Cytoskeleton and cell shape (L12) Flashcards
What defines cell shape? Why is it important?
Adjoining cells (physically shaped by neighbouring cells) cell adhesions Extracellular matrix (confines a cell like a straight jacket) Cell shape is important because it affects the function of nerves, muscles, blood etc. Cell shape is also involved in a subcellular activity like migration, phagocytosis, transport (vesicle shuttling) and cytoskeleton dynamics
What is the structure of the actin cytoskeleton?
The cytoskeleton is inside the cell (the ECM is on the outside). Its made mainly of filamentous actin (F-actin). It’s very stable under cytoplasmic conditions. Isn’t a large pool of unpolymerised filament proteins. Phosphorylation often drives disassembly, but this depends on the type. The actin polymerises into different structures e.g. lamellipodia which allow the cell to move forward, filopodia, used for sensing the environment, microvilli, cortical actin, which is responsible for dynamic changes, podosomes which anchor the cell, and stress fibres which strengthen the cell. Actin can exist as monomers when not being used. Intermediate filaments include keratin/ vimentin and laminin. Filament self-assembly is driven by the rod domain.
How are actin fibres assembled?
The initial step of actin polymerisation is energetically unfavourable, so it is slow to happen at first. However, once initiated, it will continue spontaneously. Also, actin in a solution also spontaneously forms fibres so it’s own.
There’s a nucleation phase (lag phase), then the elongation (growth phase) then the steady state (equilibrium phase)
How does actin treadmilling work?
Actin treadmilling involves recycling of actin monomers. Old actin at the pointed end of the filament gets a prolifin (which enables phosphorylation) added, and ATP is hydrolysed. ANd it then gets added back onto the barbed end of the actin. This recycling makes the filaments polar and directly regulated by ATP. There is an increased rate of monomer addition at the plus end.
What are some actin-binding proteins?
Nucleators like Arp2/3 nucleate new fibre growth from a starting base. Arp2/3 can also cause branching Gelsolin can cap/sever strands at the plus end to block growth. Alpha-actinin and filamin can cause bundling or cross-linking.
Explain signal regulation
Done by small GTPases. They are small monomeric 21kDa proteins (p21) with intrinsic GTPase activity that can bind to and hydrolyse GTP - not to be confused with heteromeric G proteins
Many GTPases have post-translational lipid modifications to target them to specific membrane sites. All small GTPases belong to a large family with the archaeal member being Ras - which is held in the membrane and interacts with RTKs.
What are the features of the Ras superfamily?
Ras has 36 genes in its subfamily in humans. They’re involved in cell proliferation, many are proto-oncogenes. Rab has 61 members and is involved in endosomal trafficking. Ran has 1 and is involved in mitotic spindle organisation. Arf has 27 genes and is involved in membrane budding . The Rho family has 20. It contains Rac, Cdc42 and Rho. Used in cytoskeleton and migration
Explain the cyclic nature of GTPase activity and how it is involved in regulation.
GTPase cycling is crucial in regulation. Guanine nucleotide exchange factor (GEF) helps catalyse GTP to GDP. If Rac1 is bound to GTO it is broken down quickly. GTPase activating protein (GAP). Guanine nucleotide dissociation inhibitor slows down dissociation of GDP. This means that GTPases serve a timing function, which is more important in signalling than the actual production of GDP. GTPases can have a switch region which means they can go through dramatic changes. Binding of nucleotide causes a structural change in the switch region. The bound nucleotide dictates signalling activity. GTP hydrolysis is an intrinsic property of the protein that turns signalling off.
What are the features of the Rho family? What is their function?
The principle Rho family of GTPases include RhoA, Rac1, Cdc42. They coordinate actin cytoskeleton organisation, which in turn ultimately controls cell morphology, cell movement and polarity. Which is crucial to the formation of multicellular organisation. Cdc42 controls the polymerisation of actin filaments and formation of actin spikes or filopodia (which are activated locally). Ras1 controls the organisation of new actin filaments into dynamic ruffling structures of lamellopodia. RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres.
Explain the outcome of different mutations in the Rho family.
You can use dominant negative and constitutively active mutant GTPases to elucidate function. Point mutations in the nucleotide binding sites of small GTPases can make the proteins always on or inhibitory. Constitutively active GTPase mutants E.g. the substitution of catalytic glutamine in switch 2 stops GTP hydrolysis. Its always GTP bound and signalling is always active. Dom negative mutants e.g. substitution of the P-loop stops nucleotide binding. Makes it nucleotide fee and therefore mops up active GEFs. Constitutively active Rho activation leads to stress fibre formation. Constitutively active Rac or Cdc42 leads to membrane ruffles or filopodia.
Explain Rho signalling.
Activated Rho proteins bind a specific 16 amino acid sequence in effector proteins - CRIB motif (Cdc42/Rac1 Interactive Binding) Rac activates WAVE proteins and Cdc42 activates WASP proteins, both of which activate Arp2/3 and causes actin filament formation. Rho activates Rho kinase, which leads to myosin phosphorylation which increases myosin contractility and leads to stress fibre formation.
