Lecture 27

Question 1

What is the results of microinjection of dominant negative Rho into active cells

Answer 1

Leads to the loss of stress fibres

Question 2

What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases

Answer 2

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

Question 3

What is the role of guanine nucleotide dissociation inhibitors

Answer 3

GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases

Question 4

What is the result of microinjection of constitutively active Rho into quiescent cells

Answer 4

Leads to the formation of stress fibres

Question 5

Why is the actin cytoskeleton not a rigid structure

Answer 5

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

Question 6

In its inactive state, GTPases are bound to GDP, what is required to activate signalling

Answer 6

Displacement of the GDP by GTP activates the GTPase and initiates signalling

Question 7

Active Rac activates WAVE proteins, what is the downstream effect of this activation

Answer 7

Activated WAVE proteins bind to Arp2/3 and lead to the formation of actin filaments associated with branching

Question 8

What is the name of the superfamily to which all small GTPases belong

Answer 8

Ras Superfamily of GTPases

Question 9

GTPases hydrolyse ATP, T or F

Answer 9

F – they hydrolyse GTP

Question 10

What is the role of RhoA

Answer 10

RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres

Question 11

Nucleotide-free GTPases are extremely energetically favourable, T or F

Answer 11

F – its extremely unfavourable

Question 12

What is the rough weight of a GTPase

Answer 12

21kDa

Question 13

Actin polymerisation occurs at both ends of the filament, T or F

Answer 13

F – actin tends to be added at one end (+ end) and subunits are removed at the other end (- end)

Question 14

What are the three members of the Rho family of GTPases

Answer 14

RhoA, Rac1 and Cdc42

Question 15

Give an example of another important actin accessory protein and its role

Answer 15

Gelsolin – involved in capping existing actin filaments as well as capping and nucleation

Question 16

What is the purpose of the post-translational lipid modifications often seen in GTPases

Answer 16

These hydrophobic lipid groups added to the proteins will target them to specific membrane sites

Question 17

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

Answer 17

GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form

Question 18

What is the result of microinjection of constitutively active Rac or Cdc42 into cells

Answer 18

Leads to the formation of membrane ruffles or filopodia respectively

Question 19

What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity

Answer 19

Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release

Question 20

What is meant by actin filaments being referred to as polar

Answer 20

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

Question 21

Profilin is an important actin-binding/accessory protein, explains its dual roles

Answer 21

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

Question 22

What are the four factors that influence cell shape

Answer 22

Adjacent cells, cell adhesions, extracellular matrix and the function of the cell

Question 23

What are the two type of actin

Answer 23

Monomeric or globular/G-actin and polymeric or filamentous/F-actin

Question 24

What is the name of the specific 16 amino acid sequence which activated Rho proteins bind to within effector proteins

Answer 24

Cdc42/Rac1 Interactive Binding (CRIB) sequence

Question 25

What is the result of the intrinsic nature of GTPases to hydrolyse GTP

Answer 25

Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state

Question 26

Describe a gain of function approach that can be used to elucidate the precise function of GTPases

Answer 26

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

Question 27

What is the role of Rac1

Answer 27

Rac1 controls the organisation of new actin filaments, particularly branched actin, into dynamic ruffling structures or lamellipodia

Question 28

What is the role of the Rho family of GTPases

Answer 28

They coordinate actin cytoskeletal organisation, which in turn ultimately controls cell morphology, movement and polarity

Question 29

Rac activation is required to precede Cdc42 activation, T or F

Answer 29

F – vice versa

Question 30

GTPases are small monomeric proteins, T or F

Answer 30

T

Question 31

What other protein family are activated by RacGTPases that go onto activate Arp2/3 and lead to actin filament formation

Answer 31

WASP

Question 32

Describe a loss of function approach that can be used to elucidate the precise function of GTPases

Answer 32

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.

Question 33

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

Answer 33

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

Question 34

Describe the phases of actin filament assembly

Answer 34

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

Question 35

Describe the four main components of the actin cytoskeleton

Answer 35

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

Question 36

What subcellular activity also plays a role in defining cell shape

Answer 36

Migration, phagocytosis, transport and cytoskeletal dynamics

Question 37

What is the role of Cdc42

Answer 37

Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia

Question 38

What can be said about the likelihood of actin polymerisation to occur

Answer 38

The initial step in actin polymerisation is energetically unfavourable and a very slow reaction

Question 39

What can be used to cause the spontaneous polymerisation of monomeric actin in vitro

Answer 39

The addition of salts stimulates actin polymerisation

Question 40

Rac stimulates the formation of new linear actin filaments whereas Cdc42 stimulates formation of branched actin filaments, T or F

Answer 40

F – vice versa

Question 41

How does Rho activation lead to the formation of stress fibres

Answer 41

Rho activates Rho kinase which in turn phosphorylates myosin to increase its contractility which ultimately leads to the formation of stress fibres