How is monomeric actin recruited to the growing actin filament?

Formins:
• Formins are dimeric proteins (>15 formin genes exist)
that have binding sites for G-actin and profilin-actin complexes
• Formins have “whiskers” which are long filaments that bind
profilin-actin

Fig 16-17
Fig 16-18
Cross-linking of filaments increases stability and strength

Fig 16- 8
 Actin filament cross-linking proteins:
• proteins that contain two actin binding sites
• used to stabilize and link actin filaments together
• some promote actin bundling

a-actinin: found in cortical actin, along stress fibers, and in

cell adhesion zones
Fimbrin and villin: stabilize actin bundles in microvilli
Filamin: links cortical actin to integrins which attach to the
extracellular matrix (focal adhesions)
Spectrin and dystrophin: bind to integral membrane proteins
and actin filaments
 Actin filament cross-linking proteins:

Fig 16-22
• actin binding domains shown in red
Fig 16-23
Microvilli actin
Fig 16-24
 Filamin mutations and disease

• Periventricular heterotropia results from mutations in

filamin A gene
• Errors in neuronal migration during development
• Causes reduced brain size and epilepsy but not mental
impairment

Fig 16-24
 Actin signaling pathways
• mediated by small G protein family Rho
• three main members Rho, Rac, Cdc42

Cortical actin Filopodia

Stress fibers
(membrane ruffles)

• all members are active bound to GTP and inactive with GDP
• inject cells with constitutively activated mutant proteins

Fig 16-84
 Dynamic Actin Rearrangement:

• most cytoplasmic F-actin turns over every few minutes

• half of cellular actin is in polymerized state
• G-actin pool concentration is 500-1000 X higher than the
concentration needed to polymerize actin
• mostly bound to other proteins
(e.g. profilin/b-thymosin/CAPs)
• cell can respond rapidly to changes requiring actin
polymerization
(e.g. cell motility at the leading edge of fibroblasts)
 Why are there so many actin binding proteins?
• many actin binding proteins are essential genes (e.g.Arp2/3,
profilin, cofilin and capping protein in yeast)
• knock out of others causes more mild effects (some
have redundant functions)
• some (dystrophin) have effects later in life if mutated
(muscular dystrophy)
• most actin binding proteins have some basic function
in all cells
• others have more defined functions in some cells or under
specific conditions
 Microtubules:
• rigid polymers of a and b tubulin that provide structural
support and tracks for movement of organelles and proteins
• microtubules composed of polymers of a and b tubulin
heterodimers
• 25 nm in diameter and can be as long as 20um in vivo
• microtubules have polarity with growing plus (+) end and a
slower growing (-) end
• plus end is peripheral and the minus end is interior (usually
anchored to microtubule organizing center (MTOC))
• MTOCs are centrosomes/centrioles (cytoplasm) or
basal bodies (axonemes in cilia)
• motor proteins move along MTs (dynein and kinesin)
 Tubulin Structure:
• a and b tubulin each bind GTP or GDP
• Dimers are very stable and rarely dissociate
• GTP on a-tubulin is not exchanged readily (usually stays
GTP)
• GTP on b-tubulin is exchanged more quickly
• when MT is formed, b-tubulin GTP is hydrolysed
• assembly of MTs is much faster with GTP-tubulin than
GDP tubulin
• association and dissociation occurs only at ends of MTs
Fig 16-42
 Structure of MTs:

• MTs are cylinders composed of longitudinally arranged

protofilaments composed of tubulin dimers
• most common MTs have 13 protofilaments but some have
more (15, 16) or less (11)
• all MTs have b-tubulin at (+) end and a-tubulin at (-) end

Mechanical properties:
If enlarged million fold, MTs would have diameter of 25mm
(same mechanical properties of steel pipe)
Fig 16-44
Fig 16-44
 Microtubules:
Dynamic Instability:
• axoneme microtubules (in cilia) are stable for days/weeks
• spindle microtubules are turned over in minutes/seconds
• undergo rapid depolymerization and then regrowth
in minutes (leads to movement of chromosomes in mitosis)
• growing and shrinking MTs exist in steady state
• MTs resist compression, thus provide cytoskeletal support
• accessory proteins can bind tubulin dimers, stabilize MTs,
associate with MT ends or sever MTs

• microtubule associated proteins (MAPs)

Dynamic Instability:
• At steady state, individual MTs grow slowly until they undergo
rapid shortening (catastrophe)
• MT polymer loses tubulin at rate of 1000 dimers/sec
• protofilaments peel away from MT
• rapid shortening is terminated by random event (rescue)
where GTP-tubulin caps the plus end which is followed by
further growth
 Dynamic Instability:
• hydrolysis of b-tubulin GTP to GDP drives dynamic instability
• Dimeric tubulin hydrolyses b-tubulin GTP slowly, but it
increases when incorporated into MT (250X)
• a-tubulin GTP does not hydrolyse rapidly, therefore MTs
usually have GDP bound to b-tubulin and GTP bound to
a-tubulin
• GTP tubulin at the MT ends stabilizes the filament (GTP caps)
through direct exchange of GTP onto b-tubulin at (+) end
• loss of GTP cap is thought to cause a catastrophe
• frequency of catastrophe is inversely proportional to
concentration of GTP tubulin dimers
Fig 16-44
Fig 16-45
Fig 16-43
 Formation of a MT:

• MTs are not formed from spontaneous nucleation from

tubulin dimers (very slow)
• cellular MTs originate from microtubule organizing centers
(MTOCs) (e.g. centrioles and centrosomes)
g-tubulin ring complex (g-TuRC) in centrioles nucleates MT and
caps their minus ends (exists separate from centrioles)
Fig 16-47
Fig 16-47
Fig 16-46
Fig 16-46
Microtubule Associated Proteins (MAPs): (panel-page 933)Table 37-3
• proteins regulate MT stability, assembly and structure
• presence of MAPs is determined by cell type and shape
• MTs composed of 80% tubulin and 20% MAPs
• many MAPs are neuronal (vertebrates)
Panel 16-4
 MAPs:
MT stabilizing:
• many MAPs bind along length of the MT
• some rod shaped MAPs (MAP1A) radiate from MT and some
lie parallel to the protofilament (e.g. tektins-found in sperm and cilia)
Tau family of MAPs:
• includes MAP2 and MAP4 and Tau (all rod shaped)
• contain several MT binding domains (18aa) separated by flexible
linkers (13aa long)
• MAP2 keeps MTs apart from each other
 MT +
MAP2

MT

Pollard
Pollard
MAP2 Tau
Fig 16-51
 Tau family of MAPs:
Tau (MT associated protein tau- or MAPT) in disease
• in presence of tau, MTs grow 3X faster and have only 2%
catastrophe rates
• tau MT binding sites can be phosphorylated (which inhibits
MT binding and destabilizes MTs)
• tau is major MAP in axons in brain
• one tau gene gives 6 isoforms (alternate splicing)
• tau gene has 16 exons: exons 2, 3 and 10 are differentially spliced:
(2–3–10–; 2+3–10–; 2+3+10–; 2–3–10+; 2+3–10+; 2+3+10+)
• including partial phosphorylation, up to 30 tau species can
be present in brain from one gene
• mice with tau knock out are fine!!
 Alzheimer’s disease and tau:
• patients have ‘neurofibrillary tangles’ that are detected by
light microscopy- more = worse diagnosis
• tau forms paired helical filaments that aggregate in tangles
• ‘tangles’ contain highly phosphorylated tau that is
proteolytically truncated and cross-linked via disulfide bonds
• resistant to proteolysis and insoluble
• may cause dementia or may be by-product of disease
• some mutations in tau are genetically inherited as dementias
Alzheimer’s disease and tau:
 CTE disease, concussions and tau:
CTE (Chronic Traumatic Encephalopathy):
• 202 deceased athletes who had played American football from a brain
donation program, CTE was neuropathologically diagnosed in 177 players
across all levels of play (87%), including 110 of 111 former National Football
League players (99%).
CTE disease, concussions, microtubules and tau:
 MT destabilizing MAPs:

1. Promote catastrophe: Stathmin

• sequesters tubulin dimers and blocks polymerization
• phosphorylated in G2/M (inactive) and dephosphorylated in G1
• some cancer cells have high levels of mutated stathmin

2. Remove tubulin subunits: MCAK (kinesin 13 family)

• kinesin like (motor protein) that bind MTs and
ATP hydrolysis of motor domain stresses MT at plus end
and leads to disassembly (catastrophe factor)
Fig 16-54
• loss of MCAK leads to block in anaphase
(mitotic centromere associated kinesin)
 MT destabilizing MAPs:
3. Actively severs MTs: katanin
• katanin is heterodimer severs MTs into short fragments (active in
mitosis to sever non-spindle MTs
• short fragments depolymerize into tubulin dimers
• severing requires ATP hydrolysis by katanin
Fig 16-52
• several anti-cancer drugs target cytoskeletal elements
Taxol (paclitaxel) inhibits MT disassembly (isolated from bark
of yew tree)
Blocks cancer cells in mitosis