Quiz 3 short answer Flashcards
A bacterium is present in the bloodstream. Explain the signalling events necessary for a bacterial peptide (chemoattractant) released from this bacterium to initiate the migration of white blood cell. (4 Marks)
Release of chemoattractant –> bind to GCPR –> PI3K and PIP3 –> Rac–> activate ARP2/3 –> lamellipodia formation –> PI3K rapidly degraded, cannot diffuse far (gives actin mesh directionality) –> GPCR activates Rho (myosin contraction) –> RAC in lead and Rho in rear gives polarity
The polarized organization of neurons involves the formation of both dendrites and axons. This involves a combination of positive and negative feedback loops that promote the formation of either an axon or dendrite. Describe the positive feedback signal that promotes formation of an axon and the negative feedback loop that tells all other neurites to become dendrites. (6 marks)
POSITIVE:
External signal (ie. GF) activates RTK –> PI3K –> PIP3
PI3K –> RAP1B (INITIATES -VE FEEDBACK LOOP targeting RAP1B for degradation in neurites that are not becoming axons)
RAP1B –> CDC42 –> actin polymerization & activates PAR complex (Par3, Par6, aPKC, TIAM1)
TIAM1 = RAS-GEF - exchange GDP for GTP on Rac1
Rac1 –> promotes actin nucleation & polymerization –> activate +ve feedback loop at PI3k level –> increase activation in pathway
NEGATIVE:
No WNT signalling –> GSK36 degrades b-catenin
(no cadherin and actin interaction to form adherens)
PTEN deactivates PIP3 = no AKT or mTOR
The activation of RhoA triggers to formation of the contractile ring during cytokinesis through 2 pathways, briefly describe these pathways. (3 marks)
1: Activates Formin → formation of filamentous actin → assembly and contraction of actin-myosin ring
2: Activate kinases (including ROCK1) → inhibit myosin phosphatase (inhibitor of regulatory myosin light-chain phosphorylation) → activate myosin II → assembly and contraction of actin-myosin ring (CONTRACTILE RING)
There are 3 models are proposed to explain the positioning of the contractile ring during cytokinesis. Name and briefly explain 2 of them. (4 marks)
Astral stimulation model: (Based on idea that MTS can carry signal to site where cleavage furrow forms)
- MT still extending to the surface of the cell, some MTs from one aster will extend closer to MT from aster of OTHER dividing cell - allows signal to be sent along MTs to midpoint between 2 dividing cells (where cleavage furrow forms)
-Sending signal from centrosomes of aster to PM on MT (minus to plus) → anterograde mediated by Kinesin.
Central spindle stimulation model
-Spindle mid zone (overlap) sends signal that transmits to cell cortex to activate Rho signaling pathway - results in formation of contractile ring
Astral relaxation model
-Local relaxation of actin-myosin produced contractile force is responsible for location of furrow formation.
-Astral MTs extend to PM and everywhere they come into contact w/ PM - relaxation of contractile force - contractile force is only exerted at top and bottom of cells in the middle (where furrow forms)
Three protein complexes are involved in the apical basal polarization of epithelial cells. Name these three complexes (1.5 Marks), and explain how the spatiotemporally regulate epithelial polarization through their interactions with the cytoskeleton and adhesion proteins (3.5 marks)
PAR complex, Crumbs complex, Scribble complex
PAR binds to early adhesions and signals their maturation to the belt-like adhesion junctions and tight junctions that localize at the apical-basal membrane border
PAR3 then excluded from PAR-complex, allows CRB to establish apical membrane and final formation of tight junctions while residual PAR-complex establishes apical-lateral border
Scrib complex defines basolateral PM domain by antagonizing the PAR and CRB complexes, restricts activity to apical region of cell, hence expansion of apical domain.
Explain the dynamics of how a neuronal growth cone is formed, include what type of receptors would link the growth cone to the ECM (5 marks)
-FILOPODIUM EXTENDS: it contacts an adhesive substance through receptors that are connected to actin cytoskeleton in cell
-COULD BE INTEGRINS: would link actin cytoskeleton to laminin or fibronectin of ECM - allow for formation of focal adhesions
-vesicle fusion at PM to support growth at plus end, (so there is space for actin filaments to polymerize and extend)
-MTS FROM CENTRAL CORE ADVANCE: once we have extended filopodia, the MT will advance into new position of core structure
-CYTOPLASM COLLAPSES TO CREATE NEW SEGMENT OF AXON: Once we have MT advancement, cytoplasm supporting lamellipodia behind growth cone collapses - do not need to maintain length of growth cone
Explain how the concept of reciprocal inhibition plays a role in determining the direction of a migrating cell.
Rho inhibits Rac
Active Rac –> WASp –> ARP2/3 –> form meshwork of lamellipodia
Rho is active in rear –> ROCK –> pMLC –> local myosin II contraction
Cdc42 –> WASp –> mDia (formin) –> nucleation ond growth of F-actin
Rac and Rho inhibit one another, causing a gradient
Rho is active in back, inhibits rac which pushes it to leading edge of the cell
Rac in lead inhibits Rho pushing it to back of cell
What is a scratch test assay and what aspect of cellular behaviour is it primarily used to examine? What is a chemo-invasion assay and what aspect of cellular behaviour is it primarily used to examine?
Scratch-test assay
-Cell migration parameters
-Wound closing, lamellposida leads in directiojsn
Chemo-Invasion assay
- Investigate how metastatic a set of cells are
-Growing cells in awell insert covered in a Matrigel and infused with GFP
-Analyze if and how much invadopodia is formed by using confocal microscopy 0 cells are stained to show actin in red
In epithelial cells the lamellipodia contains the necessary machinery for migration and plays an integral role in wound healing.
a) Is the lamellipodia formed of filamentous or branched actin? What complex is responsible for its nucleation? (1 mark)
b) If this nucleation complex was mutated how would that effect the formation of lamellipodia, filopodia and the overall process of wound healing? (2 marks)
c) What role does cofilin play in the lamellipodia during migration? (1 mark
a). Arp2/3 - Branched
b). Some movement happening but will mostly be governed by filopodia not lamellipodia
c). Is at the back, actin depolymerization (enough actin available and can move forward)
Invadopodium are protrusions of the cell membrane of some cells often called invasive feet and they are rich in actin and extend into the extracellular matrix (ECM). Briefly describe the main processes that occur during the formation of invadopodium. (4 marks)
-Localized loss of actin cortex - localized relaxation
-Blebbing forms when PM detaches from cytoskeleton or underlying actin cortex,
-Contraction of myosin in absense of opposing actin cortex allows membrane to protrude
-Actin cortex reassembles, establishes new cellular position
-As they matutre, they degrade the surrounding ECM - create pointt of entry for migration into other tissues
Here you can see that Myosin contraction and cell adhesion allow cells to pull themselves forward. Explain what is happening in each step (A, B and C). What would happen if there was a mutation in the extracellular domain of integrin beta inhibiting it from binding the ECM?
A. Actin polymerization at + end (leading end), no focal adhesions yet
*Actin polymerization force plus myosin contraction promotes focal adhesions
B. No engagement of integrins anchoring actin filaments to ECM - when myosin contracts, leading edge will retract since there is no traction to allow it to move forward
C. Engagement of integrins with ECM allows contraction of myosin to pull actin filament forward towards leading edge of cell - causes protrusion
The immunomicrograph below depicts the expression pattern of actin and myosin during the formation of the contractile ring. Which colour represents each protein? (1 mark) How does this expression pattern support the formation of the contractile ring? (2 marks)
Red: Actin
Green: Myosin
Actin at the PM but also large concentration at the contractile ring site. All of the myosin is concentrated at the contractile ring site.
No contractile force exerted anywhere else in the cell, cell cant migrate or go anywhere since all myosin is at the contractile ring site.
