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MBB342 - Genetics of Cell Growth & Division > Hettema - yeast genetics of cell growth and prolif > Flashcards

Hettema - yeast genetics of cell growth and prolif Flashcards

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1
Q

What is the diff between cell prolif and growth?

A
  • cell prolif = increase in number of cells
  • cell growth = increase in cell size
  • DIAG*
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2
Q

Are organelles, vesicles and enzs randomly distrib in cells?

A
  • no, there is more structure
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3
Q

In what way do unicellular proks have complex cellular structures?

A
  • not v homogeneous, eg. may have extensions
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4
Q

What is cell polarity necessary for?

A
  • to gen wide variety of forms to perform diverse array of functions
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5
Q

How is cell polarity important in cell movement?

A
  • if cells migrating along surface, needs diff polarities as 1 side attached to surface, top exposed to medium, front pointing in direction cell growing/moving (pm expanded in direction of movement, then membrane req for this comes from back, so transported to front of cell)
  • therefore front completely diff to back of cell
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6
Q

In budding yeast cells why do diff parts need to be specialised or diff processes to allow movement?

A
  • if haploid of mating type a and one of α (opp mating type), then will bud, form bud like structures that move towards each other = polarised growth
  • once touch each other then mate, and can exchange cyto and nuclei fuse and form new cell
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7
Q

What is cell polarity, and how is it achieved?

A
  • regions of cell have distinct port compositions and thereby can have diff capabilities and functions
  • can be achieved by organising prot and lipids on inside and surface so breaking symmetry of cell
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8
Q

Why use yeast experimentally?

A
  • simple euk –> key machineries conserved to humans
  • cheap and fast growing
  • great genetics (haploid and diploid cells can be maintained) –> can KO genes v easily and can do systematically
  • excellent targeted genetic manipulation
  • lots resources –> KO libraries, GFP tagged libs, expression profiles, genetic interaction data, prot:prot interactions, lots of mutants
  • important processes are evo conserved → 17% genes are members of orthologous gene families (direct complementation, human prot can still function in yeast cells)
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9
Q

What are eg.s of internal and external signals which cause morphological changes as a response?

A
  • internal = in response to growth and div signals

- external = in response to pheromones and nutritional signals

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10
Q

In budding yeast, where does the growth occur?

A
  • only in bud, not mother cell
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11
Q

What is the process of budding?

A
  • bud forms and grows bigger until released

- then grows until big enough to bud itself, in response to internal factors

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12
Q

What determines how 2 budding yeast can grow towards each other?

A
  • external factors
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13
Q

What is the budding yeast cell cycle?

A
  • DIAG*
  • when daughter cell reaches critical size enters cell cycle (then must undergo whole cycle, can’t go back)
  • initially small daughter cell can’t form buds and grow in polarised way
  • mother cell can go immed into start (after cytokinesis), as already big enough and wont grow bigger
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14
Q

How do budding yeast gen cell polarity, in order to grow and divide?

A
  • must choose direction for polarisation
  • build an axis
  • marking site (where budding will occur), decoding site (involves signal transduction), establishing site, maintaining site
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15
Q

What have genetic screens in budding yeast been central to elucidating about these polarity pathways?

A
  • marking the site: where on cell surface
  • decoding the site: signalling
  • establishing the site: recruitment of machinery
  • maintaining the site: remembering where machinery is and keeping it in place
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16
Q

How can budding events be followed experimentally?

A
  • by staining cells w/ fluorescent dye (calcofluor)

- visualises bud and birth scars

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17
Q

Where are bud and birth scars found?

A
  • daughter has birth scar and mother has bud sca
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18
Q

Are bud or birth scars easier to see, why?

A
  • bud scars easier to see, as thicker
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19
Q

How do no. of bud and birth scars differ?

A
  • may have multiple bud scars but only ever 1 birth scar
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20
Q

Why might you want to count the no. bud scars?

A
  • give indication of age
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21
Q

What is the process of bud site selection, when marking the site, and how does this vary between diff cells?

A
  • yeast cells bud and divide in precise spatial patterns
  • position of new bud which will grow to form new daughter cell dep on cell type (for budding yeast this refers to whether cell is haploid or diploid)
  • pattern of bud cells diff between diff cells
  • -> in haploid all adj on 1 side (axial budding)
  • -> in diploid on both sides (bipolar budding)
  • DIAG*
  • by looking at no. and distribution of bud scars can see history of cell
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22
Q

How were genes involved in identifying bud site selection identified by a genetic screen?

A
  • mutants appear spontaneously by exposure to mutagenic conditions or by directed gene deletion
  • changes in budding pattern can be observed microscopically using calcofluor staining, eg. if haploid/diploid cells budding w/ random pattern or if haploid budding in bipolar way (alt which pole cell binds at), ORA
  • genes can then be identified which allows this phenotype to be rescued
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23
Q

What genes are specifically req for yeast axial budding pattern?

A
  • Bud3, Bud4, Bud10 and septins
  • products from these genes are involved in marking the mother bud neck during 1 cycle as a site for budding in next cycle
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24
Q

What do mutations to genes req for yeast axial budding pattern result in, in diploid and haploid cells?

A
  • mutations do not have defects in diploid cells

- mutants show bipolar budding pattern in haploid cells

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25
Q

Why is it important that axial budding pattern in haploid cells dep on cues assoc w/ previous bud site?

A
  • so can recognise where budded previously and then bud next to it
  • mark site where polarised growth has to occur
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26
Q

What happens to the budding pattern if Bud3/4/10 del?

A
  • can still bud, but not in axial pattern, as don’t know where previously budded, so cant put it adj
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27
Q

What happens to budding in absence of axial pattern?

A
  • revert to bipolar pattern
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28
Q

How were genes specifically req for yeast bipolar budding pattern identified?

A
  • by a similar screen to haploid cells
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29
Q

What genes are specifically req for yeast bipolar budding pattern, and where are they located?

A
  • Bud8, Bud9, Rax2 and components of actin cytoskeleton

- Bud8 and 9 on opp ends

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30
Q

What pattern do haploid mutants in genes for yeast bipolar budding have?

A
  • still use axial pattern
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31
Q

What is the phenotype of Bud8, Bud9 and Bud8/Bud9 mutants, what does this show?

A
  • Bud8 mutants cannot bud at distal pole (opp birth scar), so all buds formed at same end as birth scar
  • Bud9 mutants cannot bud at proximal pole (adj to birth scar)
  • Bud8bud9 mutants bud randomly as diploids, but haploid cells bud normally in axial pattern –> show these proteins not req for budding in haploid cells
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32
Q

Where is Rax2 found and what is its role?

A
  • Rax2p at both poles and req to maintain bipolar budding over multiple gens –> not key factor, more of a regulator, may help stabilise bud8/9 prots
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33
Q

How are Bud 8 and 9 localised to poles?

A
  • physically
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34
Q

What genes are req for both axial and bipolar budding patterns?

A
  • Bud1, Bud2, Bud5
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35
Q

What is the role of prots encoded by Bud1, Bud2 and Bud5, and how do they function?

A
  • decode axial or bipolar marks and signal to machinery involved in gen polarity axis
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36
Q

What do mutations to Bud1, Bud2 and Bud5 cause?

A
  • random budding patterns in haploid and diploid cells –> must be working ds of marker proteins
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37
Q

How do Bud1, Bud2 and Bud5 function together?

A
  • function together in GTPase cycle
  • function as a module, composed of small Ras-related GTPase (Rsr1/Bud1), its regulatory GAP (Bud2) and GEF (Bud5)
  • Bud5 activates Bud1 only in 1 place in cell, where Bud5 is conc, which is where marker proteins are) –> Bud 2 can convert it back (so inactive)
  • DIAG*
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38
Q

What happens after cell has integrated spatial cues from budding landmarks?

A
  • this info is fed to polarity establishment machinery, which is responsible for polarisation of cell cytoskeleton and other cell components
  • polarity axis can then become established
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39
Q

What are an important group of prots involved in polarity establishment?

A
  • Rho-GTPases
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40
Q

What is the most important family of prots for polarity establishment in yeast?

A
  • cdc42

- highly conserved across evo

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41
Q

How were polarity establishment genes identified?

Hartwell

A
  • identified mutants defective in signalling for cell cycle progression
  • some couldn’t direct growth to form new bud (cdc24, cdc42, cdc43) –> just get bigger w/ no division
  • cdc42-1 Ts mutant –> at permissive temp can polarise, form bud, grow and divide and at restrictive temp show isotropic growth and cannot establish axis of polarity
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42
Q

Why might Ts for growth not be the best screen for identifying polarity establishment genes?

A
  • as also about cell prolif
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43
Q

How does cdc42 function to establish polarity?

A
  • master regulator
  • small Rho-GTPase
  • reg through cycles of activation and inactivation by it binding partners cdc24 (GEF) and several GAPs
  • cdc24 binds to active form of - Bud1 at sites marked for budding, can then activate cdc42 to allow polarity site to become established (= localised activation of Cd42)
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44
Q

How is bud site initiation coupled to cell cycle and the site established?

A
  • bud initiation takes place in late G1 and occurs only once during cell cycle –> so cell cycle control tightly linked to bud site initiation and cdc42 activity is focus of this reg
  • before bud forms, active form of cdc42 accum at site where new bud will form under pm
  • bud forms and grows to certain size –> until about 1/3 size of mother cell
  • once this size still polarised growth but grows in all directions instead of 1 (still polarised as only bud growing)
  • in late anaphase/telophase a 1° and 2° septum form –> allows sep
  • all dep on cdc42 and other landmark proteins → septins direct cdc42
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45
Q

How does cdc28 reg temporal events in cell cycle?

A
  • works on cdc24 by 3 diff mechs:
    1) +vely reg GEF
    2) blocking GAP
    3) reg availability of GEF
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46
Q

Where is cdc28 present, and in complex w/ what?

A
  • present in nucleus in complex with Far1

- but Cdc28 phos Far1 in G1, so ubiquitinated, and Cdc24 released into cytosol

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47
Q

What happens to budding w/o landmark prots or Bud1 mol?

A
  • cells form single bud and prolif

- but bud at random places

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48
Q

How is it poss that cells w/o landmark prots or Bud1 can still bud?

A
  • cdc42 can recruit cdc24 (in Bem1 complex) = recruits its own GEF
  • DIAG*
  • +ve feedback loop
  • local explosion of cdc42, even if no landmark prots
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49
Q

What is the consequence for the actin cytoskeleton when growth isn’t polarised, or when growth direction in bud reverses?

A
  • actin cytoskeleton not polarised either

- actin cytoskeleton direction also reversed

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50
Q

What are later events in actin cytoskeleton dep on?

A
  • cdc42
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51
Q

What is the gen role of the actin cytoskeleton?

A
  • forms tracks for transport of material
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52
Q

What is the role of actin cables?

A
  • delivery of vesicles to sites of polarised growth
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53
Q

What is the role of actin patches?

A
  • where endocytosis occurs (endocytosis v dep on actin in yeast)
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54
Q

What is the role of the contractile actomyosin ring?

A
  • septum formation
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55
Q

How is actin cytoskeleton diff in Ts cdc42 mutant?

A
  • no actin cables
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56
Q

How is cdc42 reg and what are its effectors?

A
  • DIAG*

- effectors = Ste20, spetins, Gic1/2, polarisome (1st), formin, actin cables, Sec3

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57
Q

What is the role of formins?

A
  • initiate assembly of actin cables
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58
Q

What is the polarisome?

A
  • complex of prots that localise to incipient bud site and to tip of newly growing bud
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59
Q

What are the known components of the polarisome?

A
  • Spa2, Pea2, Sph1
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60
Q

What does the polarisome appear to be important for?

A
  • linking Rho-GTPase signalling to actin filament assembly and for localisation of formin prots (Bni1, Bnr1), which drive actin assembly
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61
Q

What kind of prots are the septins?

A
  • landmark prots
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62
Q

What are the septins and what is their general structure?

A
  • family of structurally related prots containing GTP-binding domain and usually coiled-coil region
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63
Q

What is the role of the septins?

A
  • cytoskeletal element involved in cell polarity and cytokinesis –> establish actin cytoskeleton, then site of polarised growth
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64
Q

In yeast, which septins form the septin ring, and what happens in vitro?

A
  • cdc3/10/11/12
  • assoc to form filaments in vitro
  • in vivo form ring at mother bud neck
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65
Q

What is the role of the septin ring?

A
  • forms boundary between mother and bud during isotropic bud growth, to limit movement of material between 2 parts of cell
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66
Q

What is the result in some mutants whose septins look diff?

A
  • can’t direct their growth properly
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67
Q

How is cdc42 important in the role of septins, and what are other ds factors?

A
  • cdc42 plays role in septin ring assembly

- other ds factors are Rho GTPases

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68
Q

What is the phenotype of mutants in maintaining the site?

A
  • allow formation of new bud, but subsequent stages are aberrant
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69
Q

What critical processes are there involved in maintaining the site?

A
  • targeting of vesicles to actively growing site
  • polarised membrane growth and synthesis
  • directed deposition of new cell wall
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70
Q

What is the role of Rho prots in maintaining the site (and what is the result of mutations)?

A
  • small GTPases appear to play a role at this late stage of cell polarity dev, inc Rho1
  • Rho1 mutants arrest and lyse as small budded cells
  • Rho1 important for ensuring cell wall machinery active at sites of growth
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71
Q

What is the role of endocytosis in maintaining the site?

A
  • key aspect of cell polarity is that growth is focussed in particular area of cell –> achieved by marking the membrane
  • but new membrane being added, so how does marker itself remain polarised?
  • -> some prots could dissoc from membrane and relocalise, eg. through assoc w/ secretory vesicles
  • -> integral membrane prots, eg. Bud8, can only really be removed by endocytosis and recycled (or degrad)
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72
Q

What is the role of secretion in maintaining the site?

A
  • directed secretion critical to maintain the site
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73
Q

Why is endocytic recycling necessary to maintain cell polarity, and how does it work?

A
  • when start delivering material to bud tip, marker pushed sideways
  • if no recycling prot dispersed around bud
  • if endocytic recycling (of eg. Bud8), then can be recycled and refocussed back to bud tip
  • DIAG*
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74
Q

How does the direction of cell growth change t/o the cell cycle?

A
  • early G1 can grow in any direction
  • polarisation of secretion in late G1, leading to bud emergence
  • apical-isotropic switch in early G2, a depolarisation of growth w/in bud leading to uniform bud expansion
  • breakdown of mother-bud asymmetry in growth in late ano/telo –> all growth evenly distrib to mother and bud, instead of all to bud (as before)
  • refocusing of growth toward neck upon mitotic exit, leading to cytokinesis and cell sep
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75
Q

What does secretion and delivery of newly synthesised material follow?

A
  • where cdc42 is
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76
Q

What is the secretory pathway essential for?

A
  • for growth of pm (polarised delivery)
  • dep of cell wall prots (plants and fungi)
  • mem prots –> GF receptors, permeases etc.
  • secretion of mols to outside –> hormones, Abs, blood components, mating pheromones (fungi), ec enza (invertases, phosphatases, lipases)
  • PTMs –> glycosylation, S=S etc.
  • sorting of prots taken up by endocytosis
  • formation of vacuoles/lysozyme
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77
Q

How did pulse chase and autoradiography in mammalian cells determine the order of the secretory pathway?

A
  • gave cells radioactive AAs
  • incorp into newly synthesised prots
  • wash away AAs not taken up
  • can follow radioactive prots through their lifetime
  • made slices of certain pancreatic areas v active in secretion of enzs and prots
  • cells still active in sorting prots through secretory pathway
  • saw radioactivity levels moving from 1 part of cell to other –> gave idea of series of events that occur during secretion
  • ER –> golgi –> pm - secretory granules? (where see radioactivity)
  • but looking at still pics, no movement and trying to interpret
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78
Q

What happens to cell division, and prot and lipid synthesis when mutation affects delivery of newly synthesised material to areas of cell growth (pm)?

A
  • cell division stops
  • but prot and lipid synthesis continues
  • these cells will have diff composition, size, shape, density etc.
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79
Q

How were sec mutants identified, and what were the results?

A
  • enrichment to identify mutants in secretion –> cells blocked in secretion become dense, sep by density gradient centrifugation
  • sep mutants Ts for growth (need to be Ts as mutant should be lethal)
  • screen: release of invertase (secretion), EM, ts dec mutants blocked in secretion of invertase and acid phosphatase
  • looked at EM and found diff mutants, diff morphologies of ER –> eg. sec23, sec7
  • found 23 diff complementation groups, prob reflets 23 genes
  • 3/4 diff phenotypes visible by EM out of 23 genes, so could group genes
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80
Q

How was Sec23 morphology diff?

A
  • ER wider and more extensive, so lumen bigger, prob consequence of storage of more prots in ER that can’t be transported out
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81
Q

How was Sec7 morphology diff?

A
  • ER normal but golgi massively over exaggerated (Berkeley bodies), so delivery to golgi fine but no exit from golgi
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82
Q

What was the morphology of sec17/15 mutant?

A
  • accum of vesicles –> secretory vesicles not being delivered to correct place?
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83
Q

What can epistasis be used to indicate?

A
  • order of action of genes in a linear pathway
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84
Q

How does epistasis show the order of events?

A
  • eg. white → red → purple → black
  • if block earlier in pathway and if put later block, then phenotype of later block will not be visible (ie. if mutation in white to red step and purple to black step, then get white phenotype)
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85
Q

What did epistasis show about the sec mutants?

A
  • in sec1 saw vesicles accum
  • in sec7 saw berkeley bodies
  • if make sec1/7 double mutant get sec7 phenotype, so sec7 must act upstream of sec1
  • in sec18 some small vesicles accum, if combined w/ other phenotypes get same phenotypes, so must act upstream of all others (thus v early acting)
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86
Q

How were early acting sec mutants subdivided, and what is the result in mutants?

A
  • careful EM analysis of early mutants found 2 classes:
  • -> Class 1: sec17, 18, 22 = accum ER and vesicles
  • -> Class 2: sec12, 13, 16, 21 = only accum ER
  • all class 1/2 double mutants accum ER, so class 1 acts upstream of class 2
  • mutants in same class are synthetically lethal (ie. unable to grow at permissive temp)
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87
Q

What is essential for the glycosylation of prots?

A
  • ER and golgi
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88
Q

How is glycosylation affected in mutants blocked in ER exit?

A
  • accum invertase only w/ core glycosylation (as can’t get further glycosylation as can’t exit)
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89
Q

How is glycosylation affected in mutants blocked in transport from golgi and secretory vesicles delivery?

A
  • fully glycosylated
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90
Q

How does size of sec prots differ by amount of glycosylation they receive?

A
  • sec1 normal size of WT band
  • sec7 quite heterogeneous, prots actually reaching golgi
  • sec18 only core glycosylation (and in double mutants) –> can’t reach golgi
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91
Q

Why is the secretory pathway not linear?

A
  • also retrograde pathway and branch pathways –> makes it much more complex
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92
Q

What is the reasoning for the phenotype of mutants where vesicles only accum in bud?

A
  • these vesicles are post golgi, delivered into bud (doing polarised delivery), but can’t fuse w/ pm
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93
Q

What are the 5 diff classes of mutants in diff stages of secretion, the fate of secreted prots and the defective function?

A
  • A: accum in cytosol; defective in transport into ER
  • B: accum in RER; defective in budding of vesicles from RER
  • C: accum in ER to golgi transport vesicles; defective in fusion of transport vesicle w/ golgi
  • D: accum in golgi; defective in transport from golgi to secretory vesicle
  • E: accum in secretory vesicles; defective in transport from secretory vesicles to cell surface
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94
Q

What is sec4 and where is it found?

A
  • a Rab GTPase found on post golgi vesicles
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95
Q

What does sec4 have homology to?

A
  • Ras GTPase (GTPases are crucial in mechanisms of polarised growth)
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96
Q

What is sec4 synthetically lethal w/ and what does its overexp result in?

A
  • synthetically lethal w/ other ts mutants in late secretory group at permissive temp
  • overexp rescues mutants in late secretory group → suggests can suppress phenotype if enough sec4
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97
Q

How are actin cables orientated in the cell?

A
  • towards bud
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98
Q

What is transport along actin cables dep on?

A
  • myosin
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99
Q

What is the role of Myo2?

A
  • transport cargo, freq organelles along actin cables
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100
Q

How does Myo2 move?

A
  • forms dimer and walks along actin cables
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101
Q

In what way are cells v sensitive to actin levels?

A
  • can’t KO actin = dead cells

- can’t overexpress, add extra copy etc. = dead cells

102
Q

What is the role of tropomyosin and what is the result of a KO?

A
  • stabilises actin cables

- no phenotype if KO 1 tpm (if both KO then lethal)

103
Q

What is the experimental evidence for the fact that actin cables are req for delivery of secretory vesicles to bud tip, and how this a reversible Ts mutant?

A
  • in bud see lots of actin when tpm present, actin cables and patches
  • if shift temp w/in 2 mins no actin cables
  • actin cables fall apart v quickly when no longer stabilised and sec4 no longer polarised
  • need actin cables for polarisation of sec4 (and for polarised growth generally) –> mutants form giant cells as cannot bud
  • tpm not broken down, just not active, still swimming around in cytosol, so if switch temp back can build actin cables again (ie. its reversible ts mutant) –> after 1 min of switching temp can see its reversed
104
Q

What are the steps of depolarisation and repolarisation in response to loss and gain of tpm containing cables in tpm1-2 tpm2Δ cells?

A
  • give healthy cell temp shock –> lose all polarisation, get big cells as cannot bud
  • but if switch temp back can start budding again
105
Q

How was it tested if Myo2 is req for delivery of secretory vesicles, and what was discovered?

A
  • Ts mutation (as myo2 essential)
  • at permissive myo2 polarised
  • but at restrictive 5 mins later no longer polarised –> sec4 is marker for secretory vesicles and also loses polarisation
  • Myo2 req for polarisation of Sec4
106
Q

Where are formins found during actin cable assembly?

A
  • Bni1 mainly at bud tip, then later at bud neck

- Bnr1 mainly at bud neck

107
Q

What happens if KO Bni1 and/or Bnr1, how did this influence experiments done?

A
  • if KO 1 then other can generally compensate, not much goes wrong
  • if KO both = cells dead
  • experiments where KO 1 and did ts mutant of other
108
Q

What were the effects of formin mutations on actin cables and sec4?

A
  • at restrictive temp no actin cables, but if switch back to permissive they can form
  • effects on sec4 –> at restrictive temp no polarisation, when switch back to permissive able to polarise again
  • at restrictive temp mutants form giant cells (no budding)
  • formins req for actin cable assembly at bud tip and neck
109
Q

What does sec4 assoc w/?

A
  • secretory vesicles
110
Q

What is req for sec4 localisation to sites of polarised growth?

A
  • actin cable assembly, myosin (myo2) and sec2
111
Q

In what mutants is actin cable assembly disrupted?

A
  • formin and tpm mutants
112
Q

What is the machinery that facilitates delivery of secretory vesicles to sites of growth?

A
  • late acting Sec genes
113
Q

What is the phenotype of late acting sec gene mutants?

A
  • accum vesicles in bud after shift to restrictive temp
114
Q

What is the exocyst?

A
  • complex of many late acting sec prots
115
Q

What makes up the exocyst, and how are these prots assoc?

A
  • sec4 (small GTPase) physically interacts w/ sec15

- sec3, exo70, sec5, sec6, sec8, sec10 and sec15 physically assoc

116
Q

How are the components of the exocyst localised?

A
  • localise to bud tip

- sec3 localises indep, doesn’t need actin cables and myosin etc. (at bud tip), assoc w/ membrane –> spatial landmark?

117
Q

What diff experiments showed the components of the exocyst which are physically assoc?

A
  • 2 hybrid
  • immune precipitation
  • sucrose density centrifugation
118
Q

How is sec3 localised, and how was this shown experimentally?

A
  • time lapse localisation of sec3-GFP shows its emergence at bud tips
  • gets to bud tips indep of secretory pathway and actin
  • dep on cdc28, cdc42 and rho1
119
Q

Where is ypt31 found and what is its role?

A
  • small GTPase present at late golgi where secretory vesicles emerge from
  • ypt31 recruits sec2, GEF of sec4 (like bud1 recruiting cdc24)
  • sec4 now assoc w/ GTP and active
120
Q

How does polarised secretion occur in budding yeast, and why is this model simplistic?

A
  • sec4, w/ sec2, can recruit myosin to vesicle
  • at bud tip cdc42, polarisome and from there actin cables emerging
  • so have secretory vesicle w/ motor prot attached that can be directed to site of cdc42
  • then exocyst complex assembles on vesicles and can interact w/ sec3/exo70 complex at pm
  • v localised delivery of vesicles w/ newly synthesised enzs that have to be delivered to pm
  • myo2 also interacts w/ sec15
  • v simplistic model, as there are many more prots and prot interactions
121
Q

What is the consequence of the fact that polarisome and actin cables change position during diff stages of cell cycle?

A
  • will change where vesicles are directed
122
Q

Once delivered to a specific site, how are they able to fuse w/ cell mem?

A
  • facilitated by SNARE prots
123
Q

What is cytokinesis?

A
  • process leading to sep of cyto of dividing cells, thus leading to increase in cell no.
  • involves in excess of 100 prot components and can only be effectively studied in vivo
124
Q

Why must cytokinesis be tightly reg?

A
  • to ensure cell doesn’t divide before mitosis complete (checkpoints to ensure all chrom segregated, other parts of cell etc.)
125
Q

How is cytokinesis distinct in animals and fungi?

A
  • process in animals and fungi distinct (pm pulled in by actomyosin ring, as actin synthesised in ring like structure then contracts, then divide cell) from plants, algae and ciliates
  • latters don’t contain myosin II and do not gen contractile ring
  • cytokinesis ring evolved about 1 bil years ago
126
Q

What are the major considerations in cytokinesis?

A
  • division site positioning (marking and decoding the site)
  • contractile ring assembly (site establishment)
  • mem and septum deposition (in yeast)
  • co-ord of cytokinesis w/ nuclear cycle
  • cell sep
127
Q

What are the morphological events in cytokinesis?

A
  • if start w/ mother cell, already have polarised cytoskeleton
  • have actin myosin ring between mother and bud
  • ring contracts later in cell cycle –> after nucleus entered bud and divided
  • septins remodelled –> so 2 rings, 1 in mother and 1 in daughter
  • ring pulls in septum, so septum sep them, then build cell wall on either side, to make sure when cells split they don’t lyse
  • once ring closed no connection between mother and daughter
  • DIAG*
128
Q

What are the main steps/principles of mem depolarisation and septum assembly?

A
  • mem remodelling inherent to cell division, single pm that surrounds mother cell must be subdivided into 2 separable domains at end of cytokinesis
  • new mem also req to gen increased SA of new daughter cells
  • in yeast many components assoc w/ secretion relocate to bud neck region during late anaphase (just before cytokinesis) –> important among these are myo2 and exocyst complex, cdc42, rho1, these GTPases may coord reg of actomyosin ring and targeted membrane deposition
129
Q

What are the main morphological events in yeast?

A
  • furrow ingression coupled to septum formation
  • 2° septum deposited
  • then 1° and 2° need to be removed so cells can split
130
Q

How is the division site selected for cytokinesis, and what genes are req?

A
  • S. cerevisiae estab plane of division assoc w/ growth of new daughter which grows by localised growth to prod new bud –> site already determined right at start of cell cycle
  • thus marking site req genes that are normally involved in bud site selection
131
Q

How is cdc42 important for decoding and establishment of bud site and cytokinetic ring occur?

A
  • if a site is marked by bud 1/2/5 module, this will serve as basis of recruitment of cdc42 and hence activation of cdc42
  • but if site isn’t marked yet, yeast cells can grow in polar way and divide, but site selected random
  • for growth and division cdc42 must be activated by cdc24 at cell surface
  • cdc42 effectors = Gic1/2 prots are then req for recruitment of key structural prots in cytokinesis, the septins
132
Q

What is a model for contractile ring assembly (estab the site) in cytokinesis?

A
  • recruitment –> arrival of prots at bud neck is sequential starting from late G1 to cytokinesis
  • ring assembly –> req actin and myo1 to form, Bni1 activated to trigger actin polymerisation and final assembly of contractile ring by rho1
  • ring maturation –> bud starts to grow
  • ring contraction
133
Q

What is a model for reg of actin cable assembly at bud tip and neck?

A
  • septins form ring and thought to be scaffold for other prots to assemble on
  • formins thought to drive actin ring nucleation during cytokinesis
134
Q

What is the phenotype of septin mutants?

A
  • prevent cytokinesis and cells arrest as large budded cells
135
Q

How does cytokinesis result in cell sep?

A
  • septin ring splits in 2
  • actomyosin ring closes
  • dep of chitinous 1° septum
  • dep of 2° septum on either side
  • daughter prod chitinase to break down 1° septum
136
Q

How is cytokinesis coord w/ the nuclear cycle?

A
  • assembly of actomyosin ring and delivery of new membranes dep on entry to mitosis
  • constriction of the ring dep on proteolysis of cyclin B
  • this co-ord carried out in part by mitotic exit network (MEN)
  • this signalling cascade 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
137
Q

What direct role does MEN play in cytokinesis?

A
  • localises to the Spindle pole bodies and some of the components translocate to the bud neck
138
Q

Where is cdc42 present t/o the cell cycle?

A

DIAG

139
Q

Where is actin present t/o the cell cycle?

A

DIAG

140
Q

Where are septins present t/o the cell cycle?

A

DIAG

141
Q

What diff ways can organelles be multiplied?

A
  • growth and division
  • templated assembly/growth (eg. spindle assembled this way)
  • de novo formation
142
Q

What are the 2 main systems of organelle biogenesis, and what organelles are formed in each way?

A
  • endomembrane system = ER and ds organelles
  • autonomous organelles (can’t form de novo as would lose genome, form by fission) = mt, chloro, peroxisomes (considered part of autonomous, although dont have own genome)
143
Q

What cells are peroxisomes found in?

A
  • all euk cells
144
Q

What mem do peroxisomes have?

A
  • bound by single mem
145
Q

Are peroxisomes abundant?

A
  • no, low abundance
146
Q

What is the role of peroxisomes?

A
  • FA beta oxidation
  • several other H2O2 prod ox reactions
  • catalase degrades H2O2
147
Q

What is the old model for peroxisome biogenesis?

A
  • can grow bigger then lyse, by importing prots from cytosol, inc mem prots
  • not that far off
148
Q

What is the significance of peroxisomes, ie. implication in human disease?

A
  • Zellweger syndrome = no or v low no.
  • rare = 1:50
  • enzs mislocalised to cytosol, not functioning, or broken down completely
149
Q

What are the symptoms of Zellweger syndrome?

A
  • lots of metabolic pathways affected
  • lots tissues not functioning correctly
  • bones not formed properly
  • most die before 1 y/o → but can survive longer if milder
  • metabolism and dev affected
150
Q

How were Zellweger patients subdivided into 14 complementation groups?

A
  • cell fusion experiments
  • took cells from patient A and B –> neither have peroxisomal enzs in peroxisome
  • used catalase as marker, as should be stable in cytosol
  • incubated in PEG
  • cells fused, so cytosol of 2 cells mixed
  • saw catalase uptake when mixed certain patients
  • if fuse get complementation occuring
  • if both patients fusing have same mutation then don’t complement –> still have cytosolic catalase
  • sometimes get rescue, so 14 complementation groups doesn’t necessarily mean 14 genes
  • if mild phenotype in 1 fam, then generally inherited as mild phenotype
  • sometimes there are differences w/in fams, so genes in genetic background could affect
151
Q

Patients are generally what w/ respect to peroxisome biogenesis disorders?

A
  • heterogenous
152
Q

What are diff names for peroxisome biogenesis diseases a result of?

A
  • caused by mutations in same complementation group, just differ in severity
153
Q

How were genes assoc w/ peroxisome biogenesis orders discovered, and what mutation was found?

A
  • assays for peroxisome dysfunction in yeast
  • req peroxisomal beta oxidation to break down FA in yeast → if can’t do this cant grow on this medium (or grown v slowly)
  • many of mutants had mt defects too as this also req for growth, but these screened out
  • oleate nonutil → mutation in FA and peroxisome formation
154
Q

What diff classes of peroxisome biogenesis mutants were found?

A
  • 1 = matrix prot import: import defect, but mem prots still in mem
  • 2 = membrane biogenesis: don’t synthesise normal peroxisome membranes, also don’t have membrane or luminal prots, so don’t have any peroxisomes
    » most severe cases
  • 3 = affect shape, no. and distrib (actin/myo etc. req for transport of peroxisomes)
  • classes 1 and 2 of most interest
155
Q

How were genes involved in peroxisome biogenesis found by homology to yeast PEX genes, and what was found in patients?

A
  • identify candidate human PEX genes
  • transfect putative PEX genes into patient fibroblasts and test for complementation
  • identify mutations in patients copies of gene that complements
  • -> if dont complement, GFP in cytosol
  • -> if do complement, get a rescue
  • set up prenatal diagnosis test
  • about 70% patients have mutation in PEX1/6/26
156
Q

What else is part of the spectrum of diseases caused by mutations in peroxisome biogenesis, and what does this cause?

A
  • RCDP

- dwarfism, cartilage doesn’t form correctly, calcium crystals in joints

157
Q

Why is class 3 of peroxisome biogenesis mutants milder?

A
  • have peroxisomes, just reduced no. and shape not right
158
Q

What prots are important in cell bio of peroxisomes, and understanding how mutants are formed?

A
  • PTS1 = peroxisomal targeting signal for most peroxisomal enzs, C-ter tripeptide (SKL-COOH) –> some variation allowed (A/P for S and R for K)
  • PTS2 = peroxisomal targeting signal for some peroxisomal enzs, consensus seq close to N-ter
  • some matrix prots contain neither PTS1 or 2 → most of these are piggy back imported
  • peroxisomal mem prots (PMPs) still targeted to membranes indep of class 1 genes –> at least 3 routes for PMPs
159
Q

What is a model for prot import showing roles of pex prots?

A
  • newly synthesised prot recognised by receptor (pex5)
  • complex docks on mem
  • somehow forms pore in mem, t/ which prots can be translocated
  • pex5 ubiquitinated and extracted from mem
  • pex1/6/15 is extraction machinery (1/6/26 in humans)
  • ubiquitination of Cys = reversible
  • pore needs to be flex to fit small to large octameric prots across mem
  • PTS2 pathway is essentially the same, only diff is receptor is pex7
  • adaptations t/o evo, but this is general mech found
160
Q

What are the dynamics of peroxisomes during cell division in WT cells?

A
  • after 10 mins half peroxisomes rep in bud –> not by chance, as vol here so much smaller, so must be mech to get them into bud
  • some peroxisomes anchored, some moving = good way of making sure can segregate organelles, as anchored have to stay in mother cell
  • can see diploid cells as bipolar budding
161
Q

What does it show that peroxisomes are still present in cells w/ pex deleted?

A
  • no peroxisomes inherited
  • so can form de novo
  • reportedly come from ER
162
Q

What did a pulse chase labelling experiment show about whether WT cells form peroxisomes de novo all the time?

A
  • GFPPTS marker under Gal1 promoter (inducible)
  • 3 hour induction, then off
  • if peroxisomes multiply by fission then pre-labelled peroxisomes w/ each cycle will get dimmer but no. stays constant
  • if de novo then over time peroxisomes will keep same intensity of fluorescence, but no. of fluorescently labelled ones will decrease
  • but prot level was constant, so marker prot not being degraded, peroxisomes dimmed, but similar no. present, just fainter, suggests peroxisomes multiply mainly by fission
163
Q

How was it tested if dilution was a consequence of fission only, and what were the findings?

A
  • through simple mating experiment:
  • -> MatA cells and Matα cells pulse labelled diff
  • -> then mating
  • found peroxisomes don’t fuse, they multiply by fission
  • but are mutants where peroxisomes not present, but can form peroxisomes when put gene pack in (ie. mutants lacking peroxisomal remnants can be complemented)
  • therefore peroxisomes can form de novo, but usually multiply by fission
164
Q

What is Pex3-GFP a marker for, and what is seen if follow it?

A
  • marker for de novo peroxisome formation
  • if switch off Pex3 and follow over time
  • in pex19 mutant, pex3 appears 1st in ER
  • saw Pex3 in dots is assoc w/ ER (5-10 dots per cell) –> become peroxisomes
  • de novo formation from ER –> can’t make mem completely de novo, has to come from somewhere
  • w/o Pex19 can’t form de novo peroxisomes from ER
165
Q

How was it tested experimentally if de novo and fission pathways could happen sim in single cell?

A
  • red marker constit exp –> labels all peroxisomes
  • green marker shows material in already existing peroxisomes, so if this is in peroxisomes shows made by fission
  • pulse chase experiment
  • red only means no markers from prev peroxisomes, therefore formed de novo
  • found no peroxisomes are formed de novo, in cells already containing peroxisomes
166
Q

When are peroxisomes formed de novo, how was this found experimentally?

A
  • in cells failing to inherit them
  • used same markers as prev
  • grew cells in colony on glucose/agarose pad
  • took mutant unable to pass on peroxisomes
  • saw daughter cells had none of original green marker, but did have red marker, so had peroxisomes
167
Q

What is inps2 req for, and what happens in mutants?

A
  • inheritance of peroxisomes

- mutant can’t pass onto daughter cells

168
Q

What is dynamin and what is its role?

A
  • conserved GTPase
  • req for scission of vesicles from pm, and releases into cytosol
  • assembles into oligomeric rings, which form collar around vesicle and pinches it off
169
Q

How could dynamin related GTPases be involved in mem fission?

A
  • self assembling

- signalling GTPase (–> recruiting other enzs) or mechanoenz (–> pinching/stretching mem so vesicle released)

170
Q

What dynamin related prots are present in S. cerevisiae?

A
  • vps1p = vacuolar prot sorting
  • dnm1 = mt fission
  • mgm1 = mt inner mem fusion/remodelling
171
Q

What is the phenotype of Δvps1 cells?

A
  • 1-3 peroxisomes per cell
  • strongly reduced no. and unusual shape
  • normal size, but likes bead on a string structure
172
Q

What are the results of KO of vps1 and/or dnm1?

A
  • KO dnm1 doesn’t have much effect
  • double KO = every cell has 1 peroxisome
  • if look at mt in single KO, then see phenotype, as dnm1 machinery is only machinery that can divide mt
173
Q

How is vsp1 recruited?

A
  • still not known
174
Q

What is the result of dnm1 overexp?

A
  • rescues fission defect and corrects peroxisome abundance in vps1Δ
  • but if KO cofactors req for dnm1 function then doesn’t happen
175
Q

What 2 indep machineries are there for peroxisome function?

A
  • DIAG*

- believe Pex27 is factor X

176
Q

What mating assay was carried out for peroxisome fission, and what did it show?

A
  • pulse chase experiment
  • mating w/ cell w/o peroxisomes
  • unlabelled pex3Δ –> provide vps1p
  • saw peroxisomes divide in a vps1 dep process
  • if do same experiment in cell w/o vps1, then peroxisome doesn’t divide
  • shows vps1 req to divide peroxisomes
177
Q

What was theorised as the reason why don’t get de novo formation when peroxisomes already present?

A
  • as material fusing w/ already present peroxisomes, rather than forming new peroxisomes
178
Q

Where is newly synthesised pex3-GFP present?

A
  • assoc rapidly w/ all peroxisomes
  • 1st visible in ER (v faint)
  • visible in ER in pex19Δ cells
179
Q

Why is peroxisome no. not constant?

A
  • prolif when needed –> growth on FAs as sole carbon source to compensate for mt dysfunction
  • actively degrad when no longer req –> switch from oleate back to glucose, nitrogen starvation
180
Q

How does peroxisome phenotype differ in cells where there is only 1/few?

A
  • bigger
181
Q

What happens to peroxisomes dep on which carbon source they are grown on?

A
  • growth on glucose –> lower no. and weaker signal
  • growth on oleate –> no. peroxisomes increase and increased signal
  • growth on glucose –> pexophagy (degrad), lower no. but see dimly fluorescent structure = vacuole
182
Q

What does peroxisome prolif req?

A
  • induction of β-ox enzs

- induction of a few genes req for peroxisome biogenesis (inc Pex11)

183
Q

What is the oleate response, and how was it used experimentally?

A
  • oleate strongly induces certain genes
  • can do simple genetic screen, to identify factors involved in signal transduction and promoters containing OREs → identified oleate activating factor 1 (OAF1) and PIP2
  • these TFs work together to reg induction of OREs
  • overall oleate response is a key transcriptional activator –> oaf1/pip2 heterodimer
  • recognises oleate response elements in promoters
  • acts via a asymmetric +ve feedback loop
184
Q

How does presence of glucose affect the oleate response?

A
  • oleate doesn’t induce genes, as glucose represses all the promoters
  • ie. in presence of glucose no induction of oleate response, so no prod of peroxisomes, because cells prefer glucose to grow on
185
Q

How does the oleate response act via an asymmetric +ve feedback loop?

A
  • heterodimer forms in response to a signal (eg. presence of oleate) to reg activity of ds target
  • 1 component of heterodimer (OAF1) acts as a sensor of signal and 1 component is also target of feedback (PIP2) reg by the heterodimer itself
  • PIP2 always low conc, won’t dimerise w/ OAF1 unless OAF1 is active
  • so when add oleate in absence of glucose, get a bit of OAF1 initially activated, can dimerise w/ little bit of PIP2 thats in cell and forms heterodimer, which binds ORE
  • PIP2 also has ORE, so much more PIP2 transcribed, get more heterodimer, activate PIP2 prod further, etc.
  • system has slow induction, is v sensitive to levels of TF and there is a plateau, will never get more OAF1, even if keep prod PIP2
  • precise, tunable and robust control of responses to env stimuli
186
Q

How is the Gal4 system an eg. of symmetric +ve feedback?

A
  • Gal RE, activates Gal4, a TF that works as a homodimer, so Gal4 activates its own prod
  • so lots of Gal4 prod, then ds enzs containing Gal4 binding sites are upreg v quickly and w/in 15 mins have v high exp
187
Q

Why would a symmetric +ve feedback system not be ideal in case of OREs?

A
  • v high exp –> can be up to several % of total prot in

- this would be too much prod of organelles

188
Q

What is another eg. of an asymmetric +ve feedback loop system?

A
  • ASSURE I
189
Q

What is a summary of peroxisome biogenesis?

A
  • multiply by growth and division
  • fission med by dynamin related prots
  • prolif in response to oleate via an asymmetric +ve feedback loop
  • allows accurate reg of peroxisome abundance in response to oleate
190
Q

How does peroxisome position alt in cell cycle?

A
  • DIAG*

- certain peroxisomes retained in mother cells, others transported to bud

191
Q

What does seg of peroxisomes t/o cell cycle follow the pattern of?

A
  • where cdc42 is and how secretory vesicle are transp –> suggests actin cytoskeleton involved
192
Q

How are peroxisomes assoc w/ actin cables?

A
  • most colocalise w/ actin cables
193
Q

Are actin filaments req for directed peroxisome movement, how was this found experimentally?

A
  • traced single peroxisome movement in cell
  • saw moved around cell a lot, many of tracks towards bud
  • but some peroxisomes don’t move around much –> these are ones retained in mother
  • when drug added which means no actin cables, movement stopped –> therefore movement of peroxisomes towards bud is actin dep
194
Q

How was it discovered experimentally that Myo2 CBD overexp inhibits peroxisome seg?

A
  • genetic trace
  • used myosin mutants w/ CBD of myosin:
  • DIAG*
  • express CBD under gal promoter –> if peroxisome w/ receptors that can bind myo CBD, then if prod lots of CBD, myosin itself unable to bind any longer and should stop peroxisome movement
  • if no galactose in medium then peroxisomes well distrib between mother and daughter
  • but if add galactose, so inducing only CBD, then blocks receptors on peroxisomes for normal myosin to bind and all peroxisomes stay in mother cell
195
Q

What is the importance of Myo2 in peroxisome seg?

A
  • looked at Ts mutants
  • at permissive temp, Myo2 active and peroxisomes well distrib
  • if raise temp and look just at cells w/ newly forming buds, see buds have no peroxisomes
  • so peroxisomes rely on Myo2 to be transp into bud
196
Q

How are peroxisomes transported to be seg?

A
  • transport along actin cables
  • myo2 is the motor prot –> some peroxisomes recruit myo2, allowing it to be delivered to the bud, and others don’t so they are retained in mother cell
  • peroxisomal myo2 receptor is inp2
197
Q

Where is inp2 found, and what is its role?

A
  • in peroxisome mem
  • binds myo2 in vitro –> connect myo to peroxisomes and allows transp to occur
  • req for peroxisome seg
198
Q

What is the phenotype of a inp2 KO?

A
  • all peroxisomes in mother
199
Q

What is the role of inp1?

A
  • req for peroxisome retention
  • anchors peroxisomes to periphery of mother cell to retain them
  • also in bud to capture newly arrived peroxisomes
200
Q

What is the phenotype of a inp1 KO?

A
  • opp of inp2 KO

- all peroxisomes in bud

201
Q

What is the phenotype of peroxisomes in vps1/dnm1 del cells, why?

A
  • v elongated peroxisomes

- as retained in mother, but transp towards bud, so stretched out across bud neck

202
Q

How was the role of Pex in making new peroxisomes investigated experimentally?

A
  • made large lib of 1000s of mutants in Pex3 and transformed back into Pex3 KO
  • found group of mutants identical to Inp1 phenotype –> showed peroxisomes in these had retention defect
203
Q

Do inp1 and pex3 work together?

A
  • poss as inp1 works at periphery of peroxisome mem (not integral, so may req pex3 for recruitment of peroxisomes?)
  • inp1 had no homology to any characterised prots
  • inp1 assoc w/ peroxisomes is saturable, shows there is limited no. binding sites on peroxisome for Inp1 to bind to
204
Q

Is pex3 able to recruit inp1 to peroxisomes, what did this mean?

A
  • no
  • suggested they were binding
  • showed they interact directly in vitro
205
Q

Do pex3 and inp1 interact in vivo, how was this tested?

A
  • they did interact
  • mt targeted PEx3 recruits Inp1-GFP
  • tested using split-GFP (if 2 halves come together, ie. binding, then get fluorescence)
  • also tells you where interacts in cell
206
Q

How was it shown that pex3 is req for seq of peroxisomes, as well as formation?

A
  • if overexp inp1 and pex3, get over-retention in mother cel
207
Q

What are the 2 sep functions of Pex3?

A
  • peroxisome formation from ER

- peroxisome retention

208
Q

How does pex3 affect inp1?

A
  • anchors inp1 to peroxisomes
209
Q

Where is it now believed peroxisomes are anchored?

A
  • to pm, not ER
210
Q

How are peroxisomes seg generally?

A
  • retained in mother by anchoring to periphery of cell
  • myosins pull on peroxisomes to transport it to bud
  • dynamin related prots pinch off smaller peroxisomes, transp to bud, and anchored to bud periphery
  • if this happened w/ every peroxisome every cell cycle then would perfectly duplicate all peroxisomes into bud –> this is idealised model and prob not this well reg
211
Q

What were large scale image based genetic screens looking to find out about peroxisomes?

A
  • see if switch between growth and division and de novo formation (in yeast)
  • how peroxisomes grow (in yeast)
  • how peroxisome inheritance is reg w/ the cell cycle
212
Q

How big are de novo formed peroxisomes?

A
  • small, even in cells where peroxisome fission is blocked, but w/ time these peroxisomes grow bigger
213
Q

How was a peroxisome de novo formation assay carried out?

A
  • take cells lacking peroxisomes (eg. lacking pex 3/19 genes)
  • put under control of Gal promoter so switched on, then see small peroxisomes forming
  • in mutant that can’t divide peroxisomes, looks same initially, no peroxisomes, then forms small ones, w/ time grow bigger and no. reduces
  • did genome wide screen: synthetic genetic array
  • screen w/ KO and DAMP lib
  • -> objective: to identify factors req for growth of peroxisomes
  • -> factors that may affect peroxisome tubulation, positioning and inheritance
  • -> selected for Vps1/Dnm1/Pts1 deficient mutant
  • -> measured no. peroxisomes per cell (normally expect 1)
  • -> some cells w/ more = ones forming de novo
  • -> can also look at shape of peroxisomes
  • -> found new gene req for peroxisome inheritance, but also for vacuole inheritance
  • -> no mutants formed peroxisomes de novo except inheritance mutants
  • -> were mutants w/ smaller peroxisomes, may be affected in delivery of membranes
214
Q

What type of enz is inp3?

A
  • a kinase = kin4
215
Q

What is the phenotype of dnm1/vps1/inp1 triple KOs?

A
  • in many cases fail to retain peroxisomes in mother cells

- contrastingly, triple KO retention dominant over inheritance

216
Q

How can synthetic genetic array tech now be used in making libs of KOs?

A
  • mate query strain (selectable marker) w/ lib of known mutants, contains kanR gene
  • select diploids
  • sporulate
  • select Mat alpha haploids
  • select kanR
  • select kanR and other selectable marker
217
Q

How was a SGA screen carries out for peroxisome inheritance, growth and de novo switch?

A
  • KO both dynamin related prots = dnm1, vps2 (get single peroxisome structure)
  • used green marker for peroxisomes (GFP related)
  • crossed with 2 libs: 1 gene deletion lib w/ non essential genes KO and DAMP lib where essential genes partially inactivated
  • selecting for triple KOs
  • for most genes, no effect when mutated in Dnm1/Vps1 KO background
  • few where peroxisome no. increased
  • some where diff structure of peroxisome (ie. blob instead of sausage)
  • for each strain measured av no. per cell, av area and av circularity
  • in many cases dnm1/vps1/inp1 del fails to retain peroxisomes in mother cells, but retention dom over inheritance
  • if KO inp gene alone doesn’t cause this phenotype, it is only in dnm1/vps1 background –> prob as only 1 peroxisome present (wouldn’t matter as much is more peroxisomes in cell)
218
Q

How were DAMP libs where essential genes partially inactivated made?

A
  • by inserting fragment to make mRNA more unstable

* DIAG*

219
Q

What is inp3 partially redundant w/?

A
  • its paralog frk1
220
Q

What is the phenotype if KO inp3 and frk1 in WT background?

A
  • strong inheritance defect (only double KO gives clear phenotype)
  • if add FM4-64 dye, which is endocytosed and upon chase ends up in vacuolar mem, then see vacuoles also not inherited
  • mt inheritance doesn’t seem to be blocked
221
Q

Why was vacuole inheritance studied for kin4 and frk1 function, instead of peroxisome?

A
  • best studied organelle w/ respect to inheritance in prolif cells
222
Q

What is known about vacuole inheritance?

A
  • actin and myo2 dep
  • inheritance blocked in Vac8 and Vac17 mutants
  • Vac17 broken down upon entry of vacuoles in bud
  • Vac8 binds to vacuole and forms complex w/ Vac17 on vacuolar mem, recruits myo2
  • during inheritance
  • -> vacuole big structure in yeast cells
  • -> segregation structure formed, fuse and vacuole deposited in bud
  • -> late in cell cycle Vac17 degrad in bud
  • -> releases vacuole from Myo2
223
Q

What 2 types of reg are req for vacuole inheritance?

A
  • temporal and spatial
224
Q

How is temporal reg of vacuole inheritance achieved?

A
  • cdc28

- phos Vac17 (used analog sensitive versions, cdc28 overexp and in vitro phos assay to show interaction is direct)

225
Q

How was an experiment carried out to show cdc28 dep phos of Myo2 and Vac17 stims binding?

A
  • mutation of vac17 cdc28 phospho sites (4A) results in mild inheritance defect
  • mutation of myo2 cdc28 phospho sites results in no inheritance defect
  • double mutants block inheritance completely
  • Vac17 4A binds less well to Myo2, but still binds Vac8 well
  • Vac8 and Vac17 form complex on vacuolar mem that recruits Myo2
  • interaction of Vac17 w/ cargo binding dom of Myo2 dep on phos cdc28 (if block phos, then no inheritance)
  • contribs to reg of vacuole transport to bud
226
Q

How is breakdown of vac17 upon entry of vacuoles in the bud achieved?

A
  • suggesting release of vacuole from motor prot
  • req spatial regulated degrad
  • release of Myo2 from cargo is reg by the Dma1 ubiquitin ligase
227
Q

What did a genetic screen to identify factors re for Vac17 breakdown find?

A
  • did random mutagenesis of Vac17-GFP strain and FACS (fluorescence activated cell sorting) to enrich for cells w/ more Vac17-GFP
  • 1 mutant showed mislocalisation of vacuoles to bud neck around time of cytokinesis –> gene identified as Dma1
  • increased levels of Vac17 in large budded cells, vacuoles still contain Myo2
  • shows Dma1 req for turnover of Vac17
228
Q

What does Vac17 ubiquitination req?

A
  • req Dma1/Dma2
229
Q

What were the key findings of the experiment into the release of myo2 from cargo being reg by Dma1 ubiquitin ligase?

A
  • controls organelle localisation
  • Dma1 deletion affects position of vacuole in bud
  • Dma1 cells display increased vac17 levels
  • Dma1 recognises Cac17 via T240-phospho in PEST seq
  • Vac17 T240A is stab and affects position of vacuole in bud
  • Dma1 colocalisation dep on T240-P
  • T240 phos can take place in mother cell
  • ubiquitination activity of dma1 is req for vac17 ubiquitination and degrad
  • model for reg of vac17 degrad suggests that vac17 T240 phos occurs in mother
230
Q

Where is Dma1 recruited?

A
  • no experiments to show this

- but proposed to be either bud neck or daughter cell

231
Q

How was it discovered experimentally that spatial reg of organelle release from myosin V transport is by p21 activated kinases?

A
  • mutate thr residue in seq that destab vac17, to ala, then vac17 not broken down
  • when mutate ser222 to ala also stab vac17
  • done by kinase Cla4 or Ste20 (P21 activated kinases)
  • lot of prots in this pathway are redundant/partially redundant –> so hard to find these kinases w/ genetics
  • can’t KO both kinases = lethal
  • can KO 1 and Ts mutation in other –> at permissive vac17 levels v low as always broken down in bud, but if inactivate both, get more present
  • phos pattern also diff –> expected when KO kinases
  • inactivation of PAK kinases –> stab vac17 and repositions vacuoles to bud neck
232
Q

How was it shown in vitro and in vivo that Cla4 phos Vac17 S222?

A
  • gen Ab that specifically recognises peptides of prot when Ser/Thr phos
  • if mutation of Ser222, still get phos of Thr –> thus differentially reg
  • S222 phos is not req for vac17 T240 phos and dma1 localisation to vacuoles
  • always some dma1 present on vacuoles early in cell cycle (not in mutants)
  • S222 phos req for Dma1 dep vac17 ubiquitination
  • -> if cells w/ Dma1/2 in them, exp GFP, see if ubiquitinated
  • -> found Myc-Ubiq expression is induced by CuCl2
233
Q

What is the role of Cla4, and so where is it recruited?

A
  • cdc42 effector

- recruited to sites of polarised growth

234
Q

How does Cla4 perform its role as a cdc42 effector?

A
  • myo2 brings vacuole of bud cortex and Cla4 phos vac17-S222
  • phos of vac17 could recruit another factor, ie. binding partner which then activates dma1 to ubiquitinate vac17
  • OR vac17 could undergo conf change, allows dma1 to ubiquitinate vac17 → occurs in bud
235
Q

What type of kinase is Cla4, what does this mean?

A
  • zonal kinase
  • so only active in bud, mainly around area where cdc42 present, so only when vacuole reaches that area is vac17 broken down (and not before that)
236
Q

What happens if Cla4 pathway blocked?

A
  • get phos, may get dma1 binding, but can’t break down vac17
237
Q

What is the role of kin4?

A
  • req for peroxisome and vacuole inheritance
  • mother cell specific kinase
  • reg spindle position check point
238
Q

How does what happens to nucleus in cell div differ in yeast and mammalian cells?

A
  • in yeast stays intact

- fragments completely in mammalian cells

239
Q

How is mitotic exit reg in yeast?

A
  • don’t want mitotic exit unless nucleus in correct position –> reg by kin4 and -vely reg by Cla –> Cla4 activates Lte1, which inhibits Kin4
  • if Kin4 active in mother, blocks mitotic exit via cascade
  • when kin4 inactive (enters bud), get mitotic exit, as no longer inhibits rest of pathway
240
Q

How was role of kin4 in organelle transport investigated?

A
  • can block diff stages of reg (eg. vac17 phos, Cla4 dep phos)
  • when KO frk1/kin4 get clear vacuole inheritance phenotype = present in mother but buds mainly empty, many don’t pass on vacuoles at all
  • when KO vac17, myo2 receptor levels are down
  • hypothesis: Kin4 and Frk1 prevent Vac17 degrad in mother
    –> Kin4 in mother and Cla4 present in bud
    –> maybe Frk1/Kin4 somehow interfering w/ Cla4 activity (so w/ activation of vac17 degrad)
    –> if true expect could rescue Kin4 KO if block breakdown of vac17 via Dma1
    »> this is the case, if KO dma1, then rescue
  • if look at mutations to vac17, where dma1 can’t act (but not KO), see vac17 levels increased in Frk1/Kin4 cells upon block in DMa dep breakdown –> pathways compensate
241
Q

What is the phenotype of elm1 KO and why do not want to work too much w/ mutants like that?

A
  • in elm1 KO vac17 also v downreg and inheritance defect
  • more goes wrong as activates other kinases (not just kin4)
  • shape hyperpolarised
  • so don’t want to work much w/ mutants like this as too much wrong
242
Q

Can Cla4 and Dma1 act on vac17 in mother?

A
  • myo2-D1297N mutant (blocks transport) cannot interact w/ vac17 and this leads to defect in vacuole transport
  • mutant stops recruitment of myosin
  • get massive upreg of vac17 as not broken down in bud
243
Q

If KO frk1/kin4 what happens, and what does this mean?

A
  • breakdown of vac17
  • so Kin4 and Frk1 req to prevent vac17 breakdown in cells w/ myo2 mutant
  • Vac17 degrad in frkΔkin4Δ cells w/ myo2 mutant is Cla4 and Dma1 dep
  • so dma1 dep breakdown can occur in mother, but doesnt
  • Frk1/Kin4 act in mother cell and block dma1 dep breakdown pathway
244
Q

What is another crucial piece of evidence for the fact that Frk1/Kin4 act in mother cell and block dma1 dep breakdown pathway?

A
  • Frk1/Kin4 overexp leads to increased vac17 levels and vacuole close to site of cytokinesis (get same phenotype as if dma1 wasnt working)
  • suggests 2 pathways antagonistic (ie. Kin4 keeps dma1 inactive and Cla4 activates dma1)
245
Q

How is vacuole transport reg in a spatio-temporal manner?

A
  • Kin4 stim assembly of complex (myo2/vac17) and maintains it until leaves zone of where Kin4 active
  • then arrives in zone where Cla4 active
  • these zones can be quite sharp (a little bit of Kin4 might diffuse into bud, but then inactivated by Lt1e)
246
Q

What is the result of inhib of either dma1 recruitment or activity into frk1Δkin4 cells?

A
  • restores vacuole transport and vac17 levels
247
Q

How is organelle transport reg in spatio-temporal manner?

A
  • Kin4 active and inactive zone
  • DIAG*
  • same for Cla4
  • in mother vac17 protected from breakdown, but in bud activated for breakdown by Cla4
  • next trying to find if vac17 direct substrate of kin4 and in LT unravel gen principles of spatiotemporal reg of organelle transport involving same set of mol regulators
248
Q

How do we know vacuoles are req for cell cycle progression?

A
  • all cells have vacuoles, if not inherited can form de novo
  • pulse chase experiment, incubated w/ dye, washed and chased
  • see vacuoles
  • if look at marker constitutively expressed, see all cells have vacuoles, even if v small
  • unlike peroxisomes, cells cannot keep dividing if there no vacuoles, ie. there is a checkpoint to ensure present
249
Q

What are the 3 major events in spatio-temporal reg of vacuole transport?

A
  • assembly, transport and termination
250
Q

What is req for spatio-temporal reg of peroxisomes?

A
  • assembly, transport and termination

- specifically req inp2

251
Q

How is peroxisome transport reg in spatio -temporal manner?

A
  • if Frk1/Kin4 double KO, get lots of buds entering cells completely empty of peroxisomes, will form them later de novo
  • inp2 also downreg
  • if overexp Cla4, also get peroxisome inheritance defect (also affects vacuole inheritance)
  • in Dma1/Dma2 double KO get lots of Vac17, same true of inp2 (phenotype of inheritance defects)

MBB342 - Genetics of Cell Growth & Division (3 decks)

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