Physiology 3140A Lecture Notes - Lecture 10: Vimentin, Intercalated Disc, Intermediate Filament
Physiology 3140 Lecture 10
Cytoskeleton
September 29 2017
Cytoskeleton
- Network of protein fibers that extend throughout the cytoplasm
- Nomenclature is based on size of the structure that it takes
- Many functions:
o Cell adhesion and movement (migration)
▪ Fibroblasts migrating
▪ Lung tumor cells migrating – have stress fibers (actin)
o Cell shape and structure
▪ Mesenchymal cells vs. epithelial cells; look different due to cytoskeleton
o Endocytosis/exocytosis (secretion and recycling)he
o Organelle/ protein transport
▪ How the transferrin receptor comes into early endosome; vesicle moves along the
cytoskeleton
o Mitosis/ Cytokinesis
o Cilia and Flagella
o Muscle contraction
Categories
Structure
Diameter
Examples
Microtubules
Protofilaments
25 nm
LARGEST
Tubulin (alpha + beta subunits)
Actin (microfilaments)
Double helix (of
actin)
6-7 nm
SMALLEST
Actin
Intermediate filaments
- Two anti-parallel
helices
- Form tetramers
10 nm
Vimentin (mesenchymal cells)
Desmin (muscle cells)
Keratin (epithelial cells)
Nuclear Lamins (NOT LAMININ)
- Vimentin is a good marker for mesenchymal call
- Intercalated disks in the heart, desmosomes attached to intermediates filaments (desmin) in the
heart
- Keratin is the IF found in skin
MICROTUBULES
- Polymer of and b tubulin dimer (basic subunit)
- GTP-binding subunits come together in protofilaments (lines) to form hollow tubes
o Protofilaments come together to form a hollow tube = microtubule
o Originally, both alpha and beta tubulin are GTP bound
o As the MT grows, the GTP is cleaved off the beta tubulin subunit
▪ Get GTP-GDP-GTP-GDP pattern
- Dynamic Instability
o Balance between assembly(polymerization) + disassembly(catastrophe) + reassembly
(rescue)
o Dependent on subunit critical concentration and temperature
find more resources at oneclass.com
find more resources at oneclass.com
▪ Subunit critical concentration: need to constantly add GTP bound alpha-beta tubulin
to the positive end for catastrophe to be averted
• The second you start to lose subunits (do not pump enough GTP bound
tubulin at positive end), catastrophe occurs b/c you cleaved to GDP bound
form
▪ Temperature: any fluctuation in temperature, MT will fall apart (esp. low
temperatures)
o Dependent on GTP-b-tubulin cap
▪ Growth occurs as positive end via GTP bound alpha beta tubulin
▪ As it grows, keep adding GTP, but some get cleaved to GDP
Centrosome
- Centre of the cell, near the nucleus
- MicroTubule Organizing Center (MTOC)
- Major MTOC in cells
- Microtubules radiate outward from the MTOC
- Negative end of MT attached to the MTOC, positive end radiates outward toward the cell periphery
Microtubule Associated Proteins (MAPs)
- Can alter microtubule stability
o Can cap the positive ends to prevent them from undergoing catastrophe
- Bundle microtubules together
- Can be regulated –phosphorylation promotes disassembly
o MAP-kinase – phosphorylates MAP
MAP is a substrate in vitro for kinases and phosphatases because easily phosphorylated
+TIPs (MT + end tracking proteins)
- E.g., EB1 (End Binding protein 1)
- Possibly stabilize MTs to reduce catastrophe
- Negative end of the MT binds to MTOC, positive end that is growing EB1 adds to the very tip
- EB1 = (+) end binding protein
- Help MT grow and protect themselves
- MT that is growing, EB1 bound to it, and then it falls off MT grows a little bit, then EB1 come back
and falls off
o As the MT grows, have a concentration of EB1 off and on
o When EB1 concentrated at tip of MT = see sharp signal
o When hazy: no EB1
find more resources at oneclass.com
find more resources at oneclass.com
MT Severing Proteins
- Destabilize MTs
o Slice MT in half and it falls apart
- E.g., Katanin
Microtubules are Superhighways for transport:
- Kinesin
o MT + end motor protein
▪ Positive ends are in the periphery; these motors take vesicles and move them
toward the cell periphery (OUT)
o Many types (> 14 known classes)
o Made of two chains: heavy chain and light chain
o Heavy chains
▪ ATPase activity and bind MTs
▪ ATP dependent
• Cycle ATP-ADP-ATP-ADP; binds with ATP, cleaves the ATP, then it falls off,
conformation change, next leg moves + thats how it gets along
o Light chains recognize cargo
o Anterograde movement: movement towards the periphery of the cell (MOVE FWD –
POSITIVE END)
o ATP hydrolysis causes conformational changes which results in movement
- Dynein
o MT - end motor protein
▪ Move from the cell surface back to the center of the cell
o ATPase activity
o Interacts with another accessory protein, Dynactin, which binds to cargo
▪ Dynein associates with MT, dynactin is the substrate that binds to the cargo
▪ Dynein = heavy chain, dynactin = light chain
o Retrograde movement: moving towards the center of the cell (NEGATIVE END)
o ATP hydrolysis causes conformational changes which results in movement – just like kinesin
- Majority of organelles will bind to dynein or kinesin
o Mitochondria will bind to both because they move back and forth (not targeted to one part of
the cell versus the other, they just move along)
- MAJOR DIFFERENCES
o Kinesin:
▪ Anterograde: towards (+)ve end
▪ Light chain+ heavy chain
o Dynein:
▪ Retrograde: towards (-)ve end
▪ Interact with dynactin
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Network of protein fibers that extend throughout the cytoplasm. Nomenclature is based on size of the structure that it takes. Vimentin is a good marker for mesenchymal call. Intercalated disks in the heart, desmosomes attached to intermediates filaments (desmin) in the heart. Keratin is the if found in skin. Polymer of and b tubulin dimer (basic subunit) Dynamic instability: balance between assembly(polymerization) + disassembly(catastrophe) + reassembly (rescue, dependent on subunit critical concentration and temperature. Negative end of mt attached to the mtoc, positive end radiates outward toward the cell periphery. Can alter microtubule stability: can cap the positive ends to prevent them from undergoing catastrophe. Can be regulated phosphorylation promotes disassembly: map-kinase phosphorylates map. Map is a substrate in vitro for kinases and phosphatases because easily phosphorylated. Negative end of the mt binds to mtoc, positive end that is growing eb1 adds to the very tip. Destabilize mts: slice mt in half and it falls apart.