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Genetics of cell growth and division > Hettema - polarised growth > Flashcards

Hettema - polarised growth Flashcards

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Biology 101 flashcards
1
Q

what is cell proliferation?

A

increasing cell number by division

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2
Q

What is cell growth?

A

Cell size increasing

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3
Q

Why is cell polarity important?

A

necessary for cell form and functions
cells move by membrane recycling
budding cells mate by polarised growth towards each other

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4
Q

Why do different regions of the cell have different protein compositions?

A

can have different capabilities and functions

breaks symmetry

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5
Q

Why are yeast used?

A

• Simple eukaryote
• Cheap and fast growing
• Great genetics (haploid and diploid cells can be maintained)
• Excellent targeting genetic manipulation, huge amount of resources
o Knockout libraries
o GFP tagged libraries
o Expression profiles
o Genetic interaction data
o Protein-protein interactions
o Lots of mutants
• Important processes are evolutionarily conserved
• 1000 genes (17% ) are members of orthologous gene families heterologous complementation

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6
Q

How do budding yeast grow?

A

by budding (polarised growth event) not fission

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7
Q

What morphological changes do budding yeast respond to?

A

Internal – in response to growth and division signals

External – in response to pheromones and nutritional signals e.g. a macrophage moving towards foreign material

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8
Q

Steps of the budding cell cycle

A 
Spindle body duplication
Bud emergence
DNA replication
Spindle formation and nuclear migration
chromosomal segregation and nuclear division
cytokinesis
daughter cell growth
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9
Q

Cells must choose a direction for polarity and build an axis

A

T

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10
Q

What is a bud scar?

A

• Present on the mother
• Thicker structures that are easier to see
• Can have many
measure of ageing in yeast

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11
Q

What is a birth scar?

A
  • Present on the bud
  • Thinner structures
  • Only have one
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12
Q

How are birth/bud scars visualised?

A

staining cells with a fluorescent dye called calcofluor. binds chitin, (a component of the yeast cell wall)

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13
Q

How does bud positio depend on cell type?

A 
Haploid cells (a or α) have budding scars next to each other all on one side – axial budding 
Diploid cells (a/ α) have bud cells on both sides – bipolar budding
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14
Q

How were genes involved in budding identified?

A

Genetic screen
• Mutants appear spontaneously, by exposure to mutagenic conditions or by directed gene deletion.
• Changes in budding pattern can be observed microscopically using calcofluor staining
• Genes can then be identified which allow this phenotype to be rescued
• Genes specifically required for the yeast axial budding pattern

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15
Q

What genes were identified involved in axial budding?

A

BUD10, BUD3, BUD4 and the septins
Products from these genes are involved in marking the mother bud neck during one cycle as a site for budding in the next cycle

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16
Q

if BUD10, BUD3, BUD4 are deleted what happens?

A

Random budding

budding process not affected only position of bud

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17
Q

How does axial budding occur?

A
  1. Bud10 at bud membrane, septin at bud site
  2. bud10 at bud membrane near bud, septin at bud site
  3. septin at bud site, bud 3 on either side, bud10 on either side of that
  4. segregation
  5. septin deposited to side
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18
Q

what genes are involved in bipolar budding?

A

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

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19
Q

What is the phenotype of a Bud8 mutant?

A

All buds are formed at the same end of the cell as the birth scar (proximal pole)

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20
Q

What is the phenotype of a Bud9 mutant?

A

All buds are formed at the distal pole

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21
Q

What is the phenotype of the bud8bud9 mutant?

A

Random budding

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22
Q

What is the function of Rax2p?

A

Rax2p is at both poles and is required to maintain bipolar budding over multiple generations. It may help stabilise both bud8 and bud9 proteins

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23
Q

What genes are required for decoding the site?

A

BUD1, BUD2, BUD5

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24
Q

proteins encoded by BUD1, BUD2, BUD5 do what?

A 
decode the axial or bipolar marks and signal to the machinery involved in generating the polarity axis. 
function in a GTPase cycle
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25
Q

What do mutations in BUD1, BUD2, BUD5 cause?

A

Random budding

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26
Q

What is BUD1?

A

A ras related GTPase

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27
Q

What is BUD2?

A

GAP for bud1

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28
Q

What is BUD5?

A

GEF for bud1

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29
Q

what is cdc42?

A

Rho-GTPase

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30
Q

How were the cdc mutant identified?

A

Genetic screen

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31
Q

What is the GEF of cdc42?

A

cdc24

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32
Q

What are the GAPs of cdc42?

A

Bem2, bem3, rga1, rga2

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33
Q

how is the site established?

A

1) Cdc24 binds to the active form of Bud1 at sites marked for budding
2) Cdc24 can then activate Cdc42 to allow the polarity site to become established.

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34
Q

when in the cell cycle is but initiation ?

A

Late G1

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35
Q

what mirrors the direction of growth?

A

Cdc42 localisation

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36
Q

how is bud site initiation temporally regulated?

A

Regulates the activity of the GAP by inhibiting it
Regulates the activity of the GEF through Far1 – Far1 is bound to Cdc24 in the nucleus – Cdc28 releases Far1 by phosphorylationand Cdc24 moves out of the nucleus

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37
Q

how does positive feedback break symmetry?

A

Cdc42-GTP recruits Bem1 complex that contains Cdc24.

This can activate more Cdc42

38
Q

Why is the actin cytoskeleton redirected?

A

to direct components to the required sites

39
Q

What are the three components of teh actin cytoskeleton?

A
  • Actin cables – delivery of vesicles to sites of polarised growth
  • Actin cortical patches – site of endocytosis – endocytosis is dependent on actin.
  • Contractile actomyosin ring – Septum formation – stimulates cytokinesis
40
Q

what do cdc42 effectors do?

A

establish the site

41
Q

what establishes the site?

A 
the polarisome
septins
Rho proteins
endocytosis
secretion
42
Q

how does the polarisome establish the site?

A

the complex appears to be important for linking Rho-GTPase signalling to actin filament assembly and for localisation of the formin proteins Bni1 and Bnr1 which drive actin assembly (recruits formins)

43
Q

what are the components of the polarisome?

A

Spa2, Pea2, and Sph1

44
Q

how do the septins establish the site?

A

• The septin ring also forms a boundary between the mother and bud during isotropic bud growth to limit movement of material between these two parts of the cell – becoming two different cells

45
Q

how are the rho proteins important at establishing the site?

A

mutants arrest and lyse as small budded cells

important for ensuring cell wall machinery is active at sites of growth

46
Q

how does the endocytosis maintain the site?

A

marker must remain polarised

  1. Some proteins could dissociate from the membrane and relocalise e.g. through association with secretory vesicles
  2. Integral membrane proteins such as Bud8 can only really be removed by endocytosis and recycled (or degraded
47
Q

how does sectretion maintain the site?

A

Directed secretion is critical to maintain the site.

48
Q

what does endocytic recycling do?

A

protein gets re-focussed at the point of growth

49
Q

Cell growth during the cell cycle?

A
  1. Polarization of secretion in late G1, leading to bud emergence.
  2. The apical-isotropic switch in early G2, a depolarization of growth within the bud leading to uniform bud expansion (switch of tip growth to all over)
  3. A breakdown of mother-bud asymmetry in growth, occurring in late mitosis. Before this, all growth is directed toward the bud; afterward it is evenly directed to both mother and bud.
  4. Refocusing of growth toward the neck upon mitotic exit, leading to cytokinesis and cell
50
Q

why is the secretory pathway essential?

A
  • Growth of plasma membrane
  • Deposition of cell wall proteins (plants and fungi)
  • Membrane proteins
  • Secretion of molecules to the outside
  • Post translational modifications
  • Sorting of proteins taken up by endocytosis
51
Q

what happens is sec mutants?

A

Cell division stops
protein and lipid synthesis continues
become dense and can separate by density gradient centrifugation

52
Q

how do you screen for sec mutants?

A

o Isolated temperature sensitive mutants as these processes are essential
o Release of invertase – specificity for secretion mutants
o EM
o Ts sec mutants blocked in secretion of invertase and acid phosphatase

53
Q

what does a sec23 mutant look like?

A

ER wider and more extensive

Different morphologies as they are mutant

54
Q

what does a sec17 mutant look like?

A

Accumulation of vesicles

See small vesicles in sec17-1

55
Q

what does a sec7 mutant look like?

A

Elaboration of Golgi (Berkeley bodies)
Golgi massively exaggerated
Delivery to golgi is fine
Exit from golgi isn’t working

56
Q

what does a sec15 mutant look like?

A

accumulate vesicles

57
Q

how many sec complementation groups did they find?

A

23

58
Q

how was the pathway order determined?

A

blocking parts of the pathway

59
Q

the sec mutants were separated into how many classes

A

o Class 1: sec17,18, 22, accumulate ER + vesicles
o Class 2: sec12, 13, 16 21 only accumulate ER
o Class 2 acts upstream of class 1

60
Q

what organelles are critical for glycoslation?

A

ER and golgi

61
Q

new screen identified how nay classes of sec mutant?

A

5

62
Q

Class A sec mutant

A

Transport to ER blocked

accumulate in cytosol

63
Q

class B sec mutants

A

budding of vesicles from RER blocked

accumulate in RER

64
Q

class c sec mutants

A

fusion of transport vesicles to golgi blocked

accumulate in ER golgi vesicles

65
Q

class d sec mutants

A

transport golgi->secretory vesicles blocked

accumulate in golgi

66
Q

class e sec mutants

A

transport from secretory vesicles to the cell surface blocked
accumulate in secretory vesicles

67
Q

what is sec4?

A

Sec4 is a Rab GTPase found on post-Golgi vesicles

localises to bud tip

68
Q

what is sec4 localisation dependent on?

A

Sec2 (GEF), actin, and myosin

69
Q

Tpms are required for actin cable stability

A

Two genes for tropomyosin
Knocking out both genes kills the cell
Knocking one gene out has no phenotype
In Ts mutants Cells grow but do not divide

70
Q

which formins are required for actin cable assembly at budtip/neck?

A

Bni1: mainly at bud tip and later in neck
Bnr1: mainly in bud neck
Knockout of both is lethal

71
Q

What is the exocyst?

A

A large complex of many late Sec proteins required for exocytosis

72
Q

What are the components of the exocyst?

A

sec4, Sec3, Exo70, Sec5, Sec6, Sec8, Sec10 and Sec15

73
Q

What does sec4 interact with?

A

Sec15

74
Q

Sec 3 localises to the membrane …

A 
Independent of:
o	Secretory pathway 
o	Actin
Depends on:
o	 Cdc28
o	Cdc42 
o	Rho1
75
Q

What happens to the exocyst at the late golgi?

A

• Rab GTPase Ypt31 recruits Sec2 (GEF of Sec4)
• Sec4 is activated
• Recruitment of Sec15
• Sec4 also binds myosins
• Can now be directed to the site of Cdc42 along actin cables
• Exocyst complex on Golgi interacts with complex on exocyst
• Localised delivery of components
Myo2 also interacts with Sec15

76
Q

What is cytokinesis ?

A
  1. Cytokinesis is the process which leads to the separation of the cytoplasm of dividing cells, thus leading to an increase in cell number.
  2. It involves in excess of 100 protein components and can only be effectively studied in vivo
  3. The process must be tightly regulated in the cell cycle to ensure that the cell is not divided before mitosis is complete.
  4. Origins of cytokinesis: the process in animals and fungi is distinct from that in plants, algae and ciliates. These latter organisms do not contain myosinII and do not generate a contractile ring. Cytokinesis through function of a contractile ring evolved about 1 billion years ago.
77
Q

Major considerations in budding

A
  • division site positioning (marking + decoding the site)
  • contractile ring assembly (site establishment)
  • membrane and septum deposition (in yeast)
  • co-ordination of cytokinesis with the nuclear cycle
  • cell separation
78
Q

what are the morphological events of cytokinesis?

A

1) Get growth of a bud and relocation of septins
2) Actin ring becomes smaller and smaller until the ring closes and the septins are remodelled so there are two
3) Plasma membrane is between the two cells
4) Secondary septin separates mother and daughter
No longer a connection between mother and daughter
Stops the cells lysing
5) Get cytokinesis

79
Q

how must the membrane be remodelled in cell division

A

Membrane deposition and septum assembly

80
Q

Furrow ingression is coupled to Septum formation

A

t

81
Q

what do you get if a division site is marked?

A

polarised growth

82
Q

what do you get if a division site is not marked?

A

random budding

83
Q

Model for contractile ring assembly

A

The arrival of proteins at the bud neck is sequential starting from late G1 to cytokinesis.
recruitment: cdc42-GTP activates Gic1/2 which activates septins
ring assembly:
ring maturation:
ring contraction:
Bni1 is activated to trigger actin polymerisation and final assembly of the contractile ring by Rho1

84
Q

What do the septins do in ring assembly?

A

• The septins form a ring and are thought to be a scaffold for the other proteins to assemble on. Mutations in the septins prevent cytokinesis and cells arrest as large budded cells.

85
Q

What do the formins do in ring assembly?

A

• The formins are thought to drive actin ring nucleation during cytokinesis.

86
Q

What does iqg1 do in ring assembly?

A

• Iqg1 is thought to play a role in organising the actin once it arrives.

87
Q

Can cells lacking actin divide

A

Cells lacking an actomyosin ring can still divide by formation of a septum. Hof1 and Cyk3 are thought to co-ordinate the actin ring contractility with the growth of the septum.

88
Q

What does Inn1 do in ring assembly?

A

• Inn1, is involved in ring disassembly and possibly in co-ordinating contractility with membrane ingression.

89
Q

process of cytokinesis and cell separation

A
  • Septin ring splits in 2
  • Actomyosin ring closes
  • Deposition of chitinous primary septum
  • Deposition of secondary septum on either side
  • Daughter produces chitinase to breakdown primary septum
90
Q

how is the cytoskeleton coordinated with cekk cycle?

A
  • Assembly of an actin- myosin ring and delivery of new membranes are dependent on entry to mitosis and constriction of the ring is dependent on proteolysis of cyclin B.
  • This co-ordination is carried out in part by the Mitotic exit network (MEN). This signalling cascade is initiated when the small GTPase Tem1 is activated by Cdc5 (a Polo kinase), and culminates in release of the phosphatase Cdc14 from the nucleolus. Cdc14 dephosphorylates Cdh1 which then interacts with the anaphase promoting complex (APC) to drive cyclin B degradation.
  • MEN also plays a more direct role in cytokinesis. The MEN localises to the Spindle pole bodies and some of the components translocate to the bud neck.
  • Coupling cell cycle progression and cytokinesis

Genetics of cell growth and division (4 decks)

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