesin
  responsible for anterograde movement
• All MTs of axon are oriented with"-" ends facing
  cell body "" ends facing synaptic knobs
• Moves through ATP-dependent cross-bridge cycle
  along single MT protofilament (rate proportional
  to ATP up to 1 µm/sec) at low
  concentrations, move slowly see movement in
  distinct steps
• Each step is 8 nm in length, the spacing between
  tubulin dimers along protofilament
• Appear to move 2 globular subunits (or 1 dimer at
  a time) usually toward membrane "" ends

19
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Kinesin possesses 2 motor domains that work by
  hand-over-hand mechanism one always firmly
  attached to MT
• 2 heads of kinesin behave in coordinated manner,
  so that they are always present at different
  stages in their chemical mechanical cycles at a
  given time
• When one head binds to MT, the interaction
  induces a conformational change in adjacent neck
  region of motor protein it swings the other head
  forward toward binding site on next dimer
• Force generated by head catalytic activity leads
  to swinging movement of neck
• A kinesin molecule walks along a MT, hydrolyzing
  one ATP with each step

20
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Conventional kinesin (discovered in 1985) is only
  one member of a superfamily of related kinesins
• Mammalian genome sequence analysis leads to
  estimate that mammals make gt50 different kinesins
• Heads of kinesins have related amino acid
  sequences, reflecting common evolutionary
  ancestry their similar role in moving along MTs
• In contrast, kinesin tails have diverse
  sequences, reflecting variety of cargo different
  proteins haul

21
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Most kinesins travel toward the "" end but one
  small subfamily of kinesins (including the
  Drosophila Ncd protein) moves toward the MT "-"
  end
• one would expect that the heads of ""-
  "-"-directed would have a different structure
  since the heads contain the catalytic core of the
  motor domain
• But the heads are virtually indistinguishable
  instead differences in direction of movement are
  determined by differences in the adjacent neck
  regions of the two proteins
• When the head of a "-" end-directed Ncd molecule
  is joined to the neck-stalk portions of a kinesin
  molecule, the hybrid protein moves toward the ""
  end of a MT track
• Even if the hybrid has a catalytic domain that
  would normally move toward the "-" end of a MT,
  as long as it is joined to the neck of a "" end
  motor, it moves in the "" direction

22
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• A third subfamily of kinesinlike proteins is
  incapable of movement kinesins of this group,
  like KXKCM1, are thought to destabilize MTs
  rather than acting as MT motors

23
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Cytoplasmic Dyneins
• Dyneins - first MT-associated motor found (1963)
  responsible for moving cilia flagella
• Thought to be ubiquitous eukaryotic motor
  protein related protein found in variety of
  nonneural cells
• Cilia/flagella form of protein was called
  axonemal dynein its new relatives were called
  cytoplasmic dynein
• Huge protein (1.5 million daltons) 2 identical
  heavy chains variety of intermediate light
  chains
• Each dynein heavy chain forms large globular head
  (10X larger than a kinesin head) that generates
  force moves along MT toward "-" end

24
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Suggested roles of cytoplasmic dynein
• Force generating agent for chromosome movement in
  mitosis
• "-"-directed MT motor for Golgi complex
  positioning movement of vesicles/organelles
  through cytoplasm
• In nerve cells, cytoplasmic dynein involved in
  axonal retrograde organelle movement (toward cell
  body cell center) anterograde movement of MTs
• Fibroblasts other nonneural cells may move
  varied membranous organelles (endosomes,
  lysosomes, ER-derived vesicles going toward
  Golgi) from periphery toward cell center

25
C. 4. Microtubules Motors
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Cytoplasmic dynein does not interact directly
  with membrane-bounded cargo, but requires
  intervening multisubunit complex, dynactin that
  may regulate dynein activity help bind it to MT
• Present model may be overly simplistic kinesin
  cytoplasmic dynein move similar materials in
  opposite directions over the same railway network
• Individual organelles may bind kinesin dynein
  simultaneously although only one is active at
  given time myosin may also be present on some of
  these organelles

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C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Microtubule-organizing centers
• Alberts Panel 16-1 Fig 16-29, 30, 31, 32, 33
  Function of MT in living cell depends on its
  location orientation, thus it is important to
  understand why a MT assembles in one place as
  opposed to another
• controlled by MT-organizing centers (MTOCs)

27
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Assembly of MTs from ab-dimers occurs in 2
  distinct phases
• Nucleation - slower small portion of MT
  initially formed occurs in association with
  specialized structures in vivo called
  microtubule-organizing centers (MTOCs)
  centrosome is example
• Elongation - more rapid

28
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Centrosomes - complex structure with 2
  barrel-shaped centrioles surrounded by amorphous,
  electron dense pericentriolar material (PCM)
• In animal cells, cytoskeleton MTs typically form
  in association with centrosome
• Centrioles cylindrical 0.2 nm dia typically
  twice as long usually with 9 evenly spaced
  fibrils
• Each fibril seen in cross section to be composed
  of 3 fused MTs (A, the only complete one B
  C), A is attached to centriole center by radial
  spoke

29
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• 3 MTs of each triplet arranged in pattern that
  gives centriole a characteristic pinwheel
  appearance
• Centrioles usually in pairs at right angles to
  each other near cell center just outside nucleus
• Extraction of isolated centrosomes with 1 M
  potassium iodide removes 90 of PCM protein
  leaving behind spaghetti-like scaffold of
  insoluble fibers
• Centrosomes are sites of convergence of large
  numbers of MTs

30
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• MT polymerization disassembly - treat with
  poisons (CO, NO) or cold gt MTs disassemble much
  has been learned about their disassembly
  reassembly in cultured animal cells in this way
• Observe assembly when cells warmed or poisons
  removed fix at various times after stain with
  fluorescent anti-tubulin ABs
• Within a few minutes of inhibition removal, 1 or
  2 bright fluorescent spots seen in cytoplasm
• Within 15 - 30 minutes, number of labeled
  filaments radiating from these foci rises
  dramatically

31
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• In EM MTs radiate out from centrosome MTs don't
  actually penetrate into centrosome contact
  centrioles, but terminate in dense pericentriolar
  material residing at centrosome periphery
• PCM apparently initiates MT formation centrioles
  not involved in MT nucleation, but they probably
  play a role in recruiting surrounding PCM during
  centrosome assembly

32
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Centrosome typically situated near center of
  cell, just outside nucleus
• Columnar epithelium - centrosome moves from cell
  center to apical region just beneath cortex
  cytoskeletal MTs emanate from site, extending
  toward nucleus basal surface of cell
• Regardless of location, centrosomes are sites of
  MT nucleation polarity is always the same "-"
  end at centrosome, "" (growing) end at opposite
  tip
• Thus, even though MTs are nucleated at MTOC, they
  are elongated at opposite end of polymer

33
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Not all MTs associated with centrosome
• some animal cells (mouse oocytes) lack
  centrosomes entirely, but still make spindle
• MTs of axon are not associated with centrosome,
  which is located in cell body, but they may be
  formed at centrosome, then released from that
  MTOC carried to axon by motor proteins

34
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Basal bodies other MTOCs
• Centrosomes are not the only MTOCs in cells
  basal bodies at base of cilia flagella serve as
  origin of ciliary flagellar MTs MTs grow out
  of them
• Basal body cross-section looks like centriole in
  fact, the two can give rise to one another
• Sperm flagellum arises from basal body derived
  from sperm centriole that had been part of
  meiotic spindle of spermatocyte from which the
  sperm arose
• Conversely, sperm basal body typically becomes
  centriole during fertilized egg's first mitotic
  division of fertilized egg

35
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Plant MTOC - lack both centrioles centrosomes
  MTOCs more dispersed than those of animals
• In plant endosperm cells, the primary MTOC
  consists of patches of material situated at outer
  surface of nuclear envelope from which
  cytoskeletal MTs emerge
• MT nucleation also thought to occur throughout
  plant cell cortex

36
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• MT nucleation
• Despite diverse appearances, all MTOCs play
  similar roles in all cells
• Control number of MTs that form their polarity
• Control the number of protofilaments that make up
  their walls
• Control the time location of MT assembly

37
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• All MTOCs share a common protein component,
  g-tubulin (discovered in mid-1980s) it is
  0.005 of total cell protein while a-
  b-tubulins are 2.5 of total nonneural cell
  protein
• Fluorescent anti-g-tubulin antibodies (ABs) stain
  all MTOCs, like centrosome PCM suggests it is
  critical component in MT assembly nucleation
• Microinject anti-g-tubulin AB into living cell gt
  blocks MT reassembly after depolymerization by
  drugs or cold temperatures
• Genetically engineered fungi lacking functional
  g-tubulin gene cannot assemble normal MTs

38
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Nucleation mechanism revealed by
  structure/composition studies of PCM at
  centrosome periphery
• Insoluble fibers of PCM are thought to serve as
  attachment sites for ring-shaped structures that
  have same diameter as MTs (25 nm) contain
  g-tubulin
• Ring-shaped structures found when centrosomes
  were purified incubated with gold-labeled
  anti-g-tubulin ABs gt cluster in
  rings/semi-circles at MT minus ends (ends
  embedded in PCM)
• Isolate similar ring-shaped complexes (g-TuRCs)
  from cell extracts nucleate MT assembly in vitro

39
C. 5. Microtubules MTOCS
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Model - helical array of 13 g-tubulin subunits
  forms open, ring-shaped template on which first
  row of ab-tubulin dimers assemble
• Only a-tubulin of heterodimer can bind to ring of
  g-subunits, establishing polarity of entire MT
• 2 other tubulin isoforms d-tubulin e-tubulin
  have also been identified in centrosomes, but
  their function has not been determined

40
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Dynamic properties of microtubules
• Alberts Table 16-2 Fig 16-16, 16-17
• MTs vary markedly in stability even though
  similar morphologically - spindle/cytoskeleton
  labile mature neuron MTs less labile
  cilia/flagella very stable lability allows cell
  to respond to stimuli
• Cilia/flagella MTs are stabilized by MAP
  attachment by enzymatic modification (e. g.
  acetylation) of specific amino acid residues
  within tubulin subunits
• Labile MTs in living cells can be disassembled
  without disrupting other cell structures via a
  number of treatments

41
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Treatments that cause MT disassembly usually
  interfere with noncovalent bonds holding them
  together
• Cold temperatures
• Hydrostatic pressure
• Elevated Ca2 concentration
• Variety of chemicals (often used in chemotherapy)
  - CO, vinblastine, vincristine, NO,
  podophyllotoxin

42
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Some treatments (taxol) disrupt MT dynamic
  activity act by doing the opposite inhibit
  disassembly
• Taxol binds MT polymer thus prevents
  disassembly cell cannot build new MT structures

43
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Cytoskeletal MT lability reflects fact that they
  are polymers formed by noncovalent association of
  dimers subject to depolymerization/repolymerizati
  on as cell needs change
• Dramatic changes in MT spatial organization may
  be achieved by combination of 2 separate
  mechanisms
• Rearrangement of existing MTs
• Disassembly of existing MTs reassembly of new
  ones in different cell regions

44
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Study of MT dynamics in vitro - suggest that
  cytoskeleton can rapidly remodel respond to
  stimuli
• Early studies established that GTP binding to b
  -subunit required for MT assembly GTP hydrolysis
  not needed for binding, but it is hydrolyzed soon
  after dimer attached to MT end GDP stays bound
• After dimer is released from MT during
  disassembly enters soluble pool, GDP is
  replaced by GTP, thus recharging dimer so that it
  can add to polymer again
• A GTP molecule is also bound to a-tubulin
  subunit, but it is not exchangeable it is not
  hydrolyzed after subunit incorporation

45
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Assembly is not energetically inexpensive since
  it includes GTP hydrolysis, but it does allow the
  cell to control assembly disassembly
  independently
• A dimer being added to MT has a bound GTP dimer
  being released from MT has bound GDP
• GDP- GTP dimers have different conformations
  participate in different reactions the ends of
  growing shrinking MTs have different structures

46
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• The above facts lead to the following model
• When a MT is growing, the "" end is present as
  an open sheet to which GTP-dimers are added
• During rapid growth periods, tubulin dimers are
  added faster than GTP can be hydrolyzed
• The resultant cap of GTP-dimers on MT at
  protofilament ends is thought to favor the
  addition of more subunits hence MT growth
• However, MTs with open ends thought to undergo
  spontaneous reaction leading to tube closure

47
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• tube closure is accompanied by hydrolysis of
  bound GTP, changing tubulin dimer conformation gt
  resultant mechanical strain destabilizes MTs
• Strain is released as protofilaments curl out
  from tubule catastrophically depolymerize
• Disassembly can occur remarkably fast, especially
  in vivo, which allows very rapid MT cytoskeleton
  disassembly

48
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Study of MT dynamics in vivo dynamic character
  of MT cytoskeleton inside cell is best revealed
  by microinjecting labeled tubulin into
  nondividing cultured cell
• Inject labeled tubulin into nondividing cultured
  cell gt labeled subunits rapidly incorporated
  into preexisting cytoskeleton MTs, even in
  absence of any obvious morphological change
• Watch cell with fluorescent-labeled MTs over time
  gt some MTs grow, others shrink dynamic
• Both growth shrinkage in vivo occur
  predominantly at "" end of polymer, the end
  located opposite the centrosome (or other MTOC)

49
C. 6. Microtubules Dynamic
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Single MTs switch randomly unpredictably
  between growing shrinking (dynamic instability)
• MTs shrink faster than they grow, so in a matter
  of minutes, MTs disappear are replaced by new
  MTs that grow out from centrosome

50
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Cilia and flagella structure
• Alberts Fig 16-80, 81, 82, 83, 84
• Entire ciliary or flagellar projection is covered
  by membrane continuous with cell membrane
• Cilium core (axoneme) contains an array of MTs
  that run longitudinally through entire organelle
• Usually 9 peripheral doublet MTs surrounding
  central pair of single MTs known as 9 2
  pattern or array all MTs in array have same
  polarity ("" ends at tip, "-" ends at base)
• Doublets - 1 complete (A tubule 13 subunits) MT
  1 incomplete (B tubule) MT with 10 or 11 subunits

51
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Not all eukaryotes have them cilia flagella
  generally absent among fungi, nematodes insects
• Where they do occur, they nearly always show same
  9 2 array, a reminder that all living
  eukaryotes have evolved from a common ancestor
• Despite high degree of conservation (e. g. 9 2
  pattern) some evolutionary departures
• 9 1 array in flatworms
• 9 0 array in Asian horseshoe crab, eel,
  mayfly some lacking central elements are motile,
  some not

52
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Central MTs enclosed by projections forming
  central sheath sheath connected to doublet A MTs
  by radial spokes doublets connected by
  interdoublet bridge made of elastic protein nexin
• Pair of arms (inner outer) project from A MT in
  clockwise direction (when viewed base to tip)
• Radial spokes typically in groups of three with
  major repeat of 96 nm
• Inner outer dynein arms staggered along A MT
  length (outer arms spaced every 24 nm inner arms
  arranged to match unequal spacing of radial
  spokes)

53
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Cilia/flagellae emerge from basal bodies - 9
  peripheral fibers consisting of 3 MTs rather than
  2 (A tube complete, B/C incomplete) similar in
  structure to centrioles
• No central MTs as in centrioles also similar to
  centrioles in other ways
• A B tubules elongate to form cilia/flagella
  doublet if sheared off, regrow from basal body

54
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• The mechanism of ciliary flagellar locomotion
  sliding filament model suggested mechanism of
  ciliary/flagellar movement was sliding of
  adjacent MT doublets relative to one another
• In model, dynein arms act as swinging
  cross-bridges that generate forces needed for
  ciliary/flagellar movement dynein arms
  projecting from one doublet walk along adjacent
  doublet wall gt sliding

55
C. 7. Microtubules Flagella
A. Overview B. Experimental Methods C.
Microtubules 1. Structure 2. MAPs
3. Functions 4. Microtubule Motors 5.
MTOCs 6. Dynamic Properties 7. Flagella
and Cilia D. Microfilaments

• Sequence of events in ciliary/flagellar sliding
  motion
• Dynein arms anchored on a doublet's A MT attach
  to binding sites on B MT of adjacent doublet
• Dynein molecules undergo conformational change
  causes A MT doublet to move slightly toward basal
  end of attached B MT doublet
• Dynein then releases B tubule of adjacent doublet
• Dynein arms reattach to adjacent doublet's B MT
  closer to its base so another cycle can begin

56
D. Microfilaments
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

1. Structure
2. Polymerization/depolymerization
3. Myosin
4. Muscle Contraction
5. Non-muscle motility

57
D. 1. Microfilaments Structure
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Structure
• Alberts Fig 16-12
• Microfilaments
• 8 nm diameter
• made of globular actin subunits (G-actin)
• found in most animal cells, also higher plants
• Microfilament, actin filament, F-actin
  filaments are synonyms but F-actin often used
  for those formed in vitro

58
D. 1. Microfilaments Structure
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• In presence of ATP, G-actin polymerizes to form
  stiff filament made of 2 strands of F-actin wound
  around each other in a helical configuration
• Each subunit has polarity all subunits are
  pointed in same direction, so entire MF has
  polarity
• Depending on cell type activity in which it is
  engaged, MFs can be organized into highly ordered
  arrays, loose ill-defined networks, or tightly
  anchored bundles

59
D. 1. Microfilaments Structure
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Actin identified more than 50 years ago as one of
  major contractile proteins of muscle cells
• Since then found to be major protein in virtually
  every eukaryotic cell examined
• Higher plants animals possess number of
  actin-coding genes whose products are specialized
  for different types of motile processes
• Actin structure highly conserved evolutionarily
  (yeast actin rabbit skeletal actin 88
  identical) means that nearly all aminos are
  crucial to function actin from diverse sources
  can copolymerize

60
D. 1. Microfilaments Structure
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Actin detected microscopically
• By electron microscopy, using proteolytically
  cleaved myosin head fragments (HMM or S1
  fragments)
• HMM S1 bind actin all along MF gt see polarity
  in EM one end of MF pointed, other end barbed
• Orientation of arrowheads formed by S1-actin
  complex provides information as to direction in
  which MFs are likely to be moved by myosin motor
  protein
• By fluorescence microscopy, with fluorescently
  labeled S1 or anti-actin ABs

61
D. 2. Microfilaments P/D
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• MF polymerization/depolymerization
• Alberts Table 16 2, Fig. 16 36, 37, 38
• Before polymerization, actin monomer binds to
  adenosine nucleotide (usually ATP) like GTP in
  MTs
• Actin is an ATPase (like tubulin is GTPase) role
  of ATP in MF assembly is same as GTP in MT
• Some time after incorporation into growing actin
  filament, ATP hydrolyzed to ADP
• If filaments built at high rate, the end has
  actin-ATP cap (hinders disassembly, favors
  assembly)

62
D. 2. Microfilaments P/D
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Actin polymerization can be studied in vitro by
  labeling or viscosity studies
• In vitro with high concentration of labeled
  G-actin, both ends labeled but .....
• One end incorporates monomers at 5 - 10 times
  higher rate than the other end
• Decoration with S1 myosin fragment reveals that
  barbed ("" end) of MF is fast-growing end, while
  the pointed ("-") end is the slow-growing tip
• In lower concentrations of G-actin
• Actin-ATP subunits add to "" end actin-ADP
  subunits tend to leave from "-"
• Can be demonstrated by pulse-chase treadmilling
  experiments
• Dont know if treadmilling occurs in vivo

63
D. 2. Microfilaments P/D
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• MFs maintain a dynamic equilibrium between
  monomeric polymeric actin - can be influenced
  by a variety of different proteins
• Changes in local conditions in particular part of
  cell can push equilibrium either toward assembly
  or disassembly
• allows cell to reorganize its MFs cytoskeleton by
  controlling this equilibrium
• need such reorganization for dynamic processes
  (cell locomotion shape changes, cytokinesis)

64
D. 2. Microfilaments P/D
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Actin-binding proteins in the cell affect the
  nucleation and polymerization rate of
  microfilaments
• Formin A dimeric protein that initiates
  nucleation by capturing two monomers of actin,
  then remains associated with the plus end of a
  rapidly growing microfolament
• Thymosin A protein that binds to actin monomers
  and inhibits nucleotide exchange or
  polymerization, keeping much of the available
  actin in an unpolymerized state
• Profilin A protien that competes with thymosin
  for binding to actin monomers it binds opposite
  the ATP binding site on actin and promotes
  polymerization at the plus end of a growing
  filament

65
D. 2. Microfilaments P/D
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Inhibitors of microfilament polymerization/depolym
  erization used to study microfilament
  polymerization (Table 16 - 2

66
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Myosins
• Alberts fig 16 54, 55, 56, 57, 60, 61, 65, 68,
  69, 72
• Myosin's sole known function is as motor for
  actin
• Almost all motors known to interact with actin
  are members of myosin superfamily
• all of them move toward MF plus end (except for
  myosin VI)
• First isolated from mammalian skeletal muscle
  then from wide variety of eukaryotic cells
  protists, higher plants, nonmuscle animal cells,
  vertebrate cardiac smooth muscle tissues

67
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Structure of myosins
• All share characteristic motor (head) domain,
  which has a site that binds actin filament one
  that binds hydrolyzes ATP to drive the myosin
  motor
• While head domains of myosins are similar, tail
  domains are highly divergent
• Myosins also contain variety of low molecular
  weight (light) chains
• Based on these construction differences divided
  into 2 large groups - conventional
  unconventional

68
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Conventional (type II)
• found in various muscle tissues, and also in a
  variety of nonmuscle cells (generate tension at
  focal contacts, cytokinesis)
• Structure of myosin II molecules 6 polypeptide
  chains (one pair of heavy chains, 2 pairs of
  light chains) organized in a way that produces a
  highly asymmetric protein with 3 sections
• A pair of globular heads that contain the
  molecules catalytic site
• A pair of necks, each consisting of a single,
  uninterrupted a-helix 2 associated light chains
• A single, long, rod-shaped tail formed by the
  intertwining of long a-helical sections of the 2
  heavy chains to form an a-helical coiled-coil

69
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Immobilize isolated myosin heads (S1 fragments)
  on glass cover slip gt cause actin filament
  sliding
• Single head domain has all of the machinery
  needed for motor activity
• The fibrous tail plays a structural role,
  allowing the protein to form filaments
• Light chain phosphorylation regulates assembly of
  myosin II into thick filaments
• Tail ends of myosin molecule point toward
  filament center heads point toward ends
  (bipolar)
• Polarity of filament reverses at its center

70
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Skeletal muscle myosin II filaments are highly
  stable
• smaller myosin II filaments (most nonmuscle
  cells) often display transient construction
  (assembling when where needed, then
  disassembling)

71
D. 3. Microfilaments Myosins
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Unconventional myosins subdivided into at least
  14 different types
• Each type is presumed to have its own specialized
  functions
• Several types may be present together in same
  cell
• Some functions of unconventional myosins
• Amoeboid movement phagocytosis (myosin I)
• Movement of cytoplasmic vesicles organelles
  (myosins I, V, VI)
• Stereocilia in cochlea hair cells of inner ear
  (myosin VIIa)

72
D. 4. Microfilaments Muscle
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Muscle contraction
• Alberts 16 73, 74, 75, 76, 61, 77, 78
• Skeletal muscle cell structure - highly
  unorthodox cylindrical 10 - 100 µm thick up to
  400 mm long
• Skeletal muscle cells are multinucleate (100s),
  the result of embryonic fusion of mononucleate
  myoblasts (premuscle cells) even myoblasts from
  distantly related animals fuse in culture
• Because of their properties, these cells are more
  appropriately called muscle fibers

73
D. 4. Microfilaments Muscle
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Muscle fibers may have the most orderly internal
  structure of any cell in body
• Muscle fiber is cable made up of hundreds of
  thinner, cylindrical strands (myofibrils)
• Each myofibril is repeating linear array of
  contractile units (sarcomeres)
• Each sarcomere, in turn, has characteristic
  banding pattern that gives muscle fiber a
  striated look
• Myofibrils separated by cytoplasm with
  intracellular membranes mitochondria, lipid
  droplets, glycogen granules

74
D. 4. Microfilaments Muscle
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Banding pattern is result of partial overlap
  between thick thin filaments
• Each sarcomere extends from Z line to Z line
  (2.5 µm) contains several dark bands light
  zones there is a pair of light staining I bands
  at each end of sarcomere
• More densely staining A band is between outer I
  bands lightly staining H zone in A band center
• Densely staining M line lies in center of H zone
• I bands - only thin filaments H zone - only
  thick filaments A band outside H zone - both
  overlap

75
D. 4. Microfilaments Muscle
A. Overview B. Experimental Methods C.
Microtubules D. Microfilaments 1. Structure
2. P/D 3. Myosins 4. Muscle
Contraction 5. Nonmuscle Actin

• Composition organization of thin filaments
• Thin filaments mostly actin
• In addition to actin, thin filaments also contain
  two other proteins troponin tropomyosin
• Tropomyosin - elongated, 40 nm long fits
  securely into grooves between two thin filament
  actin chains each rod-shaped tropomyosin