Lecture 15. Microfilaments, Muscles and Myosins Flashcards
What are the microvilli in the gut epithelial cells made of?
Actin fillaments
Where are microfilaments found in fibroblast cells?
Fibroblasts need to move towards source of wound to fill in gap, microtubules found on the leading edge
Do microfilaments have to be nucleated/emerge from the centrosome?
No, microvilli microfilaments emerge from the apical membrane
What are the three different structures that are made up of actin in epithelial cells?
Microvilli: extends the plasma membrane area
Cell cortex: branched network around inside of cell - processes include endocytosis
Adherens belt: contractile belt that uses myosin motor proteins in order to respond to force from adjacent cells
All nucleated from different places
What are the four different structures that are made up of actin in migrating cells?
Cell cortex: branched network around inside of cell - processes include endocytosis
Filopodia, lamellipodium/leading edge and stress fibres all act coordinately to move in a specific direction
In a migrating cell, where are the stress fibres located?
Ends of stress fibres buried in the focal adhesion site which contracts to allow the back end of the cell to follow the forward end of the cell
What happens to actin in cells when the cell undergoes cell division?
Cell focuses on the process of cytokinesis and forming a contractile, psychokinetic active myosin ring
This constricts the two cells apart after nuclear division to enable cellular division and absolution in order to create another epithelial or fibroblast cell
What is actin?
A globular protein (G-actin) which is divided by a central cleft that binds ATP. Actin filament appears as two strands of subunits. One repeating unit consists of 28 subunits (14 in each strand) covering a distance of 72nm in length. The filament has a clockwise helical twist such that the filament has symmetry every 36nm. The ATP binding cleft always binds to the opposite side of the adjoining actin molecule. This gives the filament polarity with the actin binding cleft exposed at the minus end. Filamentous actin is called F-actin.
How does actin assembly occur?
If given ATP, ATP-G actin will start binding to both ends, preferentially at the plus end where addition is 10 times faster. The dissociation rates from both ends however are similar.
In the filament ATP hydrolyses to ADP-Pi and Pi is released slowly giving rise to a filament containing ATP-actin, ADP-Pi-actin and ADP-actin. ATP-actin is added preferentially at the plus end while ADP-actin disassembles at the minus end, giving rise to treadmilling of subunits.
What is the interaction between actin and cytochalasin D and latrunculin A?
Binds actin monomers and prevents actin polymerisation
What is the interaction between actin and phalloidin?
Binds and stabilises actin filaments. This can be labeled with a fluorescent dye for staining actin filaments in cells. Stabilises F-actin
What is the role of thymosin-β4 (monomer binding protein)?
In cells G-actin is 1000 times more concentrated than the critical concentration for actin filament formation. However most G-actin is bound to thymosin-β4 and this cannot be incorporated into filaments. Raising thymosin-β4 levels by micro-injection inhibits actin filament assembly by sequestering actin from dissociation filaments
What is the role of profilin (monomer binding protein)?
Profilin binds G-actin more weakly than thymosin-β4, but stronger than actin + ends. Uniquely it allows ADP/ATP exchange and can promote ATP replacement and promotes actin incorporation into filaments. Profilin is mostly at the plasma membrane bound to PIP₂
What is the role of cofilin (filament severing proteins)?
Cofilin (also known as Actin destabilising factor or ADF) binds to the sides of ADP-actin in the filament, inducing them to fragment. In this manner cofilin replenishes the pool of free ADP- actin which can be recharged by profilin to be used again.
Cofilin chops actin into 14 sub-filaments
How do you end up with either straight or branched actin filaments?
Formin makes straight filaments of actin
Arp2/Arp3 complex makes branched actin filaments
How does formin induce straight actin filaments?
Formin only works in the cytoplasm when Rho bound to plasma membrane
Formin is a multi -domain protein containing Rho GTPase binding domain (RBD), and profilin- ATP-actin binding domain (formin homology domain 1, FH1) and a filament nucleating domain (FH2). When not bound to Rho the RBD binds and inhibits the FH2 domain. When Rho GTPase becomes activated formin is recruited to the plasmamembrane. This causes a conformational shift which releases the FH1 and FH2 domains to trigger straight actin filament formation
How does Arp2/Arp3 complex induce branching of actin filaments?
The angle at which branch filaments are nucleated is fixed at 70º. The Arp2/Arp3 complex is frequently located near the cell membrane and can be activated by proteins such as WASp and WAVE