Lecture 27 Flashcards
What is the results of microinjection of dominant negative Rho into active cells
Leads to the loss of stress fibres
What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases
This causes a very small conformational change dictated by the presence of a final phosphate that changes the orientation of the switch 1 and switch 2 domains. This leads to an activation of signalling
What is the role of guanine nucleotide dissociation inhibitors
GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases
What is the result of microinjection of constitutively active Rho into quiescent cells
Leads to the formation of stress fibres
Why is the actin cytoskeleton not a rigid structure
Rigid structures are unstable whereas tensile cytoskeleton are much more robust. A tensile structure can temporarily adjust the application of forces to maintain its shape
In its inactive state, GTPases are bound to GDP, what is required to activate signalling
Displacement of the GDP by GTP activates the GTPase and initiates signalling
Active Rac activates WAVE proteins, what is the downstream effect of this activation
Activated WAVE proteins bind to Arp2/3 and lead to the formation of actin filaments associated with branching
What is the name of the superfamily to which all small GTPases belong
Ras Superfamily of GTPases
GTPases hydrolyse ATP, T or F
F – they hydrolyse GTP
What is the role of RhoA
RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres
Nucleotide-free GTPases are extremely energetically favourable, T or F
F – its extremely unfavourable
What is the rough weight of a GTPase
21kDa
Actin polymerisation occurs at both ends of the filament, T or F
F – actin tends to be added at one end (+ end) and subunits are removed at the other end (- end)
What are the three members of the Rho family of GTPases
RhoA, Rac1 and Cdc42
Give an example of another important actin accessory protein and its role
Gelsolin – involved in capping existing actin filaments as well as capping and nucleation
What is the purpose of the post-translational lipid modifications often seen in GTPases
These hydrophobic lipid groups added to the proteins will target them to specific membrane sites
GTPase activating proteins are responsible for catalysing the hydrolysis of the GTP bound to GTPases, thus do they act as positive or negative regulators of GTPase signalling
GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form
What is the result of microinjection of constitutively active Rac or Cdc42 into cells
Leads to the formation of membrane ruffles or filopodia respectively
What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity
Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release
What is meant by actin filaments being referred to as polar
Actin filaments have specific ends. The + end or barbed end is the faster growing end of the filament where polymerisation is favoured. The – end or pointed end is the slower growing end where depolymerisation is favoured
Profilin is an important actin-binding/accessory protein, explains its dual roles
Profilin binds to free monomeric G-actin and transports it to the correct end of the microfilament as well as catalysing the exchange of the bound nucleotides. This all acts to promote microfilament assembly
What are the four factors that influence cell shape
Adjacent cells, cell adhesions, extracellular matrix and the function of the cell
What are the two type of actin
Monomeric or globular/G-actin and polymeric or filamentous/F-actin
What is the name of the specific 16 amino acid sequence which activated Rho proteins bind to within effector proteins
Cdc42/Rac1 Interactive Binding (CRIB) sequence
What is the result of the intrinsic nature of GTPases to hydrolyse GTP
Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state
Describe a gain of function approach that can be used to elucidate the precise function of GTPases
Create a constitutively active GTPase mutant that is always on and remains in the GTP-bound form. This can be achieved by the substitution of the catalytic glutamine in the switch 2 region which perturbs GTP hydrolysis and creates an always active GTPase
What is the role of Rac1
Rac1 controls the organisation of new actin filaments, particularly branched actin, into dynamic ruffling structures or lamellipodia
What is the role of the Rho family of GTPases
They coordinate actin cytoskeletal organisation, which in turn ultimately controls cell morphology, movement and polarity
Rac activation is required to precede Cdc42 activation, T or F
F – vice versa
GTPases are small monomeric proteins, T or F
T
What other protein family are activated by RacGTPases that go onto activate Arp2/3 and lead to actin filament formation
WASP
Describe a loss of function approach that can be used to elucidate the precise function of GTPases
Create a dominant negative mutant GTPase with a point mutation in the nucleotide-binding site. This will results in a GTPase that is always off and inhibitory due to never binding to GTP. This can be achieved via substitution of the P-loop. The dominant negative effect of this mutant is due to its binding to, and mopping up of active GEFs to prevent their action on functioning GTPases. By binding to these inhibitory mutant GTPases, the GEFs are no longer available to activate other functions wild type GTPases.
Which actin-binding protein acts as a nucleator to promote the formation of both new actin fibres and the branching of existing fibres and how does it do this
Arp2/3 is a nucleator of actin filament formation that mimics and actin nucleus. It consists of two subunits which resemble actin monomers and promote new actin filament formation either de novo or on the side of existing microfilaments
Describe the phases of actin filament assembly
The initial phase, known as the nucleation or lag phage occurs when all actin is in the G-actin state prior to nucleation. Once the core is established it is elongated rapidly during the growth phage where actin monomers are added at either end. Then the actin polymerisation reaches a plateau at a the critical concentration where the equilibrium phase ensues. The critical concentration is where removal of actin monomers is preferred and polymerisation stops, here, the rate of addition equals rate of removal
Describe the four main components of the actin cytoskeleton
Stress fibres – stretch across the cells to link anchor points and provide stability. Cortical actin – involved in amoeboid migration. Lamellipodium – branched network of actin filaments that push the membrane out. Filopodium – rod like structures that push the membrane out but are unstable due to their rigidity
What subcellular activity also plays a role in defining cell shape
Migration, phagocytosis, transport and cytoskeletal dynamics
What is the role of Cdc42
Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia
What can be said about the likelihood of actin polymerisation to occur
The initial step in actin polymerisation is energetically unfavourable and a very slow reaction
What can be used to cause the spontaneous polymerisation of monomeric actin in vitro
The addition of salts stimulates actin polymerisation
Rac stimulates the formation of new linear actin filaments whereas Cdc42 stimulates formation of branched actin filaments, T or F
F – vice versa
How does Rho activation lead to the formation of stress fibres
Rho activates Rho kinase which in turn phosphorylates myosin to increase its contractility which ultimately leads to the formation of stress fibres
