Cell Polarity

Question 1

What is cell polarity?

Answer 1

Cell polarity is the organisation of proteins inside and at the cell surface such that regions of the cell have distinct protein compositions and the cell can thereby have different capabilities and morphologies.

Question 2

What are the 4 stages of cell polarity?

Answer 2

1. Marking the site (via internal or external cues)
2. Decoding the marked site
3. Establishing the site
4. Maintaining the site (or depolarising it)

Question 3

Why is budding yeast a good candidate for investigating cell polarity?

Answer 3

It undergoes many morphological changes, it has to respond to internal cues - growth and division signals, and external cues - pheromones and nutritional signals. It is genetically tractable.

Question 4

Describe haploid and diploid budding

Answer 4

Haploid cells are either mata or matα and must mate with the opposite sex. The mother and daughter cells bud in axial pattern adjacent to the site of the previous budding event.
Diploid cells do not require a mate to bud, they bud in a bipolar pattern. The mother and daughter cells bud from opposite ends of an ellipsoidal cell.

Question 5

Which stain is used to observe budding events

Answer 5

Calcofluor, a fluorescent dye, binds to chitin, which is particularly prominent at birth scars.

Question 6

Which genes are required for axial, bipolar budding and both?

Answer 6

Axial: BUD3,4,10 and Septins. These protein products mark the mother bud neck as the site of budding in the next cell cycle.
Bipolar: BUD8,9. These proteins mark the poles of diploid cells. RAX2 and components of the actin cytoskeleton are also involved.
Both: BUD1,2,5. The protein products are involved in decoding the axial and bipolar landmarks. Mutations lead to random budding patterns.

Question 7

Which proteins are involved in each stage of cell polarity development in budding yeast?

Answer 7

1. Marking the site: Septin, BUD3,4,10 mark the axial landmark while BUD8 & 10 mark the bipolar landmark
2. BUD1 GTPase cycle is key in decoding the landmarks. BUD5 acts as a GEF and BUD2 acts as a GAP.
3. The GTPase CDC42 is key in establishing the site. It’s GEF is Cdc24 and GAPs are Rga1/2 and Bem3.

Question 8

What are the downstream impacts of Cdc42-GTP?

Answer 8

a) Bni1 is activated - nucleates actin filaments, ensures the cytoskeleton is polarised.
b) Sec3 is activated - part of the exocyst complex, ensures membrane trafficking is polarised.
c) Cla4/Ste20 activation - PAK family kinases, links cell polarity to the cell cycle, aligns microtubules to the mitotic axis.

Question 9

Tell me about the process of mating in budding yeast

Answer 9

Mata cells secrete the pheromone a-factor, Matα cells secrete the pheromone α-factor.
Mata cells have a Ste2 receptor which binds α-factor while Matα have the Ste3 receptor which binds a-factor.

Question 10

What is the role of the Ste2/3 proteins?

Answer 10

Ste2/3 are GPCRs which control a downstream signalling pathway. They activate Far1 which activates the GEF Cdc24. They also activate a MAPK cascade leading to transcription cell cycle arrest.

Question 11

Tell me about organelle inheritance in budding yeast

Answer 11

Daughter yeast cells have different properties than their mothers, this is achieved through:
a) Myosins (Myo2 and Myo4) are key in asymmetric inheritance of factors, proteins, mRNA
b) Cargo proteins are degraded in the daughter cell

Question 12

Tell me about Candida albicans

Answer 12

Candida albicans can cause
a) candidiasis, a mucosal surface and skin infection
b) candidaemia, a bloodstream infection with 30-50% fatality rate.
They have the ability to switch between yeast growth and filamentous hyphal growth. This is stimulated by 37° and neutral pH, although this switch often occurs in macrophages resulting in macrophage lysis. Using this ability, candida are able to escape into tissues where they can switch back to yeast growth causing systemic infections.

Question 13

What is the difference between intrinsic and extrinsic cell fate determination?

Answer 13

Intrinsic is when polar mothers cells produce two daughter cells with different inherited components.
Extrinsic is where a mother cell divides yielding 2 identical daughter cells that become different due to different environmental signals

Question 14

What are the important steps when establishing cell fate?

Answer 14

1. Establish an axis of polarity
2. Mitotic spindles positioned along the axis
3. Cell fate determinants differentially distributed to each daughter cell

Question 15

Tell me about asymmetric division in C. elegans

Answer 15

The zygote is known as the P0 cell. When the egg is fertilised by sperm it delivers a Microtubule Organising Complex (MTOC) which defines the anterior-posterior axis.
The first division of the zygote yields a larger anterior AB cell and a smaller posterior P1 cell.

Question 16

Tell me about the Par proteins and their role in establishing polarity in the C. elegans P0 cell

Answer 16

Par proteins discovered in the Par genetic screens (Par for partitioning defective when mutated) form the core of the polarity networks in animals. It is centred around mutual antagonism, when a protein strays from it’s domain it is acted upon by other Par proteins, typically kinases.
p) The MTOC recruits Par1 and Par2 to the posterior domain, they antagonise anterior Par proteins.
a) Cdc42, Par3, Par6 and PKC3 localise to the anterior domains, they antagonise posterior Par proteins.
b) Par4 and Par5 are localised to the anterior-posterior boundary
It is worth noting that LGL localises to the posterior domain and causes greater pulling of mitotic spindles towards the posterior domain

Question 17

What are neuroblasts in Drosophila?

Answer 17

Neuroblasts are CNS progenitors in Drosophila. They are located in a region of the epithelial monolayer known as the ventral neuroectoderm.

Question 18

Describe Neuroblast division

Answer 18

Neuroblasts delaminate from the neuroectoderm where they undergo several rounds asymmetric divisions
- Initially, neuroblasts divide into small basal cells called Ganglion Mother Cells (GMCs) and larger apical cells.
-The GMC divides again into a neuron and glia cell while the apical cell continues to divide asymmetrically producing another apical cell and a GMC

Question 19

Describe neuroblast polarity establishment and cell fate determinant distribution

Answer 19

When neuroblasts delaminate, Cdc42, Par3 (Bazooka in Drosophila) and Par6 are found in a stalk extending into the epithelium. After delamination they are found in the apical region.
- Bazooka anchors the Insc/Pins complex to the membrane, which along with the Scribble complex, orient and align the mitotic spindles
- Cell fate determinants are transported in a basal direction to the GMC which includes factors such as Prospero and Staufen which regulate gene expression in the GMC

Question 20

Tell me about the activities involved in cell migration

Answer 20

In animals, excluding sperm, all locomotion occurs via crawling along substrate.
1. Protrusion, pushing out the plasma membrane in the direction of travel
2. Attachment, actin cytoskeleton inside the cell is attached via interacting proteins across the plasma membrane and the extracellular matrix
3. Traction, the bulk of the cell body is drawn forward via contraction

Question 21

Tell me about the role of actin structures in cell migration

Answer 21

a) Lamellipodia, sheet-like broad structures are controlled by Rac GTPases
b) Filopodia AKA microspikes are dense cores of bundled actin filaments which are controlled by Cdc42 GTPase
c) Stress fibres, bundled actin filaments involved in contractions required to move the cell body forward. Controlled by Rho GTPases

Question 22

What is chemotaxis, give a detailed example?

Answer 22

The movement of cells away or towards a diffusible chemical signal.
Neutrophils move towards towards the site of bacterial infections.
1. GPCRs on neutrophils bind bacterial peptides
2. Rac is activated leading to the generation of Lamellipodial protrusions
3. Rac also activates Rho which activates myosin-based contraction moving the cell body forward

Question 23

Describe epithelium organisation

Answer 23

It is the first tissue that emerges during the development of an embryo with key roles in embryo morphogenesis and tissue development.
The apical side faces the external environment (lumen of tissue) while the basal side faces the basement membrane.
The lateral sides face each other through homophilic adhesion molecules such as E-cadherin.

Question 24

Describe the role of polarity in epithelium organisation

Answer 24

Polarised actin cytoskeleton allows the apical surface to constrict, key in gastrulation and tubulation.
They are able to orient their mitotic spindles to divide either in the plane of the epithelial sheet to elongate it or perpendicular to the plane of the epithelial sheet to produce different daughter cells.

Question 25

What is EMT/MET?

Answer 25

Epithelial cells are able to rapidly lose and reacquire their phenotype through Epithelial-Mesenchymal transition and Mesenchymal-epithelial transition.

Question 26

Which proteins are involved in establishing epithelial polarity?

Answer 26

a) Crumbs complex: CRB and Stardust protein
b) Scribbled complex: SCRIB, LGL (Lethal Giant Larvae), DLG (Disk large homologue)
c) Crumbs and Scribbled interact with Par proteins and Cdc42.