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Advanced Molecular Biology > Cell Polarity > Flashcards

Cell Polarity Flashcards

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1
Q

What is the definition of cell polarity?

A

Cell polarity is the organization of proteins inside, and at the surface of cells, such that regions of the cell have distinct protein compositions and the cell can thereby have different capabilities, morphologies and functions

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2
Q

What does cell polarity allow?

A

Cell polarity allows different cells to have different functions.

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3
Q

What are 4 key functional requirements to polarise a cell?

A
  1. Marking the site
    >Allows cell to differentiate one part of itself from another.
  2. Decoding the site
    >Signalling that the site needs to grow
  3. Establishing the site
    >An axis is made
    >Key proteins come to the site organising cytoskeleton to send materials here via membrane trafficking.
  4. Maintaining the site
    >How does the site continue to function while material is added, how does it not just cover the whole cell (controlled by feedback loops)
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4
Q

What is Saccharomyces cerevisiae?

A

Budding yeast.

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5
Q

Why do we use yeast to study cell polarity?

A

> Yeast undergoes significant morphological changes in response to both internal and external signals.
And we know the entire genome.

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6
Q

When does yeast bud from internal signals?

A

In response to growth and division signals e.g. growth of a bud (growth from a single point)and cytokinesis (whole cell division)

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7
Q

When does yeast bud from external signals?

A

In response to pheromones (for mating) and nutritional signals (cells can elongate so can move colony away from poor nutritional area to better areas)

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8
Q

What method was used to study polarity pathways in yeast?

A

Genetic screens

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9
Q

What can staining a budding yeast with fluorescent dye show us?

A

Birth scars which mark the sites of previous cell separations to be viewed as bright rings on the cell wall. So we can see the site of polarisation.

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10
Q

How do a) Haploid b) Diploid yeast bud and what is the advantage for both?

A

a) Haploid (1n DNA)
>Bud in AXIAL pattern
>Both mother and daughter cells form buds immediately adjacent to the previous site of separation so both of their buds can mate.

b) Diploid (2n DNA)
>Bud in a BIPOLAR pattern
>Bud from ends of cells so moves cells away to explore different environments (don’t need to be next to other buds to mate)

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11
Q

What determines the position of the new bud which grows to from a new daughter cell on a budding yeast?

A

Whether the cell is haploid or diploid.

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12
Q

What are the 3 genes necessary for axial budding and what protein is needed?

A

> genes identified include BUD10, BUD3, BUD4. Products of these genes mark the mother bud neck during one cycle as a site for budding in the next cycle

> Septins (GTP-binding protein) necessary for cell division and cell polaity.

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13
Q

What are the 3 genes involved in bipolar budding and what other mechanisms are involved?

A

> BUD8, BUD9, RAX2 and components of the actin cytoskeleton are involved.

> Products of these genes mark the ends of diploid cells.

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14
Q

Would mutations in BUD10, BUD3, BUD4 genes effect bipolar budding?

A

No, only Axial wouldn’t work anymore

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15
Q

Would mutations in BUD8, BUD9, RAX2 genes effect Axial budding?

A

No, only Bipolar couldn’t work anymore

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16
Q

What 3 genes are required for both axial and bipolar budding patterns and what is their function?

A

> BUD1, BUD2, BUD5.

> Proteins encoded by these genes decode the axial and bipolar marks and signal to the machinery involved in generating the polarity axis.

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17
Q

What is the effect of mutations in BUD1, BUD2, BUD5 genes.

A

Mutations in these genes cause a random budding pattern in both haploid and diploid cells.

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18
Q

How do Bud1 (Ras), Bud2 and Bud5 proteins work together in a GTPase cycle?

A

> Bud1 (Ras) when in active GTP form can recruit polarity machinery.

> But it is Bud2 and 5 that mediate when Bud1 is active or inactive.

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19
Q

What enzyme is most the important marker protein for polarity and what family does it belong to?

A

Cdc42 is a member of Rho GTPase family, is most important marker protein for polarity

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20
Q

What is the process of establishing cell polarity by Cdc42 in 3 steps?

A
  1. Bud1 protein when active recruits Cdc24 which activates Cdc42
  2. Cdc43 sticks the active Cdc42 onto membrane (lipid moiety)
  3. Active Cdc42 then recruits a number of proteins and activates them
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21
Q

While establishing cell polarity, what 3 proteins does Cdc42 recruit and activate?

A
  1. Bni1 which generates new actin filaments (all end or start at this point),
  2. Sec3 (Exorcyst) where vesicles can dock (growth is driven by fusion of vesicles to membrane),
  3. Kinases links polarity to cell cycle.
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22
Q

How does the actin cytoskeleton help with cell polarisation?

A

Actin cytoskeleton is polarised along the axis helping to move organelles into the right buds, trafficks certain mRNAs, recruits vesicles to dock for growth, captures ends of microtubules to help aline mitotic spindle axis so when nucleus divides the chromosomes are separate along the right axis.

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23
Q

Why is budding yeast producing mating projections chemotropic?

A

> Haploid yeast cells can polarise and redirect their growth to a tip axes in order to facilitate mating with a partner ( the growth of organisms navigated by chemical stimulus from outside of the organism)

> The response is chemotropic due to mating pheromones being secreted by different cell types. The receptors which bind to pheromones are GPCRs that cause the downstream polarity pathway.

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24
Q

How do budding yeast produce mating projections in 4 steps?

A
  1. Mating pheromone and pheromone receptor
  2. Heterotrimeric G protein releases beta gamma complex.
  3. Far1 (kinase cascade module)

4.Cdc24-> Cdc42-> Bni1 ->Sec3 (same polarity establishment machinery recruited despite different stimuli)

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25
Q

Why do yeast only mate during early G1 stage of the cell cycle?

A

Only mate during early G1 stage of cell cycle as if they mate part way through the cell wouldn’t be able to fuse DNA with another bud. So cell cycle stops at G1

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26
Q

Why do daughter yeast cells have different properties/ polarity from their common mother cell?

A

> As each receives different mRNAs from the mother

27
Q

How do different daughter cells receive different mRNAs from a common mother cell?

A

Certain myosin filaments can travel across actin filaments carrying mRNAs with them. Leads to different mRNAs in the daughter cells causing different polarity

28
Q

Why can’t yeast be used to understand mechanisms involving intercellular interactions (between cells)?

A

As yeast are unicellular.

29
Q

How has studying budding yeast helped us treat disease from pathogenic fungus?

A

The genes in this pathogenic fungus have a very high level of homology to those in Saccharomyces cerevisiae. So we can understand their polarity pathways while causing disease in us.

30
Q

How can the ability to switch between yeast and hyphal form be important for Candida’s virulance?

A

> Yeast cells taken up by macrophages can switch to filamentous growth and lyse the macrophage.

> Switching to Yeast cells as are carried more effectively in the bloodstream promoting fungal dissemination in the body.

31
Q

Why are Candida found more in Hyphal form over Yeast form in mammals?

A

As Hyphal formation is stimulated at 37°C by serum or neutral pH.

32
Q

What is the effect of polarity in development?

A

A cell can generate daughter cells that are intrinsically different from one another.

33
Q

What are the 2 main routes of cell diversity?

A
  1. Intrinsic approach: Diversity generated by mechanisms intrinsic to a cell, polar mother cells divide to generate different daughter cells in two ways:
    i. Localised determinants on the polar ends of the mother cell leads to the daughter cells containing different molecules.
    ii. Mitotic spindle is not located in the centre of the cell, leads to asymmetric division plane producing daughter cells of different sizes.
  2. Extrinsic (diversity due to external factors):Daughters could be equal at ‘birth’ but then become different by exposure to different environmental signals.
    >These signals can be from other cells or from the sister cells themselves. Both lead to diversity of the daughter cells.
34
Q

What are 3 important steps in generating polarity and therefore cell fate decisions?

A

1) Establishment of an axis of polarity (this involves marking a site, signalling and establishing – as discussed in last lecture)

2) Mitotic spindle is positioned along the axis
>So cell division occurs in a direction

3) Cell fate determinants are often distributed differentially to daughter cells

35
Q

What is important in allowing diversity to occur in cells?

A

Cell polarity of a mother cell drives diversity between daughter cells.

36
Q

How do PAR proteins form cell polarity networks?

A

PAR proteins are localized asymmetrically to establish the apical and basal domains of a cell. If a protein strays into the wrong domain it will be re-attached to the correct membrane.

37
Q

How does polarisation occur during fertilisation in C.elegans in 4 steps?

A
  1. Polarisation starts with entry of sperm into the oocyte (egg), the position of entry defines the posterior end of the zygote (fertilised egg).
  2. Zygote divides asymmetrically along the anterior-posterior axis due to off centre-mitotic spindle.
  3. Produces larger anterior cell and smaller posterior cell.
  4. The differently sized daughter cells are committed to different cell fates (which was determined by the polarity of their mother cell).
38
Q

At what point does cell polarity influence cell fate during development?

A

From the first division, cell fates are determined by polarity

39
Q

What defines the location of mitotic spindle?

A

Position of nuclei

40
Q

In C-elegans what do the PAR genes encode for?

A

Proteins Par1-6 as well as the 7th Par protein which is atypical protein kinase C

41
Q

How does sperm contact with oocytes in fertilisation create an asymmetric division plane in 6 steps?

A
  1. Sperm delivers a microtubule organising centre (MTOC) to the oocyte, this site becomes the posterior pole (defines the axis of polarity).
  2. Microtubules recruit (transport) Par1 (kinase) and Par2 to the posterior side.
  3. Par3, 6, protein kinase C, and Cd42 stay localised at the anterior side
  4. Par 4 and 5 are found in the middle of the cell defining the boundary.
  5. This protein set up allows mitotic spindles to extend from microtubules and have a greater pulling force on the cortex in the posterior direction.
  6. This sets up the cell division plane to be asymmetrical in the whole zygote, as the spindles are pulled closer to the posterior side.
42
Q

How does sperm contact with oocytes in fertilisation create localised determinants in a zygote?

A

Actin and the motor proteins myosin carry cell fate determinants towards the posterior end polarised by sperm contact.

43
Q

Describe neuroblast cell division in Drosophila in 4 steps (example of cell fate determined by localised determents)

A
  1. Neuroblast delaminates from epithelial monolayer (ventral neuroectoderm)
  2. Can divide so one daughter cell is more apical and one is more basal (smaller, called ganglion mother cell)
    >Requires neuroblast to send different cell fate determinants to ganglion mother cell and daughter apical cell so they have different cell fates.
  3. Ganglion mother cell divides once more to give a neuron and glial cell
  4. The apical daughter cell can give rise to another ganglion mother cells (each time will give rise two one neuron and one glial cell)
44
Q

How do Drosophila have asymmetric division in neuroblasts due to localised determinants in 4 steps?

A
  1. Polarity is established when the cell is still in the neuroectoderm layer. When neuroblasts delaminate, Cdc42, Par3, Par6 are found in the stalk region inside the neuroectoderm layer.
  2. After delamination, Cdc42, Par3, Par6 continue to be localised at the apical region.
  3. Par3 anchors another complex at the membrane in order to orient the mitotic spindle
  4. Cell fate determinants are transported in a basal direction to the ganglion mother cell, giving it a different cell fate due to inheritance of different determinants than the apical daughter cell.
45
Q

Where is a lot of branched actin found in a cell and what is it involved in?

A

A lot of branched actin filaments under cortex, involved in cell movement (crawling)

46
Q

What are the 3 main steps of cell movement?

A
  1. Protrusion – the pushing out of the plasma membrane in front of the cell
    E.g. lamellipodium forms.
  2. Attachment – the actin cytoskeleton inside the cell is attached via interacting proteins across the plasma membrane to the substratum (e.g. extracellular matrix), adhere to extracellular surface
    E.g. integrin protein
  3. Traction – the bulk of the cell body is drawn forward through a process of contraction via actin stress fibres.
47
Q

What are 3 actin structures involved in cell migration?

A
  1. Filopodia or microspikes are a dense core of bundled actin filaments
    >Is a protrusion
  2. Lamellipodia are sheet-like broad structures.
    >Is a protrusion
  3. stress fibres that are bundles of actin filaments
48
Q

What are stress fibres and what is their function?

A

Stress fibres that are bundles of actin filaments that are involved in the contractility required to move the body of the cell forward (pulls the cell). Also have to be involved in dissembling adhesions of cell to surface.

49
Q

What enzyme type controls cell migration and what experiment shows this?

A

> Rho small GTPases

> Injected Rho caused many stress fibres, so pathway involved in moving cell body uses Rho

50
Q

What is chemotaxis and an example of this in our body?

A

> This is the movement of cells towards or away from a signal such as a diffusible chemical

> An example is the movement of a neutrophil moving towards a site of bacterial infection.
Bacteria shed parts of cell walls, their peptides are slightly different so can be detected.

51
Q

How is the polarised localisation of a) Rac b) Rho important for cell motility?

A

a) Rac is found at the front of the cell, a chemoattractant binds to a GPCR it activates Rac which stimulates Lamellipodia protrusions towards the chemoattractant.

b) Rho is found at the back of the cell, when activates it contracts actin stress fibres to bring the trailing end of the cell towards where the front has moved.

52
Q

What is the first tissue that emerges during development?

A

The epithelium is the first tissue that emerges during development of the fertilized egg. The epithelium has key roles in embryo morphogenesis and organ development.

53
Q

What are the 5 key properties of the epithelium?

A
  1. The apical side faces the external environment or lumen of the tissue; the basal side faces the basement membrane.
  2. Lateral sides of epithelial cells adhere to each other through homophilic (bind to each other) adhesion molecules, such as E-cadherin
  3. Epithelial cells have polarised actin cytoskeleton – allows apical surface to constrict (important for gastrulation and tubulation forming villi)
  4. Epithelial cells can orient their mitotic spindle to allow division in the plane of the epithelial sheet to increase their number or perpendicular to the sheet to generate different daughter cells.
    >Axis of polarity can cause mitotic spindle to orientate in perpendicular to sheet
  5. Epithelial cells can rapidly lose the epithelial phenotype (epithelial mesenchymal transition – EMT) and re-acquire it (Mesenchymal epithelial transition – MET)
54
Q

How are epithelium layers made?

A

After formation of the zygote instead of dividing into many cells, large number of nuclear divisions: Many nuclei line up against edges of syncytial blastoderm (one big cell with nuclei lining the edge), cellularization occurs

55
Q

How are epithelium layers made?

A

After formation of the zygote instead of dividing into many cells, large number of nuclear divisions: Many nuclei line up against edges of syncytial blastoderm (one big cell with nuclei lining the edge), cellularization occurs (epithelium cells form at the edges where the nuclei are).

56
Q

What is cellularisation?

A

Cellularisation= part of membrane comes down to form epithelium between nuclei, forming a cellular blastoderm (a layer of epithelium surrounding the cell)

57
Q

What is the Bazooka gene in Drosophila another name for?

A

Par3

58
Q

How is Par3 involved in establishing epithelial polarity?

A

When nuclei are close to membrane, centrioles form microtubules (role of actin cytoskeleton) to help traffic proteins such as Par3 and begin forming intracellular interactions such as Par3 binding to E-Cadherin in adjacent cells allowing adherence junction formation, allows for epithelial with apical and basolateral face.

59
Q

What is the role of the actin cytoskeleton in polarity of cells?

A

Microtubules used to transport proteins to different ends of the cell.

60
Q

What is a difference between the polarity of epithelial cells in invertebrates and vertebrates?

A

After all other polarities have bene set up in vertebrate epithelial cells, tight junctions form between adjacent epithelial cells which is critical to maintain a full barrier between the apical and basolateral face (so things like pathogens have to go through the cell itself and not around).

61
Q

What is the epithelial mesenchymal transition (EMT)?

A

EMT is a critical process during development and is also associated with cancer metastasis

62
Q

What occurs during epithelial to mesenchymal transition (EMT) in 3 steps?

A
  1. EMT triggered by signals that lead to loss of E-cadherin (usually holds adjacent epithelial cells together).
  2. Epithelial apical-basal polarity axis is converted to a migration axis with a front-rear polarity.
  3. Now apical becomes the front end of the cell, containing Rac stimulating Lamellipodia protrusions, while the basal becomes the back of the cell, containing Rho for contraction of actin stress fibres, allowing mesenchymal cell motility.
63
Q

What is the a) Actin Cytoskeleton b) Extra cellular Matrix?

A

A) Network of actin filaments found in cytoplasm

b) Network of proteins like elastin and collagen.

64
Q

What is the process of haploid cell polarity generation for mating?

A

MATa cells secrete a-factor, MAT-alpha cells secrete alpha-factor:

  1. MATa cells have Ste2 receptor that recognise alpha-factor
  2. MAT-alpha cells have a Ste3 receptor that recognise a-factor
  3. When the receptors recognise their respective pheromones, gorwth is activated.

Advanced Molecular Biology (9 decks)

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